METABOLISM OF POLYSACCHARIDES

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

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

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".

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.

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|Peripheral nerve damage (polyneuritis beriberi) or central nervous |

| | |and α-ketoglutarate dehydrogenases, |system lesions (Wernicke-Korsakoff syndrome) |

| | |transketolase; poorly defined function in |Concentration of pyruvate is increased in the patient's blood, the |

| | |nerve conduction |most of which is excreted with urine |

|B2 |Riboflavin |Functional part of coenzymes FAD, FMN in |Epithelial, mucosa, cutaneous, corneal lesions: lesions of corner of|

| | |oxidation-reduction reactions |mouth, lips, and tongue; seborrheic dermatitis |

|B3 |Niacin, nicotinic |Functional part of coenzymes NAD+, NADP+ in|Pellagra: photosensitive dermatitis, glossitis (tongue |

|(PP) |acid, nicotin-amide |oxidation-reduction reactions |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 |Numbness in the toes, burning sensation in the feet, the defeat of |

| | |acyl carrier in Krebs cycle, fatty and |mucous membranes of internal organs, early graying, hair loss, |

| | |other carboxylic acid metabolism) and |various disorders of the skin: the development of small cracks in |

| | |phosphopantetheine (acyl carrier protein in|the corners of the mouth, the appearance of white patches on various|

| | |fatty acid synthesis) |parts of the body. There may also be depressed mood, fatigue. |

|B6 |Pyridoxine, |Functional part of coenzyme PLP in |Dermatitis of the eyes, nose, and mouth. There is mental confusion, |

| |pyridoxal, |transamination and decarboxylation of amino|glossitis and peripheral neuropathy, convulsions (due to lack of |

| |pyridox-amine |acids and glycogen phosphorylase |inhibitory neurotransmitter GABA) |

|B7 |Biotin |Coenzyme in carboxylation reactions in |Seborrheic dermatitis, anemia, depression, hair loss, high blood |

|(H) | |gluconeogenesis and fatty acid synthesis |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 |Megaloblastic anemia: red tongue, anemia, lethargy, fatigue, |

| | |transfer of one-carbon fragments |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 |Vitamin B12-deficiency anemia (in other words pernicious anemia or |

| | |adenosylcobalamin (Methylmalonyl Co A |Addison–Biermer anemia) is one of many types of megaloblastic |

| | |mutase) and methylcobalamin (Methionine |anemias with degeneration of the spinal cord, anemia, fatigue, |

| | |synthase) in transfer of one-carbon |depression, low-grade fevers, diarrhea, weight loss, neuropathic |

| | |fragments and metabolism of folic acid |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 |Scurvy: general weakness, subcutaneous hemmorhages (frequent |

| | |hydroxylation reaction for: |hemorrhages from internals and mucous membranes), gingival |

| | |- collagen synthesis (prolyl- and lysyl |hemmorhages, loss of teeth, formation of spots on the skin, spongy |

| | |residues are hydroxylated by prolyl |gums, yellow skin, fever, neuropathy |

| | |3(4)-hydroxylase and lysyl 5-hydroxylase |Multiple hemorrages in the places of clothes friction are possible |

| | |respectively; |if a person often experiences acute respiratory infections |

| | |- catecholamines and steroid hormone | |

| | |synthesis; | |

| | |It has properties of antioxidant; enhances | |

| | |absorption of iron | |

|A |Retinol |Functional part of visual pigments |Vision impairment hemeralopia (night blindness), xerophthalmia; |

| | |(rhodopsins and iodopsins) in the retina; |keratinization of skin |

| | |regulation of gene expression and cell | |

| | |differentiation; β-carotene (provitamin A) | |

| | |is an antioxidant | |

|D |Calciferol |Stimulation of Ca2+ absorption through |Rickets = poor mineralization of bone; osteomalacia = bone |

| | |intestinal wall, maintenance of calcium |demineralization |

| | |balance and mobilization of bone mineral |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, |Coenzyme in formation of γ-carboxyglutamate|Impaired blood clotting, hemorrhagic disease, osteoporosis and |

| |mena-quinones |residues in structure of: |coronary heart disease |

| | |- factors II (prothrombin), VII, IX, X, |Intestinal dysbacteriosis occurs hemorrhagic syndrome |

| | |XIV, protein S (blood coagulation system); | |

| | |- bone matrix proteins | |

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.

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);

- 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.

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 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.

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? | |

| |Vitamin A | |

| |Thiamine | |

| |Vitamin K | |

| |Vitamin E | |

| |Vitamin C | |

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|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? | |

| |Lack of vitamins PP, H | |

| |Lack of oleic acid | |

| |Lack of linoleic acid, vitamins A, D, E, K. | |

| |Lack of palmitic acid | |

| |Low caloric value of diet | |

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|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? | |

| |Folic acid | |

| |Thiamine | |

| |Ascorbic acid | |

| |Riboflavin | |

| |Pyridoxine | |

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|4. |A patient suffers from vision impairment hemeralopia (night | |

| |blindness). What vitamin preparation should be administered the | |

| |patient in order to restore his vision? | |

| |Pyridoxine | |

| |Retinol acetate | |

| |Vicasol | |

| |Thiamine chloride | |

| |Tocopherol acetate | |

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|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? | |

| |K | |

| |C | |

| |D3 | |

| |PP | |

| |B12 | |

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|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: | |

| |Carboxylation of glutamic acid residues | |

| |Polymerization | |

| |Partial proteolysis | |

| |Glycosylation | |

| |Phosphorylation of serine radicals | |

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|7. |Most participants of Magellan expedition to America died from | |

| |avitaminosis. This disease declared itself by general weakness, | |

| |subcutaneous hemmorhages, falling of teeth, gingival hemmorhages.| |

| |What is the name of this avitaminosis? | |

| |Biermer's anemia | |

| |Polyneuritis (beriberi) | |

| |Pellagra | |

| |Rachitis | |

| |Scurvy | |

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|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? | |

| |Deficiency of vitamin B12 | |

| |Decreased production of thyroid hormones | |

| |Deficiency of vitamin K | |

| |Decreased production of parathyrin | |

| |Deficiency of iron-containing enzymes | |

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|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? | |

| |NAD-dependent dehydrogenases | |

| |Cytochrome oxidases | |

| |FAD-dependent dehydrogenases | |

| |Peptidases | |

| |Aminotransferases | |

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|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? | |

| |Tocopherol acetate | |

| |Riboflavin | |

| |Folic acid | |

| |Ergocalciferol | |

| |Thiamine chloride | |

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|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? | |

| |K | |

| |E | |

| |D | |

| |A | |

| |Carotene | |

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|12. |Concentration of pyruvate is increased in the patient's blood, | |

| |the most of which is excreted with urine. What avitaminosis has | |

| |the patient? | |

| |Avitaminosis B1 | |

| |Avitaminosis B2 | |

| |Avitaminosis E | |

| |Avitaminosis B9 | |

| |Avitaminosis B3 | |

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|13. |Hydroxylation of endogenous substrates and xenobiotics requires a| |

| |donor of protons. Which of the following vitamins can play this | |

| |role? | |

| |Vitamin C | |

| |Vitamin E | |

| |Vitamin P | |

| |Vitamin A | |

| |Vitamin B6 | |

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|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? | |

| |Activation of tissue thromboplastin | |

| |PP hypovitaminosis | |

| |Fibrinogen deficiency | |

| |Vitamin K insufficiency | |

| |Hypocalcemia | |

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|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 | |

| |B2 | |

| |B1 | |

| |B6 | |

| |С | |

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|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? | |

| |Vitamin U | |

| |Vitamin B5 | |

| |Vitamin K | |

| |Vitamin B1 | |

| |Vitamin C | |

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|17. |Ultraviolet radiation can be used in the form of physical | |

| |treatments. What vitamin formation is activated under UV light in| |

| |the skin: | |

| |Vitamin B6 | |

| |Vitamin A | |

| |Vitamin E | |

| |Vitamin C | |

| |Vitamin D3 | |

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|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: | |

| |Dopamine | |

| |Histamine | |

| |Serotonin | |

| |DOPA | |

| |GABA | |

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|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: | |

| |Thiamin | |

| |Riboflavin | |

| |Bioflavonoids | |

| |Pantothenic acid | |

| |Cholecalciferol | |

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|20. |A patient presents with twilight vision impairment. Which of the | |

| |following vitamins should be administered? | |

| |Cyanocobalamin | |

| |Ascorbic acid | |

| |Nicotinic acid | |

| |Retinol acetate | |

| |Pyridoxine hydrochloride | |

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|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 | |

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|22. |In clinical practice tuberculosis is treated with izoniazid | |

| |preparation – that is an antivitamin able to penetrate into the | |

| |tuberculosis bacillus. Tuberculostatic effect is induced by the | |

| |interference with replication processes and oxidation-reduction | |

| |reactions due to the buildup of pseudo-coenzyme: | |

| |FMN | |

| |NAD | |

| |CoQ | |

| |FAD | |

| |TPP | |

| | | |

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| | | |

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|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? | |

| |They inhibit the absorption of folic acid | |

| |They are allosteric enzyme inhibitors | |

| |They are allosteric enzymes | |

| |They are antivitamins of paraaminobenzoic acid | |

| |They are involved in red-ox processes | |

| | | |

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|24. |A 20-year old male patient complains of general weakness, rapid | |

| |fatigability, irritability, decreased performance, bleeding gums,| |

| |petechiae on the skin. What vitamin deficiency may be caused of | |

| |these changes? | |

| |Riboflavin | |

| |Ascorbic acid | |

| |Retinol | |

| |Thiamine | |

| |Folic acid | |

| | | |

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|25. |A number of disorders can be diagnosed by evaluation activity of | |

| |blood transaminases. What vitamin is one of cofactors for these | |

| |enzymes? | |

| |B6 | |

| |B1 | |

| |B5 | |

| |B2 | |

| |B8 | |

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|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: | |

| |Lysine | |

| |Threonine | |

| |Tryptophan | |

| |Arginine | |

| |Histidine | |

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|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? | |

| |Cyanocobalamin | |

| |Tocopherol acetate | |

| |Pyridoxine hydrochloride | |

| |Thiamine | |

| |Retinol acetate | |

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|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: | |

| |Cytochrome oxidase | |

| |Cytochrome B | |

| |Cytochrome A1 | |

| |Cytochrome C | |

| |FAD or FMN | |

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|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? | |

| |K | |

| |B12 | |

| |B9 | |

| |C | |

| |P | |

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|30. |A 6-year-old child suffers from delayed growth, disrupted | |

| |ossification processes, decalcification of teeth. What can be the| |

| |cause? | |

| |Vitamin D deficiency | |

| |Hyperthyroidism | |

| |Vitamin C deficiency | |

| |Decreased glucagon production | |

| |Insulin deficiency | |

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|31 |A patient is diagnosed with chronic atrophic gastritis attended | |

| |by deficiency of Castle`s (intrinsic) factor. What type of anemia| |

| |does the patient have? | |

| |B12-deficiency anemia | |

| |Iron-deficiency anemia | |

| |Hemolytic anemia | |

| |Protein-deficiency anemia | |

| |Iron refractory anemia | |

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|32 |During regular check-up a child is detected with interrupted | |

| |mineralization of bones. What vitamin deficiency can be the | |

| |cause? | |

| |Calciferol | |

| |Riboflavin | |

| |Tocopherol | |

| |Folic acid | |

| |Cobalamin | |

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|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 | |

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|34 |The avitaminosis of ascorbic acid is named as: | |

| |A. Cushing`s syndrome | |

| |B. Addison`s disease | |

| |C. Kwashiorkor | |

| |D. Hemolytic anemia | |

| |E. Scurvy | |

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|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 | |

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|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 | |

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|37 |Choose the vitamin, which is a powerful natural antioxidant: | |

| |A. Retinal | |

| |B. Tocopherol | |

| |C. Ergocalciferol | |

| |D. Riboflavin | |

| |E. Pyridoxine | |

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|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 | |

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|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 | |

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|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 | |

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| | | |

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 |Usually attached to bones by |Motion, posture, heat |

|dark bands within ﬁbers that are visible under a light microscope). |tendons. |production, protection. |

|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. | | |

|Cardiac muscle tissue |

|Branched, striated ﬁbers with usually only one centrally located nucleus |Heart wall. |Pumps blood to all parts of |

|(occasionally two). Attach end to end by transverse thickenings of plasma | |body. |

|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 |Iris of eyes; walls of hollow |Motion (constriction of blood |

|smooth). Smooth muscle ﬁber is a small spindle-shaped cell thickest in |internal structures such as blood|vessels and airways, |

|middle, tapering at each end, and containing a single, centrally located |vessels, airways to lungs, |propulsion of foods through |

|nucleus. Gap junctions connect many individual ﬁbers in some smooth muscle |stomach, intestines, gallbladder,|gastrointestinal tract, |

|tissues (for example, in wall of intestines). Can produce powerful |urinary bladder, and uterus. |contraction of urinary bladder|

|contractions as many muscle ﬁbers contract in unison. Where gap junctions | |and gallbladder). |

|are absent, such as iris of eye, smooth muscle ﬁbers contract individually,| | |

|like skeletal muscle ﬁbers. | | |

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 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 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), 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.

[pic]

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 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.

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).

[pic]

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 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 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.

[pic]

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 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.

[pic]

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 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.

[pic]

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. 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.

[pic]

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 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.

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

(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.

[pic]

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 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.

[pic]

Figure 8. Creatine metabolism.

Clinical significance of creatine and creatinine determination. The normal concentrations of creatine and creatinine in human serum and urine are:

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 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:

[pic] [pic]

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 polypeptide synthesis: If 14C-labeled 4-hydroxyproline is administered to a rat, the collagen synthesized is not radioactive, whereas radioactive collagen is produced if the rat is fed 14C-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.

[pic]

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-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.

[pic]

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).

[pic]

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 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).

[pic]

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 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.

[pic] [pic]

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

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: | |

| |Ovoalbumin | |

| |Collagen | |

| |Myosin | |

| |Troponin | |

| |Mucin | |

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|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? | |

| |Hydroxylation of proline | |

| |Detaching of N-ended peptide | |

| |Formation of polypeptide chains | |

| |Glycosylation of hydroxylysine residues | |

| |Removal of C-ended peptide from procollagen | |

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|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 | |

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|4 |Name the polysaccharide represented in connective tissue: | |

| |A. Collagen | |

| |B. Elastin | |

| |C. Laminin | |

| |D. Hyaluronic acid | |

| |Е. Fibrillin | |

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|5 |It is established that there is specific system of energy supply | |

| |in muscular 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 | |

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|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? | |

| |Glucose | |

| |Common lipids | |

| |Uric acid | |

| |Creatinine | |

| |Mineral salts | |

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|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? | |

| |Lactate dehydrogenase | |

| |Glutamate dehydrogenase | |

| |Adenylate cyclase | |

| |Pyruvate dehydrogenase | |

| |Creatine phosphokinase | |

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|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: | |

| |Albumin | |

| |Hemoglobin | |

| |Collagen | |

| |Fibrinogen | |

| |Keratin | |

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|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 | |

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|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 | |

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|11 |Choose the product of guanidoacetate transmethylation from | |

| |following list: | |

| |A. Chlorine | |

| |B. Hydroxyproline | |

| |C. Creatinine | |

| |D. Creatine | |

| |E. Glutathione | |

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|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 | |

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|13 |Triple helix is seen in one compound listed bellow. Choose it: | |

| |A. Collagen | |

| |B. Fibrinogen | |

| |C. Histones | |

| |D. Serum amylase | |

| |E. F-actin | |

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|14 |Name the immediate source of energy for muscular contraction. | |

| |A. Glycogen | |

| |B. ATP | |

| |C. Creatine phosphate | |

| |D. Glucose | |

| |E. Pyruvate | |

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|15 |What does cardiac muscle prefer as source of energy? | |

| |A. Fatty acids | |

| |B. Glucose | |

| |C. Ketone bodies | |

| |D. Glycogen | |

| |E. Fructose | |

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|16 |Hydroxylation of proline to 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 | |

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|17 |What is the product of guanidoacetic acid transmethylation? | |

| |A. Acetylcholine | |

| |B. Choline | |

| |C. Creatinine | |

| |D. N-methyl nicotinamide | |

| |E. Creatine | |

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|18 |Creatine is formed metabolically using one compound listed below.| |

| |Choose it: | |

| |A. Tryptophan | |

| |B. Phenylalanine | |

| |C. Lysine | |

| |D. Valine | |

| |E. Leucine | |

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|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 | |

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|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 | |

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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.

[pic]

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 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.

• 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).

[pic]

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.

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

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

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 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.

[pic]

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

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 |[pic] |[pic] |

| |Glutamate |GABA |

| |[pic] | |

| |Acetylcholine |[pic] |

| | |Glycine |

|neuromodulator |[pic] |[pic] |

| |Nor-epinephrine |Adenosine |

| | |[pic] |

| |[pic] |Dopamine |

| |Serotonin | |

Amino acids may be precursors for formation of neurotransmitters. α-Decarboxylation of histidine gives histamine:

[pic]

Biological role of histamine

• 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.

[pic]

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.

[pic]

In figure 6 amino acids phenyl alanine and tyrosine may be discussed as precursors for dihydroxy phenyl alanine (DOPA), dopamine, norepinephrine and 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.

[pic]

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;

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.

[pic]

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.

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.

[pic]

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

• 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:

[pic]

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.

[pic]

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) 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: | |

| |Acetylcholine | |

| |Serotonin | |

| |Dopamine | |

| |Noradrenalin | |

| |Adrenalin | |

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|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: | |

| |Fumarate | |

| |Succinate | |

| |Oxaloacetate | |

| |Malate | |

| |Citric acid | |

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|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: | |

| |Urea | |

| |Histamine | |

| |Glutamine | |

| |Ammonia | |

| |Bilirubin | |

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|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: | |

| |Phosphatidic acid | |

| |Cholesterol | |

| |Sphingolipids | |

| |Neutral fats | |

| |Higher fatty acids | |

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|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 | |

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|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)? | |

| |Oxidative deamination | |

| |Carboxylation | |

| |Addition of an amino group | |

| |Removal of methyl group | |

| |Hydrolysis | |

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|7. |An inhibitory mediator is formed by the decarboxylation of | |

| |glutamate in the CNS. Name it: | |

| |Asparagine | |

| |Serotonine | |

| |Histamine | |

| |GABA | |

| |Glutathione | |

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|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: | |

| |TDP | |

| |GABA | |

| |THFA | |

| |Pyridoxal phosphate | |

| |ATP | |

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|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 amine | |

| |generated as a from histidine amino acid across: | |

| |Methylation | |

| |Isomerization | |

| |Phosphorylation | |

| |Decarboxylation | |

| |Deamination | |

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|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? | |

| |Decarboxylation | |

| |Microsomal oxidation | |

| |Transamination | |

| |Desamination | |

| |Formation of paired compounds | |

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|11. |Cerebral trauma caused the increase of ammonia formation. What | |

| |amino acid takes part in removal of ammonia from cerebral | |

| |tissue? | |

| |Tryptophan | |

| |Lysine | |

| |Glutamic acid | |

| |Valine | |

| |Туrosine | |

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|12. |Glutamate decarboxylation results in the formation of inhibitory| |

| |transmitter in CNS. Name it: | |

| |Glutathione | |

| |Gamma amino butyric acid | |

| |Serotonin | |

| |Histamine | |

| |Asparagine | |

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|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? | |

| |Lysine | |

| |Glutamic acid | |

| |Histidine | |

| |Proline | |

| |Alanine | |

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|14. |Neurotransmitter serotonin is derived from one amino acid. | |

| |Choose it: | |

| |A. Phenylalanine | |

| |B. Serine | |

| |C. Tryptophan | |

| |D. Cysteine | |

| |E. Proline | |

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|15. |In the brain ammonia is converted to product from following | |

| |list. Point out it: | |

| |A. Aspartate | |

| |B. Glutamine | |

| |C. Alanine | |

| |D. Histidine | |

| |E. Urea | |

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|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 | |

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|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 | |

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|18. |This enzyme may be promoter of Glutamate utilization in direct | |

| |reaction, and in opposite reaction it can utilize toxic ammonia | |

| |in nervous tissue. Name it: | |

| |Glutaminase | |

| |Glutamine oxidase | |

| |Glutamate dehydrogenase | |

| |Glutamate decarboxylase | |

| |Glutamate reductase | |

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|19. |Tetrahydrobiopterin (THBP) is in need for hydroxylation | |

| |reactions of some amino acids to produce neurotransmitters in | |

| |nervous tissue. Name these neurotransmitters: | |

| |Dopamine, Serotonin | |

| |Glycine, Glutamate | |

| |GABA, Glycine | |

| |Histamine, Tryptamine | |

| |GABA, Histamine | |

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|20. |Insulin-regulated glucose trans-porters are found in neuronal | |

| |cell bodies in the brain cortex of humans. Name them: | |

| |GLUT4, GLUT8 | |

| |GLUT1 | |

| |GLUT2 | |

| |GLUT1, GLUT2 | |

| |GLUT5 | |

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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.

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 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).

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.

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

|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 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:

- 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, 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

[pic]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.”

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.

[pic]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.

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:

1. This reaction is catalyzed by Xenobiotic medium-chain fatty acid:CoA ligase:

[pic]

Benzoic acid Benzoyl-CoA

2. The reaction is catalyzed by Glycine transferase:

[pic]

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 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,

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 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)?

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 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.

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? | |

| |The anaerobic glucose metabolism predominates in muscles | |

| |The gluconeogenesis is increased in the liver | |

| |The breakage of glycogen is increased in the liver | |

| |The gluconeogenesis velocity in the liver is decreased | |

| |The anaerobic breakage of glucose is increased in muscles | |

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|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? | |

| |Uric acid | |

| |Сreatinine | |

| |Ammonium salts | |

| |Hippuric acid | |

| |Amino acids | |

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|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? | |

| |Bilirubinuria | |

| |Increase of ALT, AST level | |

| |Heperbilirubinemia | |

| |Cholemia | |

| |Urobilinuria | |

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|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 | |

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|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 | |

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|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 | |

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|7. |What enzyme is activated in liver at physiological hypoglycemia:| |

| |A. Glycogen phosphorylase | |

| |B. Glutaminase | |

| |C. Glutamate decarboxylase | |

| |D. Creatine kinase | |

| |E. Glycogen synthase | |

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|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 | |

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|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 | |

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|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 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 | |

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|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 | |

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|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 | |

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|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 | |

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|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 | |

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|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 | |

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|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 | |

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|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 | |

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|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 | |

| |E. UDP - glucoronyl transferase | |

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|19. |Disulfiram is widely used in medical practice to prevent | |

| |alcoholism, it inhibits aldehyde dehydrogenase. Increased level | |

| |of what metabolite causes aversion to alcohol? | |

| |Acetaldehyde | |

| |Ethanol | |

| |Malonyl aldehyde | |

| |Propionic aldehyde | |

| |Methanol | |

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|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: | |

| |Aerobic oxidation of glucose | |

| |Anaerobic oxidation of glucose | |

| |Gluconeogenesis | |

| |Pentose phosphate cycle | |

| |Glycogenesis | |

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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.

[pic]

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 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 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.

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 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.

[pic]

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); 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 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.

[pic]

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), 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.

• 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 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:

[pic]

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, 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''-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.

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: | |

| |Cytochrome aa3 | |

| |Cytochrome C oxidase | |

| |Cytochrome P-450 | |

| |Cytochrome C1 | |

| |Cytochrome b | |

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|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? | |

| |Catalase | |

| |Glutathione reductase | |

| |Peroxidase | |

| |Superoxide dismutase | |

| |Glutathione peroxidase | |

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|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? | |

| |Indole | |

| |Ornithine | |

| |Acetacetate | |

| |Butyrate | |

| |Biotin | |

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|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 | |

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|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 | |

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|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 | |

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|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 | |

| |D. Carbomoyl phosphate synthetase | |

| |E. UDP - glucoronyl transferase | |

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|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 | |

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|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 | |

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|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? | |

| |Catalase | |

| |Glutathione reductase | |

| |Peroxidase | |

| |Superoxide dismutase | |

| |Glutathione peroxidase | |

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|11. |Point out the donor of sulfate group in the conjugation phase of| |

| |xenobiotics transformation: | |

| |Glutathione | |

| |UDP-glucuronic acid | |

| |Adenosine 3́-phosphate-5́-phosphosulfate (PAPS) | |

| |Acetyl-CoA | |

| |S-adenosylmethionine | |

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|12. |All of the following may have a physiological antioxidant role | |

| |except | |

| |Lipoic acid | |

| |Vitamin C | |

| |Selenium | |

| |Iron | |

| |Vitamin E | |

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|13. |Point out the chemical nature of prosthetic group of cytochrome | |

| |P450: | |

| |Nucleotide | |

| |Fe3+ | |

| |Fe2+ | |

| |Phosphate | |

| |Haem | |

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|14. |Choose the exogenous factor (the drug) that can induce the | |

| |UDP-glucuronosyltransferase gene expression in the liver: | |

| |Calcitriol | |

| |Thyroxine | |

| |Riboxin | |

| |Phenobarbital | |

| |Thiamine diphosphate | |

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|15. |Choose one wrong continuation of a phrase: Phase I of | |

| |xenobiotics transformation: | |

| |Is carried out by enzymes of endoplasmic reticulum | |

| |Demands presence of NADPH | |

| |Results in increase of polarity of a substance | |

| |Occurs in anaerobic conditions | |

| |Proceeds at participation of cytochrome Р450 | |

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|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 | |

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|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 | |

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|18. |Which of following cytochrome participates in drug metabolism? | |

| |A. Cytochrome aa3 | |

| |B. Cytochrome c1 | |

| |C. Cytochrome P450 | |

| |D. Cytochrome c | |

| |E. Cytochrome b | |

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|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 | |

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|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 | |

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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

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.

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 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 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 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 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.

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 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.

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) 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.

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 |Normally, present in plasma in very low |

| |comparison to tissues |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, |ALT, AST, CK, LDH, alkaline phosphatase, acid |

| |Lipoprotein lipase and pseudo- choline esterase |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 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

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.

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.

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.

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).

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

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? | |

| |Viral hepatitis | |

| |Diabetes insipidus | |

| |Collagenosis | |

| |Diabetes mellitus | |

| |Myocardial infarction | |

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|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? | |

| |Absolute hyperproteinemia | |

| |Relative hyperproteinemia | |

| |Absolute hypoproteinemia | |

| |Disproteinemia | |

| |Paraproteinemia | |

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|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? | |

| |Proteinuria | |

| |Hypoproteinemia | |

| |Hypoglobunemia | |

| |Hyperalbuminemia | |

| |Paraproteinemia | |

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|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? | |

| |Metabolic alkalosis | |

| |Metabolic acidosis | |

| |Respiratory alkalosis | |

| |Respiratory acidosis | |

| |E. Any changes won't happen | |

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|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? | |

| |R-glycosidase | |

| |Acid phosphatase | |

| |Lipoproteins | |

| |General cholesterol | |

| |Additive glycosaminoglycans | |

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|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? | |

| |Myocardial infarction | |

| |Hepatitis | |

| |Pancreatitis | |

| |Rheumatism | |

| |Cholecystitis | |

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|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? | |

| |In the heart muscle {initial stage of myocardium infraction} | |

| |In skeletal muscle {dystrophy, atrophy} | |

| |In kidneys and adrenals | |

| |In liver and kidneys | |

| |In connective tissue | |

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|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. | |

| |Diabetes mellitus | |

| |Skeletal muscle dystrophy | |

| |Myocardial infarction | |

| |Acute pancreatitis | |

| |E. Viral hepatitis | |

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|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? | |

| |Disturbance of genetic apparatus of cells | |

| |Cellular breakdown | |

| |Disorder of enzyme systems of cells | |

| |Disturbance of cellular interrelations | |

| |E. Disturbed energy supply of cells | |

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|10 |A worker has decreased buffer capacity of blood due to exhausting| |

| |muscular work. What acidic substance that came to blood caused | |

| |this phenomenon? | |

| |3-phosphoglycerate | |

| |1,3-bisphosphoglycerate | |

| |Lactate | |

| |α-ketoglutarate | |

| |Pyruvate | |

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|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? | |

| |Reduced contents of erythrocytes | |

| |Increased contents of erythrocytes | |

| |Increased contents of leukocytes | |

| |Increased plasma proteins | |

| |Reduced contents of thrombocytes | |

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|12 |Examination of a 43 y.o. anephric patient revealed anemia | |

| |symptoms. What is the cause of these symptoms? | |

| |Folic acid deficit | |

| |Vitamin B12 deficit | |

| |Reduced synthesis of erythropoietins | |

| |Enhanced destruction of erythrocytes | |

| |Iron deficit | |

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|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? | |

| |Iron-deficiency anemia | |

| |B12-folate-deficiency anemia | |

| |Hemolytic anemia | |

| |Aplastic anemia | |

| |Chronic posthemorrhagic anemia | |

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|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: | |

| |Albumin | |

| |Immunoglobulin G | |

| |Immunoglobulin E | |

| |C-reactive protein | |

| |Prothrombin | |

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|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: | |

| |Ceruplasmin | |

| |Bilirubin | |

| |Antitrypsin | |

| |Bence Jones protein | |

| |Haemoglobin | |

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|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: | |

| |Leucine aminopeptidase | |

| |Xanthine oxidase | |

| |Alcohol dehydrogenase | |

| |Ceruloplasmin | |

| |Carbonic anhydrase | |

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|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 | |

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|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: | |

| |γ-globulins | |

| |Albumins | |

| |α1-globulins | |

| |β-globulins | |

| |α2-globulins | |

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|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? | |

| |Increased proteolysis | |

| |Decrease in the concentration of amino acids in blood | |

| |Albuminosis | |

| |Increase in the oncotic pressure in the blood plasma | |

| |Increase in low-density lipoprotein level | |

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|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 | |

| |B. Ceruloplasmin, transferrin | |

| |C. a2-macroglobulin, a1-antitrypsin | |

| |D. Cryoglobulin, interferon | |

| |E. Hemopexin, haptoglobin | |

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|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 | |

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|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 | |

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|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 | |

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|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 | |

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|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? | |

| |A. Hemolytic anemia | |

| |B. Megaloblastic anemia | |

| |C. Siclemia | |

| |D. Iron-deficiency anemia | |

| |E. Pernicous anemia | |

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|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 | |

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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

[pic]

Figure 1. A structural unit of kidney tissue - a nephron.

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).

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.

[pic]

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.

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.

[pic][pic]

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:

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 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.

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.

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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 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 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:

[pic]

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)

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.

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+. Ca2+, Mg2+, SO42-, HCO3 -, HPO42-, H2PO4-, PO43- 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.

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.

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.

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.

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? | |

| |Liver dysfunction of protein formation | |

| |Hyperosmolarity of plasma | |

| |Proteinuria | |

| |Hyperproduction of vasopressin | |

| |Hyperaldosteronism | |

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|2. |Examination of a 43 y.o. anephric patient revealed anemia | |

| |symptoms. What is the cause of these symptoms? | |

| |Folic acid deficit | |

| |Vitamin B12 deficit | |

| |Reduced synthesis of erythro-poietin | |

| |Enhanced destruction of erythrocytes | |

| |Iron deficit | |

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|3. |A biochemical urine analysis has been performed for a patient | |

| |with progressive muscular dystrophy. In the given case muscle | |

| |disease can be confirmed by the high content of the following | |

| |substance in urine: | |

| |Urea | |

| |Porphyrin | |

| |Hippuric acid | |

| |Creatine | |

| |Creatinine | |

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|4. |Kidney insufficiency in patient is accompanied with: | |

| |Excess levels of urea in the blood plasma | |

| |Excess levels of potassium ions in the blood plasma | |

| |Disturbed clearance | |

| |Disturbed filtration and reabsorption processes | |

| |All that is placed above | |

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|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 | |

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|6. |Point out main source of ammonia in kidney tissue: | |

| |A. Urea | |

| |B. Aspartate | |

| |C. Glutamine | |

| |D. Glutamate | |

| |E. Uric acid | |

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|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 | |

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|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 | |

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|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 | |

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|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 | |

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|11. |Appearance of albumins in the urine of diseased person may be | |

| |at: | |

| |Acute nephritis | |

| |Chronical nephritis | |

| |Severe form of diabetes mellitus | |

| |Pyelonephritis | |

| |All that is placed above | |

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|12. |Choose the main biochemical tests for diagnostics of kidney | |

| |diseases: | |

| |Urea content in the blood plasma and in the urine | |

| |Creatinine content in the blood and urine | |

| |Sodium ions content in the blood and urine | |

| |N-acetyl-beta-D-glucose-aminidase activity (blood serum, urine) | |

| |All that is placed above | |

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|13. |What organic compounds accumulate in final urine at severe form | |

| |of diabetes mellitus? | |

| |Albumins | |

| |Glucose | |

| |Ketone bodies | |

| |Bilirubin conjugated | |

| |All that is placed in positions A, B, C | |

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|14. |Kidney insufficiency development will cause the infringements in| |

| |those processes: | |

| |Erythropoietin synthesis and secretion | |

| |Calcitriol synthesis | |

| |Mineralization of bone tissue | |

| |Creatine synthesis | |

| |All that is placed | |

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|15. |The infringement in glomerular filtration mostly is associated | |

| |with appearance in the urine of this class compounds. Name it: | |

| |Lipids | |

| |Proteins | |

| |Amino acids | |

| |Keto acids | |

| |Carbohydrates | |

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|16. |Renal clearance may be calculated using this compound | |

| |concentration value in the blood serum and in urine of patient. | |

| |Name it: | |

| |Inulin | |

| |Creatine | |

| |Free ammonia | |

| |Ammonia salt | |

| |Indican | |

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|17. |Utilization of excess protons in renal tubule lumen may be due | |

| |to: | |

| |Aspartic acid | |

| |Creatinine | |

| |Uric acid | |

| |Ammonia | |

| |Water | |

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|18. |This compound impossible to find out in the urine of healthy | |

| |person: | |

| |Globulin | |

| |Alanine | |

| |Pyruvate | |

| |Oxaloacetate | |

| |Carbonic acid | |

| | | |

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|19. |Acute tubular necrosis is associated with increase of this index| |

| |in the blood serum of patient. Name it: | |

| |Creatine | |

| |Free amino acids | |

| |Pyruvate | |

| |Cholesterol total | |

| |Urea | |

| | | |

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|20. |This vitamin derivative is produced in renal tubules mainly to | |

| |control calcium ad phosphate ions levels in the blood. Name it: | |

| |FAD | |

| |NAD+ | |

| |Calcitonin | |

| |Calcitriol | |

| |Angiotensin II | |

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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.

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.

Answers to tests tasks:

The role of water-soluble and fat-soluble vitamins in the metabolism of humans. Vitamin similar substances. Antivitamins

|1 |2 |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 |

|Answers to tests tasks………………………………………………………... | |185 |

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