1. Veterinary Pathophysiology Student’s Lectures, 5th Semester Department of Internal Medicine, Faculty of Veterinary Science Szent István University Pathophysiology of defence mechanisms 3. Altered antioxidant mechanism Additional reading: Continuing Education paper

2. Exam question 23. Effect of free radicals, disturbances of the antioxidant system.

3. DEFINITION OF A FREE RADICAL Electron-orbitals of most chemical compounds have paired electrons with opposite-directed spin. Free radicals are molecules or molecule-fragments which have one unpaired electron on their external electron-orbital, independently from the number of protons. Their symbol is: X  Free radicals are chemicaly reactive, their life time is short. A "biradical" free radical: has two unpaired electrons on its external electron-orbital. (i.e.: molecular O 2 ) The spin of these electrons are parallel. Therefore O 2 is a relatively weak oxidant.

4. Stable and radical molecules Molecule A: paired electrons on the outer orbitals → stable Radical molecule: unpaired electrons → unstable

5. 5 Birth of a free radical Free radicals ar „electron hunters”

6. OXYGEN-DERIVED FREE RADICALS Facts: 1. O 2 is a weak oxidant, but 2. O 2 -derived free radicals are very reactive UNIVALENT (=1-electron) reduction of O 2 : +e +e O 2  O 2 -  H 2 O 2  OH  H 2 O +2H + +H + +H + molecularsuperoxide hidrogen hidroxyl oxigen anion rad. peroxide radical water reactive oxygen intermediers/species (ROI, ROS) Tetravalent (= 4-electron) reduction of O 2 ie. terminal oxidatition: O 2 +2H 2  2 H 2 O

7. 7 ROI or ROS – reactíve oxygen intermediers/ species Gwinner and Grone, 2000

8. EFFECT OF SUPEROXIDE DISMUTASE (SOD), an antioxidant enzyme SOD 2 O 2  + 2 H +  H 2 O 2 + O 2 Dismutation: a reaction in which two molecules of the same compound interact to yield one oxidized and one reduced product. H 2 O 2 is less dangereous than superoxid anion and is handled by other antioxidant enzymes as catalase and glutathione-peroxidase

9. HABER - WEISS REACTION O 2  + H 2 O 2  O 2 + OH - + OH  superox.hydrogenmolec.hydroxidehydroxyl radicalperoxideoxygenionradical This process is slow as H 2 O 2 is eliminated by the enzymes (CAT, GPX) Development of hydroxide ion and hydroxil radical:.. Water molecule H : O : H...... H + : O : H H.. O : H.... hydrogenhydroxide hydrogen hydroxil ionion, OH - atom radical, OH 

10. 10 Antioxidant enzymes: hand in hand SOD: superoxide dismutase POx: peroxidases including catalase and glutathione-peroxidases

11. FENTON REACTION On the effect of metal catalysis OH  production accelerates: 1) O 2  + metal n+1  metal n + O 2 2) H 2 O 2 + metal n  metal n+1 + OH - + OH  Sum of 1) + 2) = Haber-Weiss reaction, i.e. O 2  + H 2 O 2  O 2 + OH - + OH  Metal catalysis is supplied by transient metals such as iron

12. FREE RADICAL REACTIONS IN THE NATURE Exogenous free radical reactions (not only oxygen-derived radicals) occur during: -engine function -smog formation -metal corrosion -ranciding (peroxidation) of fat, butter, oil -halogenation of some carbohydrates Endogenous free radical reactions occur during: –In controlled, physiological conditions (electron-transport chain, autooxidation of biomolecules, myelo-peroxidase enzyme activity of the phagocytes) –In pathological, enhanced free radical reactions

13. LIPID PEROXIDATION (LPO) A. Natural oxidation of lipids (fatty acid part of lipids): oxidation Lipid + molecular O 2  oxidized lipid (became rancid) L+O 2 LO 2 slow reaction ! B. Free-radical mediated oxidation of lipids : 1., Initiation lipid + free radical  lipid free radical L  lipid free radical + O 2  lipid-peroxy radical LOO  fast reaction! 2., Propagationchain reactions (lipids, DNA-s, amino acids, proteins) 3., Terminationtissue damages, formation of stabile endproducts

14. Structures of linoleic acid and similar compounds (PUFAs) Further target fatty acids: C20:4, C20:6. The more duble bonds the more sensitive to free radical effect

15. LIPID PEROXIDATION General scheme for the oxidation of lipids (LH) by a free radical chain mechanism.

16. PHYSIOLOGICAL LPO REACTIONS - aging - melanin formation - phagocytosis activity of thrombocytes - arachidonic acid, prostaglandin metabolism - synthesis of the hormones of adrenal cortex - wound healing

17. PATHOLOGICAL LPO PROCESSES IN HUMAN DISEASES - radiation disease; effect of UV light - inflammation - carcinogenesis - rheumatic diseases - autoimmune diseases - atherosclerosis - infarct - ischaemia - reperfusion - lung emphysema, asthma bronchiale - alcoholic liver disease - uraemia - muscular diseases - diseases of the CNS (Parkinson’s d., Alzheimer’s d.)

18. PATHOLOGICAL LPO PROCESSES IN THE DEVELOPMENT OF THE DISEASES OF ANIMALS - nutritional muscle degenerations/myopathies Se and vitamin E deficiency (calf, lamb, foal etc.) - myoglobinuria/azoturia/Monday morning disease (horse) - PSS (Porcine Stress Syndrome) (swine) - encephalomalacia, exsudative diathesis (fowl) - toxicoses: "oxygen poisoning", DDT (attacking CNS, causing carcinogenesis) trichlorphon (liver) paraquat (Gramoxon) (lungs) halothan

19. 19 Free radical mediated diseases White muscle disease (lamb heart) Encephalomalacia („crazy chick”) PSE – an inherited disorder of swine

20. 20 Antioxidant deficiency - liponecrosis Rabbit subcuteneous fat Tortoise abdominal fat Crocodile abdominal fat By courtesy J. Gal

21. 21 White muscle disease Flaccid tetraparalysis Necropsy finding

22. 22 White muscle disease: Se-deficiency www.upei.ca

23. Free-radical caused membrane damage (Freemann)

24. 24 Lipid peroxidation of cell mebranes Normal sideDamaged side

25. DEFENCE AGAINST FREE RADICALS

26. 26 ”E” numbers on food E 300: ascorbic acid (Vitamin C) E 330: citric acid E 320: butil-hydroxyl anisol (BHA) E 306-309: tocopherols E 321 butil-hydroxyl toluol (BHT)

27. 27 Vitamin E Royal Canine leaflet

28. 28 Antioxidant network 1. (Bulger and Helton, 1998) VE: vitamin E VC: vitamin C

29. 29 Antioxidant network 2. Ascorbate regenerates vitamin E by a nonenzymic mechanism, whereas glutathione regenerates vitamin E enzymatically. (Chan,1993; Ward & Peters 1995).

30. 30 Chemical antioxidants: act by donating an electron to a free radical (”electron hunter”) and converting it to a non-radical form. However, chemical antioxidant defense is a double-edged sword.

31. 31 When an antioxidant scavenges a free radical, its own free radical is formed. Antioxidant free radical

32. 32 Examples Free radical-producing DRUGS as target molecules: CCl 4 → CCl 3 ● Halothane → trifluoroethyl ● radical Paraquat → paraquat ● radical Paracetamol → N-acetyl-p-benzoquinone-imine (NAPQI) ● radical (cats are extremly sensitive!) Defending ANTIOXIDANTS: Ascorbate → dehydroascorbate ● (DHA) radical Tocopherol-OH → tocopherol-O ● (tocopheroxyl) radical GSH → thiyl ● radical