ISCHAEMIA, REPERFUSION, FREE RADICAL REACTIONS Prof. Elizabeth Rőth

1. ISCHAEMIA, REPERFUSION, FREE RADICAL REACTIONS Prof. Elizabeth Rőth Department of Experimental Surgery University of Pécs

2. Reactive radicals and intermediers originating from oxygen ( O O ) Σ 1O2Δ1O2 ( O O ) Sigmasinglet oxygen excitation Deltasinglet oxygen O2 ( O O ) Molecular oxygen e - ( H O O ) HO2 O2- ( O O - ) e - Perhidroxi radical Superoxid anion H2O2 ( H O O H ) reduction Hydrogen peroxid e - OH ( O H ) Hydroxil radical e - H2O ( H O H ) water

3. Proposed mechanism of formation of oxygen-derived radicals and their metabolites 1. Electron reduction of oxygen O2 + e  O2–. ( szuperoxid anion) 2. Spontaneous dismutation of O2- 2O2– + 2H+  H2O2 + O2 (or 1O2) 3. Haber-Weiss reaction O2– + H2O2 O2 (or 1O2)+HO– + HO. 4. Fenton reaction O2_ + Fe3+  Fe2+ + O2 Fe2+ +H2O2  Fe3+ +HO _ + HO. O2_ +H2O2  O2 + HO_ +HO. 5. Myeloperoxidase - hydrogen peroxide - chloride system H2O2 + Cl_  OCl_ + H2O OCl_ + H2O2  H2O + Cl_ + 1O2 OCl_ + RNH2  RNHCl + HO_ OCl_ + RNHCl  RNCl2 + HO_ MPO

4. REACTIONS OF RADICALS I. Radical plus radical O2- + NO• ONOO- (peroxynitrite) Peroxyinitrite damages proteins directly produces toxic product: nitrogen - dioxid ( NO2• ) hidroxyl radical ( OH• ) nitronium ion ( NO2+ )

5. REACTIONS OF RADICALS II. Radical plus non - radical When a free radical reacts with a non - radical, a free radical chain reaction results and new radicals are formed. Polyunsaturated fatty acids (PUFA) - lipidperoxydation DNS chain brake ( strand-brake mutations ) carbohydrates ( receptors)

6. Lipid peroxidation O2 C + X XH + C C + O2 C C O2 H O2H Lipid peroxide C + C + C C + , stb. O2 C Reactive radical (such as NO2• ,OH• or CCl3O2• ) abstracts atom of hydrogen from polyunsaturated fatty-acid side-chain in membrane or lipoprotein This leaves unpaired electron on carbon (hydrogen atom has only one electron, so its removal must leave spare electron. H Carbon radical reacts with oxygen: Resulting peroxyl radical attacks adjacent fatty-acid side chain to generate new carbon radical: And chain reaction continues : Overall, attack of one reactive free radical can oxidise multiple fatty-acid side-chains to lipid peroxides, damaging membrane proteins, making the membrane leaky, and eventually causing complete membrane breakdown. O2

7. Antioxidant protection I. Intracellular -superoxid dismutase (SOD) 2O2- + 2H• H2O + O2 Mitochondria : Mn-SOD cytosol: Cu-Zn-SOD - catalase - H2O2 elimination localisation: peroxisome - glutathion peroxidase (Gpx) 2 GSH + H2O2 GSSG + H2O localisation: cytosol, mitochondria - repair enzymes : eliminate oxidated fatty acids ; repairs DNA damage caused by free radicals

8. I • - transferrin, lactoferrin plasma iron-transport-protein • - coeruloplasmin plasma copper-transport-protein • - haemopexine bind free heme and • - haptoglobin heme proteins • - albumin contents sulphydril groups, • binds copper ions • - GSH, urate, ascorbate non catallitically react with free radicals Antioxidant protection II. Extracellular types III Extra- and intracellular types - α -tocopherol blocks the chain reaction of lipid peroxidation

9. Possible methods for detection of free radical reactions 1. Determination of radicals -spectrophotometry -NMR (radical capture) -ESR (radical capture) -chemiluminescence 2. Detection of end-products of radical reactions -lipidperoxidation-malondialdehid -exspired carbohydrates (etan, pentan) -conjugated diens 3. Decrease of free radical reactions specific:SOD, catalase relative specific:allopurinol, desferroxamine, MTDQ-DS non specific:mannitol, DMSO

10. IN NORMAL TISSUE FREE RADICALS: SCAVENGERS: O2 - H2O2 OH• 1O2 Superoxid dismutase Glutathion peroxidase Katalase Endogen thiols Vitamins Lipid peroxidation on base level hyperoxia hypoxia Decreased scavenger contents Permanent or decreased scavenger level Unchanged or increased free radical production Increased production of free radicals Increased lipid peroxidation Membrane destruction

11. NEUTROPHILS O2 NADPH NADPH OXIDASE 1O2 O2•- + NO• H2O2 NADP MYELOPEROXIDASE H2O2 + Cl- HOCl L-arginin VASCULAR ENDOTHELIUM xanthine dehydrogenase ATP xanthine xanthine oxidase NO-syntase O2 uric acid NO. O2•- + H2O2 MITOCHONDRIAL ELECTRON TRANSPORT CHAIN O2 O2•- + H2O2

12. Physiological free radicals I. Superoxide radical O2- -poorly reactive - product of autooxidative processes - constituents of mitochondrial electron transport chain(1-2%) -regulator of enzymes II. Nitric oxide NO. - regulator of blood pressure and vascular tone - mediators of phagocytes function in the brain -regulates neural signaling and cellular immune response Neither superoxide nor nitric oxide is highly reactive chemically, but under certain circumstances they can generate more toxic product.

13. reoxygenation ATP AMP xanthine dehydrogenase ischemia adenoine xanthine oxidase inosine hypoxanthine O2- O2 McCord J. M. The biology and pathology of oxygen radicals.J Of Int. Medicine 1978.69

14. ATP MICROVASCULAR INJURY OXIDANTS PROTEASES AMP NEUTROPHILS PAF, LTB4, TNF ADENOSINE XANTHINE LOOH DEHYDROGENASE LH INOSINE •OH Fe 3+ HYPOXANTHINEXANTHINE OXIDASE URATE + O2- + H2O2 O2 REPERFUSION ISCHEMIA

15. I. The trigger of reperfusion injury is the endothelial cell dysfunction -marked reduction of NO. release -release chemotactic factor (PAF, LTB4, C5A ) -PMN accumulation, adherence to the dysfunctional endothelium -endothelial activation of adhesive molecule ligands (ICAM-1, P-selectin) -extravasation of leukocytes, dangerous effect of free radicals, proteases Reperfusion injury II. Overproduction of free radicals: xanthine-oxidase derived oxidant - O2- activated neutrophils - NADPH oxidase, myeloperoxidase lipid mediators - PAF , LTB4 , polypeptide mediators ( C5A )