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 1O Δ1O2 ( O O )
excitation
Sigmasinglet
oxygen
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
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
C X
XH C C
+ O2 O2
Carbon radical reacts with oxygen:
Resulting peroxyl radical attacks adjacent fatty-acid side chain to generate new carbon radical: O2 C + H
O2H
Lipid peroxide
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 C + O2
, stb.

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) GSH + H2O GSSG + H2O
localisation: cytosol, mitochondria
- repair enzymes : eliminate oxidated fatty acids ;
repairs DNA damage caused by free radicals

8. Antioxidant protection
- 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 hyperoxia hypoxia 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. L-arginin NO-syntase NO. NEUTROPHILS O2 NADPH NADPH OXIDASE 1O2 O2•- +
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. ATP
AMP
xanthine dehydrogenase
ischemia
adenoine
xanthine oxidase
inosine
hypoxanthine
O2- O2
reoxygenation
McCord J. M. The biology and pathology of oxygen radicals. J Of Int. Medicine

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

15. Reperfusion injury
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
II. Overproduction of free radicals:
xanthine-oxidase derived oxidant O2-
activated neutrophils - NADPH oxidase, myeloperoxidase
lipid mediators - PAF , LTB4 , polypeptide mediators ( C5A )