ISCHAEMIA, REPERFUSION, FREE RADICAL REACTIONS

Prof. Elizabeth Rőth

Department of Experimental Surgery

University of Pécs

Reactive radicals and intermediers originating from oxygen

( O O ) Σ 1O2 Δ1O2 ( O O )

( O O )

( H O O ) ( O O - )

OH

H2O

HO2 O2-

( O H )

O2

H2O2 ( H O O H )

( H O H )

e -

e -

e -

e -

Sigmasingletoxygen

Perhidroxi radical

Deltasinglet oxygen

Molecular oxygen

Superoxid anion

Hydrogen peroxid

Hydroxilradical

water

redu

ctio

nex

cita

tion

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

Proposed mechanism of formation of oxygen-derived radicals and their metabolites

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+ )

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)

REACTIONS OF RADICALS

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• •

Carbon radical reacts with oxygen:C•

+ O2 C

O2•

Resulting peroxyl radical attacks adjacent fatty-acid side chain to generate new carbon radical:

O2•

C + C

H

C•

+ C

O2H

Lipid peroxide

And chain reaction continues :

O2

•

C•

+ O2 C , stb.

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.

Antioxidant protection

Mitochondria : Mn-SODcytosol: Cu-Zn-SOD

- catalase - H2O2 elimination localisation: peroxisome

- repair enzymes : eliminate oxidated fatty acids ; repairs DNA damage caused by free radicals

- glutathion peroxidase (Gpx) 2 GSH + H2O2 GSSG + H2O localisation: cytosol, mitochondria

I. Intracellular

-superoxid dismutase (SOD) 2O2- + 2H• H2O + O2

III Extra- and intracellular types

- α -tocopherol blocks the chain reaction of lipid peroxidation

II. Extracellular types

Antioxidant protection

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 reactionsspecific:SOD, catalaserelative specific:allopurinol, desferroxamine, MTDQ-DSnon specific:mannitol, DMSO

IN NORMAL TISSUE

FREE RADICALS: SCAVENGERS:

Superoxid dismutaseGlutathion peroxidaseKatalaseEndogen thiolsVitamins

O2 -

H2O2

OH•1O2

Lipid peroxidation on base level

hypoxia hyperoxia

Increased production of free radicals

Permanent or decreased scavenger level

Unchanged or increased free radical

production

Decreased scavenger contents

Increased lipid peroxidation

Membrane destruction

NEUTROPHILS

NADPH OXIDASE1O2 O2•-

NO• H2O2

O2 NADPH

NADP

+

MYELOPEROXIDASE H2O2 + Cl- HOCl

VASCULAR ENDOTHELIUM

ATP xanthine

uric acid

xanthine oxidase

xanthine dehydrogenase

O2

O2•- + H2O2

MITOCHONDRIAL ELECTRON TRANSPORT CHAIN

O2 O2•- + H2O2

L-arginin

NO-syntase

NO.

Physiological free radicalsI. 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.

O2-

reoxygenation

ATP

AMP

adenoine

inosine

hypoxanthine

xanthine dehydrogenase

xanthine oxidase

O2

isch

emia

McCord J. M. The biology and pathology of oxygen radicals. J Of Int. Medicine 1978.69

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

IS

CH

EM

IA

Reperfusion injury

II. Overproduction of free radicals:

xanthine-oxidase derived oxidant - O2-

activated neutrophils - NADPH oxidase, myeloperoxidaselipid mediators - PAF , LTB4 , polypeptide mediators ( C5A )

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