Free radicals and antioxidants• What is „free radical“?

• Reactive oxygen and nitrogen species (RONS)

• Are the RONS always dangerous?

• Well known term „oxidative stress“ - what is it?

• Antioxidants - types and appearance

• Markers of oxidative stress

• Disorders Associated with Oxidative stress

Free radical - what is it?

Atom: proton, neutron, electronic shell (orbital)

Free radical

• particles with an unpaired electron spinning around the nucleus. (can be atom, ions, molecule).

• tend to reach equilibrium, plucks an electron from the nearest intact molecule.

• most of biomoleculs are not radicals

Free radical and „science“

• chemistduring the thirties - there is superoxide

• biochemist during the sixties - make a discovery of superoxid dismutase (SOD)

• doctorfree radicals are associated with many disorders

Mechanism of radical reactions

Radicals are highly reactive species

Three distinc steps

• initiation (homolytic covalent bonds cleavage)

• propagation (chain propagation)

• termination

ROS (reactive oxygen species)

Free radicals

superoxide, O2 · -

hydroxyl radical, OH ·

peroxyl, ROO ·

alkoxyl, RO ·

hydroperoxyl, HO2 ·

Particals, which are not free radicals

hydrogen peroxide, H2O2

(Fenton´s reaction)

hypochlorous acid, HClO

ozone, O3

singlet oxygen, 1O2

RNS (reactive nitrogen species)

Free radicals

nitrogen(II) oxide, NO .

nitrogen(IV) oxide, NO2 .

Particals, which are not free radicals

nitrosyl, NO+

nitrous acid, HONO

nitogen(III) oxide, N2O3

peroxynitrite, ONOO -

alkylperoxinitrite, ROONO

The main sources of free radicals

membranes enzymes and/or coenzymes with flavine structures, hem coenzymes, enzymes containing Cu atom in an active site

1. respiratory chain mitochondria : mainly superoxide and then H2O2

• approx 1- 4% O2 entres into resp. chain (mainly complexes I a III)

The main sources of free radicals II

2. Endoplasmic reticulum

superoxide creation (by cytochrome P- 450)

3. special cells (leukocytes)

superoxide creation by NADP-oxidas

4. hemoglobin to methemoglobin oxidation

(erytrocyte is „full“ of antioxidants)

Free radicals physiological function

Used by oxides a oxygenes

• cytochromoxidase (toxic intermediates, H2O2 and superoxide, are bound to an enzymu)

• monoxygenases - activate O2 in liver ER or in adrenal gland mitochondria ; hydroxylation

Free radicals physiological function II

ROS a RNS against bacteria

• enzyme complex NADPH-oxidase of leukocytes

• myeloperoxidase - catalysis of the following reaction

H2O2 + Cl- + H+ = HClO + H2O

Free radicals physiological function III

• Signal moleculesfirst messenger second messenger information net

This info net function is affected by the redox state of cells

• redox state : antioxidant capacity, reduction equivalent availlability, RONS rate

ROS: second messenger

Immunity vs. regulation

a massive production of ROS as immunity instrument

x

an induction of the changes low concentration ROS, which are probably regulation

mechanism

Antioxidant defence system

3 levels

inhibition of production the abundance of RONS

capture of radicals (scavengers, trappers, quenchers)

correction mechanism of destroyed biomoleculs

Antioxidants and scavengers review

1. Endogennous antioxidants

• enzymes (cytochrome c,SOD, GSHPx, catalase)

• nonenzymatic - fixed in membranes ( -tocopherol, -

caroten, coenzym Q 10)- out of membranes (ascorbate, transferrin,

bilirubin)

Antioxidants and scavengers review II

2. Exogennous antioxidants

• FR scavengers

• trace elements

• drugs and compounds influence to FR metabolism

Enzymes defence mechanism

Superoxid dismutase (EC 1.15.1.1, SOD)

2O2. - + 2H+ H2O2 + O2

SOD - is present in all oxygen-metabolizing cells, different cofactors (metals)

an inducible in case of superoxide overproduction

Superoxid dismutase

Mn 2+ SOD (SOD1)

tetramer

matrix mitochondria

lower stability then Cu, Zn - SOD

Superoxid dismutase

Cu 2+/Zn 2+ SOD (SOD 2)

dimer, Cu = redox centr

cytosol, intermitochondrial space

hepatocyt, brain, erytrocyte

high stability, catalysation at pH 4,5-9,5

Glutathion peroxidases

elimination of intracellular hydroperoxides and H2O2

2 GSH + ROOH GSSH + H2O + ROH

• cytosolic GSH - glutathionperoxidasa (EC 1.11.1.9, cGPx)

• extracelullar GSH - glutathionperoxidasa (eGSHPx)

• phospholipidhydroperoxide GSH - peroxidase (EC 1.11.1.12, PHGPx)

Catalasa

(EC 1.11.1.6, KAT)

2 H2O2 2 H2O + O2

high affinity to H2O2 : peroxisomes hepatocytes mitochondria, cytoplasm of erytrocytes

tetramer with Fe, needs NADPH

High-molecula endogennous antioxidants

• transferrin

• ferritin

• haptoglobin

• hemopexin

• albumin

Low-molecule endogennous antioxidats I

Ascorbate (vitamin C)

collagen synthesis

dopamine to epinephrine conversion

reduction agent

Fe absorption

antioxidant = reduction O2 · -

OH ·, ROO·, HO2 ·

tocopheryl radical regeneration

prooxidant

Alfa-tocopherol a vitamin E

localise in membranes

produces hydroperoxides, which are changes by GSHPx

Ascorbic acid and its metabolites

Low-molecule endogennous antioxidats II

• ubiquinone (coenzyme Q)

electron carrier in respisratory chain

co-operates with tocopheryl

• carotenoides, -caroten, vitamin A

removing the radicals from lipids

Low-molecule endogennous antioxidats III

• glutathione (GSH, GSSG)

in all mammalian cells (1-10 mmol/l)

important redox buffer

2 GSH GSSG + 2e- + 2H+

ROS elimination, stabilisation in reduction form ( SH- groups, tocopheryl and ascorbate regeneration)

substrate of glutathione peroxidases

Low-molecule endogennous antioxidats IV

• Lipoic acid (lipoate)

PDH cofactor tocopheryl and ascorbate regeneration

• melatonin

lipophilic ; hydroxyl radicals scavenger

Low-molecule endogennous antioxidats V

• uric acid (urates)

• bilirubin

• flavonoids

Trace elements influence to FR metabolism

Seleniuminfluence to vitamin E resorption, part of selenoproteins of Se = insufficient immun. respons, erytrocytes hemolysis, methemoglobin synthesis

Zinc cell membrane stabilisation Fe antagonist

Oxidative stress

Equilibrium failure between creation and a elimination of RONS leads to

oxidative stress

Be carefull - this equilibrium can be disbalance in both sides

Oxidative damage to lipid

Damage

• unsaturated bonds loss• arising of reactive

metabolites (aldehydes)

Sequel

• changes in fluidity and permeability of membranes

• membranes integral enzymes are influenced

The peroxidation of linoleic acid

Oxidative damage to proteins

Damage

• agregation, fragmentation and cleveage

• reaction with hem iron ion

• functional group modification

Sequel

• changes in: enzymes activity, ions transport

• proteolysis

Oxidative damage to DNA

Damage

• saccharide ring cleveage

• bases modification• chain breakeage

Sequal

• mutation• translation mistakes• protoesynthesis

inhibition

Oxidative stress markers

Free radicals detection

• very difficult, because of chem-phys. properties

Oxidative stress products detection

• more simple, a wide range of techniques

Oxidative stress markers II

Lipoperoxidation markers:

malondialdehyde (MDA), conjugated diens, isoprostanes

Oxidative damage to protein markers :

protein hydroperoxides

Oxidative damage to DNA :

modified nucleosides

Antioxidants determination

ascorbate

tocopheryl

SOD

GSHPx

glutathion

Disorders Associated with Oxidative stress

Neurological

Alzheimers Disease

Parkinson‘s Disease

Endocrine

Diabetes

Gastrointestinal

Acute Pancreatitis

Disorders Associated with Oxidative stress

Others conditions

Obesity

Air Pollution

Toxicity

Inflammation

Literature

Štípek Stanislav a kol.: Antioxidanty a volnéradikály ve zdraví a nemoci, Grada, 2000

Free radicals and antioxidant protocolsedited by Armstrong D., Methods inMolecular biology, volume 108, HUMANAPRESS, Toronto, New Yersey, 1998