Transcript Slajd 1

Free radical theory
Oxidative stress and
antioxidants in the human
body
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The nature of free radicals
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Reactive oxygen, nitrogen and chlorine species
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The oxidative stress
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Important cellular sources of oxidative stress
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Exogenous sources of free radicals
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Pathological processes involving oxygen free radical reactions
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The defense system against free radicals and reactive oxygen
species:
 - antioxidant enzymes,
 - endogenous antioxidants,
 - dietary antioxidants.
The nature of free radicals
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Free radicals are highly reactive and thereby destructive molecules.
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A free radical is any chemical species, capable of independent (although
extremely short) existence with one or more unpaired electrons.
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Free radicals seek electrons in order to pair their unpaired electrons.
Because most of the molecules in living organism do not have unpaired
electrons, free radicals steal electrons from normal molecules.
This process is called oxidation.
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Free radicals cause a chain reaction of oxidation.
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The rate of radical chain reactions is incredibly high – from one billionth of
a second to less than ten seconds.
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Radicals attack all molecules: lipids, sugars, proteins, DNA, and newly
damaged molecules start a chain reaction.
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Many of these molecular species are oxygen (and sometimes nitrogen and
chloride) centered. The molecular oxygen we breathe is a free radical.
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Oxidation causes physiologic damage. Free radicals are part of life,
but when produced in large quantities, they can be dangerous
and extremely damaging.
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Free radical oxidative damage has been implicated in majority of chronic
diseases.
The oxidative stress in living organism is defined as:
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the physiological state of free radicals (FR) and reactive oxygen
species formation (ROS) (Helmut Sies, 1985)
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increased production of free radicals and reactive oxygene species
causes an imbalance in the concentrations of prooxidants and
antioxidants
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damage to intact cells and organs caused by FR and ROS
Sources of FR and ROS
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Free radicals are formed naturally in the body – for example, as
byproducts of normal metabolism, by the breakdown of bacteria by
white blood cells, in enzymatic reactions.
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Endogenous sources:
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reduction of O2 (respiration)
peroxidation of lipids
enzymatic reactions
oxidation of low molecular weight components of cells
oxidation of proteins and nucleic acids in mitochondria, peroxisomes,
inflammatory cells
Sources of FR and ROS
(cont.)
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They are also form outside the body and enter the body through the
skin, respiration and other ways.
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Exogenous sources:
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environmental pollution (heavy metals, nitric oxides, smoking,
ozone,…)
radiation (electromagnetic field, UV light, ionization)
sonification
xenobiotic oxidation
Pathological processes involving ros and fr formation
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The effects of free radical damage – many free radicals and
reactive oxygen species have been implicated in disease
development:
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arthritis,
kidney disease,
cataracts,
cardiovascular disease,
atherosclerosis,
cancer and other malignancies,
aging,
degenerative neurological diseases,
ischemia/reperfusion injury …..
Free radicals and reactive oxygen species overview
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Free radicals:
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Singlet O2 - extremely reactive
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Triplet O2
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Alkoxyl radical RCO·
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Alkoxyl peroxide radical RCOO·
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Reactive oxygen species (ROS):
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Hydrogen peroxide H2O2
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Hydroperoxyl radical HO2·
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Superoxide anion O2· 
Peroxide O22
Hydroxyl radical HO·
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Ozone O3
Reactive nitrogen and chlorine species overview
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Reactive nitrogen species (RNS):
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Nitric oxide NO, NO2
Peroxynitrite ONOO-
Reactive chlorine species (RClS):
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Hypochlorous acid HOCl, hypochlorite anion OCl-
Product of oxygen reduction
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Oxygen molecule reduction and ROS synthesis
Molecular oxygen is reduced to water in four single-electron steps.
Reactive oxygen species are products of individual one-electron reactions.
Four electron reduction of oxygen molecule
Reactice oxygen species (cont.)
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superoxide anion O2· - - less reactive than HO·, travels in the
blood and attacts a number of biological targets, acts as
vasodilator, may have a role in intracellular signaling and
growth regulation,
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hydrogen peroxide H2O2 - crosses cellular membranes easily
and may cause expression of virus genes, has only a few cellular
targets but can result in the production of hydroxyl radicals,
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hydroxyl radical HO· - highly reactive which can attack all
biological molecules,
Production in vivo of hydroxyl radical:
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Haber-Weiss reaction:
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Fenton reaction:
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H2O2 + O2· - → HO· + OH- + O2
Fe2+ + H2O2 → HO· + OH- + Fe3+
O2· - + Fe 3+ → O2 + Fe 2+
Summerizing the Fenton reaction the H-W reaction is obtained:
Fe 2+ / Fe 3+
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H2O2 + O2· -
→
HO· + OH- + O2
Wolne rodniki …
Overview of mitochondrial ROS production
ROS production by mitochondria can lead to oxidative
damage to mitochondrial proteins, membranes and DNA,
impairing the ability of mitochondria to synthesize ATP and
to carry out their wide range of metabolic functions,
including the tricarboxylic acid cycle, fatty acid oxidation,
the urea cycle, amino acid metabolism, haem synthesis and
FeS centre assembly that are central to the normal operation
of most cells.
Mitochondrial oxidative damage can also increase the
tendency of mitochondria to release intermembrane space
proteins such as cytochrome c (cyt c) to the cytosol by
mitochondrial outer membrane permeabilization (MOMP)
and thereby activate the cell's apoptotic machinery.
In addition, mitochondrial ROS production leads to induction
of the mitochondrial permeability transition pore (PTP),
which renders the inner membrane permeable to small
molecules in situations such as ischaemia/reperfusion injury.
Consequently, it is unsurprising that mitochondrial oxidative
damage contributes to a wide range of pathologies. In
addition, mitochondrial ROS may act as a modulatable redox
signal, reversibly affecting the activity of a range of functions
in the mitochondria, cytosol and nucleus.
Biochem J. 2009 January 1; 417(Pt 1): 1–13.
Oxidation of unsaturated fatty acids in lipids
Product of protein oxidation:
Product of DNA oxidation:
Reactive nitrogen species
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Nitric oxides and peroxynitrite:
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NO,
NO2,
ONOO-
NO is a free radical produced in our body in:
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the vascular endothelial cells which form the lining of blood vessels,
the phagocytes which are part of the immune system,
some brain cells
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NO acts on smooth muscle cells in vessel walls causing relaxation
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NO can react with O2·- and produce peroxinitrite anion ONOO-, strong
oxidant, not stable at physiological pH, but its half-life time is 1 second,
and it means that it can diffuse in cells
The defense system against free radicals and reactive oxygen
species
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The body is able to prevent free radical and reactive oxygen species
damage by producing antioxidant molecules and enzymes.
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They neutralize free radicals (oxidants) by donating an electron to
them or robbing one from them.
Antioxidants
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Antioxidants are a group of compounds which, when present at low
concentrations, in relation to oxidizable substrates, inhibit or delay
oxidative processes, while often being oxidized themselves.
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The role of antioxidants is to interact with free radicals and “quench”
them, or render them harmless.
Antioxidants are involved in the prevention of cellular
damage
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Preventive antioxidants – prevent the formation of new FR and ROS:
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Scavenging antioxidants – remove ROS once formed, thus prevent radical chain
reactions:
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ceruloplasmin
albumin
transferrin, ferritin, myoglobin, haptoglobin
metallothioneine
enzymes: SOD, GPx, GR, CAT, paraoxonase (PON), metalloenzymes
small molecules (lipophilic, hydrophilic): GSH, vitamin C, E, A (β-carotene), bilirubin,
uric acid, flavonoids
Repair enzymes – repair or remove ROS-damaged biomolecules:
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DNA repair enzymes,
methionine sulphoxide reductase
lipase
proteases
transferases
Naturally occurring antioxidants, of high or low molecular weight, can
differ in their composition, their physiological and chemical properties
and in theirmechanism and site of action.
They are divided into following categories:
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enzymes – attenuate the generation of reactive oxygen species by
removing potential oxidants or by transforming ROS/RNS into
relatively stable compounds.
Superoxide dismutase - Cu,Zn-SOD, Mn-SOD, Fe-SOD
SOD catalyzes the transformation of the superoxide radical into hydrogen
peroxide, which can be further transformed by the enzyme catalase into water and
molecular oxygen:
O2· - + O2· - + 2 H+ → H2O2 + O2
2 H2O2 → H2O + O2
Naturally occurring antioxidants (cont.)
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Glutatione peroxidase (GPx) reduces lipid peroxides (ROOH), formed by the
oxidation of polyunsaturated fatty acids (PUFA), to a stable nontoxic molecule –
hydroxyl Fatty acid (ROH).
ROOH + 2 GSH → ROH + H2O + GSSG
Together with phospholipases, GPx can also convert phospholipids hydroperoxides
(PL-OOH) into phospholipids hydroxide (PL-OH)
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Glutathione reductase (GR):
NADPH + H+
+ GSSG
→
NADP+ + 2 GSH
Naturally occurring antioxidants (cont.)
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high molecular weight proteins:
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albumin,
ceruloplasmin,
transferrin, haptoglobin
All are present in plasma, bind free metal ions active in the redox reactions
and limit the production of metal-catalyzed free radicals.
Albumin and ceruloplasmin can bind copper ions, and transferrin binds iron
ions. Haptoglobin binds heme-containing proteins and can clear them from the
circulation.
Naturally occurring antioxidants (cont.)
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low molecular weight antioxidants subdivided into:
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lipid-soluble antioxidants (tocopherols, carotenoids, quinines, bilirubin,
some polyphenols)
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water-soluble antioxidants (ascorbic acid, uric acid, some polyphenols)
They delay or inhibit cellular damage mainly through their free radical
scavenging property.
Intact cell and the cell under FR and ROS attack
Antioxidants as important markers of disease
FR and ROS circulate freely in the body with access to all organs and tissues, which can
have seriuos repercussions throughout the body.
The body utilises antioxidant reserves to cope with FR and ROS and monitoring antioxidant
levels may be conducive to the early detection of disease.
Monitoring both individual antioxidant components and the overall status of the antioxidant
system on a range of automated instruments include:
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Total antioxidant status (TAS)
SOD activity
GPx and GR activity
Iron level/ total iron binding capacity (TIBC)
Concentration of:
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transferrrin
ferritin
albumin
bilirubin
uric acid
Monitoring of TAS level, SOD and GPX activity,
level of selenium in some diseases
Biomarkers of the oxidative stress
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Lipids’ damage markers:
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Proteins’ damage markers:
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lipid peroxides
4-hydroxyalkenals
malonyldialdehyde (MDA)
oxysterols
isoprostanes, isoleucotriens
carbonyl groups (>C=O, CO)
adducts of CO with carbohydrates,
derivatives of Tyr, Trp, Phe, His, Met, Lys, Leu, Ile, Val oxidation, nitration,
chlorination
oxidation of –SH groups: sulphoxides = S(O)2
Markers of DNA (purine and pyrimidine bases) damage:
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deamination products,
adducts with CO
nitration and oxidation products