1
12B. Biological oxidations.
Effects of free radicals to organisms.
Lipoperoxidactions,antioxidants.
Patobiochemistry 2016
12B-biological oxidation
• Oxidative stress appears during large accumulation of
arising reactive forms of oxygen/nitrogen, when the
organism is unable to dispose of them.
• Oxidative stress harms the cells (especially cell
membranes), proteins, enzymes, genetic material and
contributes to development of infectious and
degenerative diseases.
• Is supposed to participate in development of
atherosclerosis, diabetes, tumour diseases, degenerative
nervous diseases, aging, …
• Doesn´t have only undesirable effect – under the
supervision of white blood cells, it serves for killing of
bacteria, parasites, viruses, tumour cells…
Oxidative stress
212B-biological oxidation
TEST
Free radicals
Any molecule/atom capable of independant
existence with 1/more unpaired electrons
Atom: proton, neutron, electron shell (orbital)
Radical: contains free unpaired electron in outer orbital
(it can be atom or molecule, neutral or ion)
-homolytic cleavage of covalent bond (energetically
difficult, not often in biolog. systems)
- by reduction, oxidation
• majority of biomolecules are not radicals
312B-biological oxidation
Radical reactions
Radical: effort for pairing of electrons,
mostly significant reactivity
Generally three stages
• initiation
• propagation
• termination
412B-biological oxidation
Reactive forms of oxygen and nitrogen
(ROS, RNS)
• Overall term for free radicals and some non-radical
compounds (RONS)
• Significant physiological functions in organism
• Toxical under certain conditions
• Transformations catalysed by ionts of transition metals
• Fenton´s reaction: H2oO2 + Fe2+ …. HO. + HO- + Fe3+
• Regeneration Fe2+: O2-. …
• Haber-Weiss reaction
• Transition metals: first row of d-elements has unpaired electrons which can be
considered as free radicals, except Zn
• The most significant: Fe, Cu, Mn and Zn
• In organism bound in depot forms, inactive, transferin,
feritin, ceruloplasmin 512B-biological oxidation
TEST
ROS (reactive oxygen species)
free radicals
superoxid, O2
· hydroxyl
radical, OH ·
peroxyl, ROO ·
alkoxyl, RO ·
hydroperoxyl, HO2
·
are not free radicals
hydrogen peroxide, H2O2
(Fenton´s reaction)
hypochlorus acid, HClO
ozone, O3
singlet oxygen, 1O2
6
TEST
12B-biological oxidation
RNS (reactive nitrogen species)
free radicals
nitric oxide, NO .
nitrogen dioxide, NO2
.
are not free radicals
nitrosyl, NO+
nitrous acid, HONO
dinitrogen oxide, N2O3
dinitrogen tetroxide, N2O4
peroxynitrite, ONOO alkylperoxinitrite,
ROONO
hypochlorous acid, HOCl
hypochlorite ClO-
712B-biological oxidation
TEST
8
Hlavní zdroje volných radikálů
Dýchací řetězec v mitochondriích
• 1 - 4 % O2 při oxidativní fosforilaci přeměněno na superoxid a H2O2
Biotransformace v endoplazmatickém retikulu
• reakce katalyzované Cyt P450 vedoucí ke vzniku volných radikálů
Bílé krvinky
• ROS jako ochrana při napadení bakteriemi (enzym NADPH - oxidáza)
Fentonova reakce přechodových kovů
Fe++ + H2O2Fe+++ + HO– + HO.
Fyzikální faktory
• UV a X-ray záření
12B-biological oxidation
TEST
9
Oxidative stress
• antioxidative processes can´t manage the elimination of excessive free radicals
Oxidative
processes Antioxidants
Oxidative
stress
OS protection
OS protection
Oxidative
stress
12B-biological oxidation
Where do free radicals come
from?
Main ROS producers: membrane bound enzymes
alternatively coenzymes with flavine structure, hem
coenzymes, enzymes with Cu in active center
1. Mitochondrial respiratory chain: especially
superoxide, subsequently H2O2
• about 1- 4% O2 entering the respiratory chain
(especially complexes I and III)
1012B-biological oxidation
TEST
1112B-biological oxidation
Where do free radicals come
from?
2. endoplasmatic reticulum
formation of superoxide (cytochrom P- 450)
3. specialized cells (leukocytes, macrophages)
production of superoxide by NADP-oxidase
4. oxidation of hemoglobine to methemoglobin
(erythrocyt is „charged“ by antioxidants)
1212B-biological oxidation
TEST
13
Respiratory chain
~
Reactive forms of oxygen
12B-biological oxidation
14
Nutrients are reduced forms of carbon
because of prevailing low oxidation numbers of carbon
O
OH
OH
OH
OH
CH2OH
I
0
0
0
0
-I
H3C
COOH
-II
-III
III
H3C CH
NH2
COOH
-III III0
glucose: 6,7 % H
average ox.n. C = 0,0
alanine: 7,9 % H
average ox. n. C = 0,0
stearic acid: 12,8 % H
average ox. n. C = -1,8  carbon is the most reduced
12B-biological oxidation
15
Two ways of ATP formation in cell
95 % of ATP is formed by aerobic phosphorylation (in
presence of O2):
ADP + Pi + H+ gradient energy  ATP
5 % is formed by substrate phosphorylation:
ADP + macroergic phosphate ⇄ ATP + second product
ADP + 1,3-bisPglycerate ⇄ ATP + 3-P-glycerate (glycolysis)
ADP + phosphoenolpyruvate  ATP + pyruvate (glycolysis)
GDP + [succinyl-CoA + Pi ⇄ succinylphosphate] ⇄ GTP + succinate + CoA (citrate
cycle)
ADP + creatine-P ⇄ ATP + creatine (muscle, direction of reaction is influenced by
current concentration)
higher / comparable energetic contain as ATP
12B-biological oxidation
16
RC is system of redox processes in inner mitochondrial membrane,
begining with NADH oxidation and ending with O2 reduction to
water.
Transfer of electrons in inner mitochondrial membrane is connected
to transfer of protons through membrane to intermembrane space.
Proton gradient is used to ATP synthesis.
H+
H+
H+
H+
– – –
+ + +
ADP + Pi
ATP
NADH+H+ O2 2H2ONAD+
H+
H+ H+
H+
H+H+
H+
H+ H+
H+
H+H+
H+
H+
H+
H+
H+
H+
H+
e–
proton gradient
Matrix (negative)
MMP (positive)
12B-biological oxidation
17
Components of respiratory chain
• substrates (NADH+H+, FADH2)
• enzyme complexes (I – IV)
• cofactores bound to enzymes of complexes (FMN, FAD, Fe-S,
hem)
• individual components between complexes (ubiquinone,
cytochrome c)
Distinguish:
hem (cyclic tetrapyrrole chelating Fe ion) × cytochrome (hem protein)12B-biological oxidation
18
Collecting points for reduction
equivalents
CC
-oxidace
Q
I.
II.
člunek
NADH + H
NAD
+
matrix
cytoplazma
FAD
FAD FAD
glycerol-P DHAP
sukcinát
fumarát
alkanoyl-CoA
alkenoyl-CoA
I.
acyl-CoA
enoyl-CoA
pyruvate, CC, keto
compounds
12B-biological oxidation
19
Enzyme complexes in RC
Name Cofactors Oxidation Reduction
I. NADH-Q oxidoreductase* FMN, Fe-S NADH  NAD+ Q  QH2
II. succinate-Q reductase FAD, Fe-S, cyt b FADH2  FAD Q  QH2
III. Q-cytochrome-c-reductase Fe-S, cyt b, c1
QH2  Q cyt cox  cyt cred
IV. cytochrome-c-oxidase cyt a, a3, Cu cyt cred  cyt cox O2  2 H2O
* also called NADH dehydrogenase
12B-biological oxidation
20
• about 98 % of O2 is consumed in RC (cytochrome-c-oxidase)
• except of water, reactive forms of oxygen (ROS, reactive oxygen species) are
formed
• complexes I and III are main sources of ROS (formation of superoxide)
• production of superoxide increases if flow of electrons in RC slows down or
turns around
• mitochondria contain plenty of antioxidants (GSH, QH2, superoxide
dismutase)
Mitochondria and oxidative stress
respiratory chain
cyt cROS
mitochondrial
dysfunction apoptosis
necrosis
Lipoperoxidation of
HFA
diseases, aging
mtDNA
mutation
defect
proteins cytochrome c release
TEST 12B-biological oxidation
21
Mitochondria and apoptosis
• apoptosis is regulated process of cell extinction with minimal response to
surrounding tissue
• apoptosis is important for natural tissue regeneration
• regulatory apoptotic proteins belong to Bcl-2 family (B-cell lymphoma 2),
• Some of them are anti-apoptotic (Bcl-xl), others pro-apoptotic (Bax, Bak)
• Bax and Bak proteins oligomerize to form a pore in outer mitochondrial
membrane
• cytochrome c is released to cytosole, binds to inactive caspases and
other proapoptotic factors –apoptosom is formed – that triggers
executive stages of apoptose (caspase cascade)
12B-biological oxidation
22
Reactive forms of oxygen in organism
Radicals Neutral, anionts, cationts
Superoxide ·O2
Hydroxyl
radical ·OH
Peroxyl radical* ROO·
Alkoxyl radical RO·
Hydroperoxyl radical HOO·
Nitric oxide NO·
Hydrogen peroxide HOOH
Hydroperoxides* ROOH
Hypochlorous acid HClO
Singlet oxygen 1O2
Peroxynitrite ONOONitronium
NO2
+
* Derivates of phospholipides during lipoperoxidation: PUFA-OO·, PUFA-OOH
23
Hydroxyl radical HO·
• The most reactive free radical, reacts
immediately with molecules in place of formation
• Reacts with all the molecules in living organisms
• Extremly strong oxidation agent
• Formation: Fenton´s reaction, homolytic cleavage
of O-O bond in H2O2, ionizing radiation, in
reaction of HOCl with O2-., ultrasound, in
lithotripsia and lyophilisation
12B-biological oxidation
24
Strongly reactive hydroxyl radical •OH is
formed in Fenton´s reaction
H2O2 + Fe2+  •OH + OH- + Fe3+
H2O2 + •O2
-  •OH + O2 + OHor
from hydrogen peroxide and superoxide, catalyzed by Fe2+
ions:
12B-biological oxidation
25
Superoxide anion-radical ·O2
•
is formed by one electron reduction of dioxygen
• relatively little reactive
• acts as oxidation and reduction agent (reduction of cytoch.
C x oxidation of ascorbate)
• dirrect damage of biomolecules highly selective
• indirrect facilitates HO formation.
• formation of peroxynitrile after reaction with NO.
O2 + e-  ·O2
[this
is not a reaction, only one redox pair]12B-biological oxidation
26
Superoxide formation in
organism
• so called respiratory inflammation (NADPH
oxidase, phagocytizing leukocytes)
2 O2 + NADPH  2 ·O2
- + NADP+ + H+
• spontaneous oxidation of hemoproteins
hem-Fe2+ + O2  hem-Fe3+ + ·O2
[these
are the reactions, combination of two redox pairs]12B-biological oxidation
27
Singlet oxygen1O2
• Excitated state of triplet dioxygen, molecular O2 with paired spins, more
reactive than common O2,
• Formed in photochemical reactions, also after light absorption by some
pigments (porphyrins)
• Causes biologic damage (damage of retina, porphyria)
• Treatment of neonatal hepatitis, psoriasis
• Interaction with other molekules
• Chemical reactions (formation of hydroperoxides, endoperoxides from
compounds with one or more double bonds/conjugated systems
• Formation of carbonyl compounds from tryptophane
• Transfer of excitation energy (quenching)
3O2  1O2
12B-biological oxidation
28
Hydrogen peroxide H2O2
• in vitro relatively unstable compound, easily
decomposed to water and oxygene
• In organism is formed in AA/amines deamination
• also in xanthinoxidase reaction
• two electron reduction of O2
• can oxidize -SH groups of enzymes, produce
hydroxyl radical, …
• Little reactive, toxic in high concentrations
12B-biological oxidation
29
Oxidative deamination of aminoacids
provides ammonia, oxoacid and hydrogen
peroxide
R CH
NH2
COOH
FAD FADH2
R C COOH
NH
O2H2O2
katalasa
H2O + O2
H2O
R C COOH
O
NH3
iminokyselina
H2O + ½ O2
12B-biological oxidation
30
Xanthinoxidase produces hydrogen
peroxide
hypoxanthin + O2 + H2O  xanthin + H2O2
xanthin + O2 + H2O  uric acid + H2O2
Majority of tissues, mainly livers
12B-biological oxidation
31
Compare: reduction of
dioxygen
Reduction type Partial reaction (redox pair)
Four electron O2 + 4 e- + 4 H+  2 H2O
One electron O2 + e-  ·O2
Two
uelectron O2 + 2 e- + 2 H+  H2O2
12B-biological oxidation
32
Hypochlorous acid HClO
• Formed in neutrophilic granulocytes from
hydrogen peroxide and chloride anion
• Reaction is catalyzed by myeloperoxidase
• HClO has strong oxidative and bactericidal
effects
• Damage of biomolecules:
• Damage of proteins (transforms Met to Met
sulphoxide, chloration of Tyr to form 3-chlortyrosine,
damage of -SH group of membrane proteins
• Chloration of DNA bases (especially pyrimidines)
H2O2 + Cl- + H+  HClO + H2O
12B-biological oxidation
33
Nitric oxide NO· formation from arginine
has 1free electron, free radical
• Free diffusion between cells 1-10s, in blood catched by erythrocytes,
produced by NO synthase: nNOS, eNOS, iNOS
• Exogenous sources: medicaments, vasodilatatie
• Phys. function (vasodilatation, neurotransmitter, macrophages-bactericidal
effect
• NO· binds to guanylatecyclase  cGMP  relaxation of smooth muscles
(especially vessels) and other effects…
• NO· is radical and provides other reactive metabolites: formation of
peroxynitrite and others RNS and nitrosothiols
NO· + ·O2
-  O=N-O-O-  O=N-O-O-H (peroxonitrous acid)
H+
NO2
+ + OH- ·NO2 + ·OH
tyrosine
nitration
NO3
- (plasma, urine)
peroxonitrite
nitrosylation
12B-biological oxidation
34
NO releasing compounds
CH2 O NO2
CH
CH2
O NO2
O NO2
O
OO
O NO2
O2N
CH CH2 CH2
H3C
H3C
O N O
Na2[Fe(CN)5NO]
natrium nitroprusside (natrii nitroprussias)
disodium pentacyanonitrosylferrate
ruby red crystals
extremly efficiant, i.v. infusion
glycerol trinitrate (glyceroli
trinitras)
yellowish oily liquid
classic medicament, fast action
sublingual tablet, spray, plaster
isosorbide dinitrate (isosorbidi dinitras)
more advantageous pharmacokinetic
properties
amyl-nitrite (amylis nitris)
volatile liquid, inhalation use
CH
H3C
H3C
CH2 O N O
isobutyl-nitrite
volatile liquid, new drug
poppers, rush, liquid aroma …
12B-biological oxidation
Peroxynitrite ONOO•
Strong cytotoxic oxidation agents
• Toxic effects:
• Deplexation of –SH groups and other antioxidants
• Oxidation of lipids
• DNA breaks, nitration and deamination of DNA bases (G)
• Nitration of aromatic AA (Tyr, Phe, Trp) 3-nitrotyrosine (inaktivation
of enzymes, interference with signal transduction)
• Oxidation of Met to sulphoxide
NO· + ·O2
-  O=N-O-O-
peroxonitrite
nitrosylation
3512B-biological oxidation
Sulphurous radicals
• In vivo –SH –antioxidants, but thiols can be source
of free radicals as well
• Formation of thiol radicals GS.
• Formation of potential cytotoxic radicals
3612B-biological oxidation
Exogenous causes of free radicals formation
• Ultraviolet or ionizing radiation (UV light, g
radiation, X- ray)
• Smoking
• Air pollution
• Intoxication (PCB, CL4, chloroform, alcohol
• Food (thermal processing, crushing, light
influence)
37
TEST
12B-biological oxidation
Endogenous causes of free radicals
formation
• Reaction catalyzed by XOD (injuries,
necrosis)
• Decay of phagocytes and macrophages
(inflammations, sepses, burns)
• Synthesis of prostaglandins
• Hyperglycemia
• Reperfusion after previous ischemia
(oxygen debt)
3812B-biological oxidation
TEST
Function of free radicals in healthy orgamnism
I Tool of oxidases and oxygenases
• cytochromoxidase (toxic intermediates, H2O2 and
superoxides, bount to enzymes)
(mitochondrial respirátory chain)
• monoxygenases (oxygenases with mixed function) activate
O2 in liver ER or in gland mitochondria;
hydroxylation
(cytochrome P450, oxidation of wide range of substrates
using O2, liver P450- metabolism of xenobiotics)
3912B-biological oxidation
I Tool of oxidases and oxygenases
• Xanthinoxidase (XOD) –oxidation of xanthinu
to uric acid
• Proline and lysinehydroxylases (hydroxylate
Pro and Lys in colagen synthesis)
• Tyrosinehydroxylase (hydroxylates Tyr,
begining of synthesis of dopamine, adrenaline,
noradrenaline)
Synthesis of thyroid gland hormons
-Thyreoperoxidase
Oxidation of I- to I2 by hydrogen peroxide ---mono
and di iodotyrosine, thyroxine T4, triiodothyronine
T3 4012B-biological oxidation
Ovum fertilization
Sperm
Disruption of ovum membrane during
penetration –O2- production.
Ovum
Prevention of other sperms penetration –
production of H2O2 ..formation of
transverse bonds in membrane
4112B-biological oxidation
Function of free radicals in healthy organism
II
ROS and RNS against bacteria, phagocytosis
Form of protection against extraneous particles and
microorganisms
Macrophages, neutrophils, NK cells
Enzymes participating in disabiling of absorbed microorganisms
in phygocyte
• enzyme complex NADPH-oxidase of
leukocytes and macrophages (respiratory
inflammation)
• myeloperoxidase – catalysis of reaction
H2O2 + Cl- + H+ = HClO + H2O
• iNOS: NADPH dependent (Arg-Citrulin..NO)
4212B-biological oxidation
Function of free radicals in healthy
organism III
• signal molecules
primary messenger  secondary messenger 
info net
• redox state of cell influences function of that net
• redox state: capacity of antioxidative system,
accesibility of reduction equivalents, intensity of
oxidation load (RONS)
ROS: secondary messengers
NO (neurotransmitter, vascular endotel-relaxation
vascular walls, NO in phagocytizing cells) 4312B-biological oxidation
Immune protection
vs.regulation
massive production of ROS as a tool of
immune protection
x
Induction of changes in low ROS levels,
which are probably regulatory
mechanism
4412B-biological oxidation
45
Positive effects of oxygen radicals
• Intermediates of oxidase and oxygenase
reactions (cyt P-450), during reactions radicals are
bound to enzyme so they don´t harm surrounding
tissues
• bactericidal effect of phagocytes, respiratory
inflammation (NADPH-oxidase)
• signal molecules (primary messengers), proven
in NO· so far, some other radicals are supposed to
have similar effects12B-biological oxidation
Oxidative stress
When balance between formation and
elimination of RONS is broken,
oxidative stress happens
balance can be broken on both sides!!
Causes of oxidative stress formation:
-excessive formation of RONS,
- insufficient activity of antioxidative defense
system,
- combination
4612B-biological oxidation
Damage of lipids - attack to unsaturated FA
Damage
• loss of multiple bonds
• Formation of reactive
metabolits
(aldehydes)
Effect
• change of fluidity,
permeability of
membranes
• effect to membrane
bound enzymes 47
Peroxidation of lipids
- Chain reaction
- enzyme peroxidations of lipids (synthesis of prostanoids,
leukotrienes, active center of hydro- and endoperoxidases
(COX and lipoxygenases)
- non-enzyme peroxidations of lipids – patological process,
intermediates of lipoperoxidation, binding to proteins,
influence of fluidity
Peroxidation of
linoleic acid
48
peroxyl radical
hydroperoxide
Peroxide radical– alkanals,
alkenes….modification, lungs
malondialdehyde
12B-biological oxidation
Damage of proteins
Damage
• agregation and networking,
• fragmentation and cleavage
• reaction with hem Fe
• modification of functional
groups
Consequence
• changes in ion transport
• changes in aktivity of enzymes
• proteolysis, activstion of proteases
and phospholipases by Ca21
accumulation in cytosol
• formation of new antigen
determinants with subsequent
autoimmune reactions
49
Dirrect damage of proteins by RONS influence
-oxidation, hydroxylation, nitration, chloration AA, no chain
reaction
Indirrect damage of proteins by products of lipid peroxidation
-alkoxyl and peroxyl radicals
Malondialdehyde and 4-hydroxynonenal, formation of transverse
bonds between neighbouring chains, formation of carmobyne
compounds
DNA damage
Damage
• cleavage of
carbohydrate cycle
• modification of
bases
• breaks of chain
Consequence
• mutation
• translation errors
• Inhibition of
proteosynthesis
• missmatching
50
• Hydroxylation of purine bases
- 8-hydroxyadenin, 8-hydroxyG, 8-oxoG, FapyG, FapyA
• Hydroxylation of pyrimidine bases
-thyminglycol, uracilglycol,…
• Hydroxylation of carbohydrate residues
-oxidation and fragmentation – releasing of bases,
interruption of DNA chain and malondialdehyde formation
51
Damage of biomolecules
Compound Damage Consequences
Lipids
- oxidation of PUFA (loss of
double bonds)
-formation of reactive compounds
(aldehydes and ROO·)
-oxidation of cholesterol
- change in membrane permeability
- damage of membr. enzymes,
proteins
- change in membrane fluidity
Proteins
- modification of -SH and
phenyl (arom.) AA
- Formation of transverse bonds
between chains-networking
and agregation
- fragmentation + cleavage
changes in ion transport
Ca2+ enter to cytosol
changes in aktivity of enzymes
formation of new antigen
determinants
activation of proteases and
phospholipases
DNA
modification and cleavage of
deoxyribose
modification of bases
breaks of chain
formation of transverse bond
between DNA a protein chains
mutations
missmatches
translation errors
inhibition of proteosynthesis
TEST
How can we quantify oxidative stress?
Detection of free radicals
• quite difficult because of phys. chem.
properties
Measuring of oxidative stress products
• simplier, wide range of oxidative stress
markers
5212B-biological oxidation
Markers of oxidative stress
Appraisal of lipoperoxidation:
malondialdehyde (MDA), conjugated dienes,
isoprostans
Appraisal of protein damage:
protein hydroperoxides
appraisal of DNA damage:
determination of modificated nucleosides
(8-oxoG)
5312B-biological oxidation
TEST
Determination of antioxidants
ascorbate
tocoferol
SOD
GSHPx
glutathion
5412B-biological oxidation
Diseases connected to oxidative stress
Neurologic
Alzheimer disease
Parkinson disease
Endocrine
Diabetes
Gastrointestinal
Acute pankreatitis
Vascular
Aterosclerosis
Other
Obesity
Organe transplantation, cancer 5512B-biological oxidation
Antioxidative protective system
Three types of protection
• inhibition of excessive RONS production
• capture and elimination of radicals (catcher)
• reparative mechanisms of damaged
biomolecules
5612B-biological oxidation
TEST
Summary of FR antioxidants and catchers
1. Endogenous antioxidants
• enzyme (cytochrome c, SOD, GSHPx, catalase)
• non-enzyme
- membrane ( -tocoferol, -carotene, coenzyme Q 10)
- non-membrane (ascorbate, urates, transferine, bilirubin)
2. Exogenous antioxidants
• inhibitors of FR formation (regulation of enzyme activity)
• scavengers of formed FR (enzymes,non-enzymes)
• trace elements (Se, Zn)
57
TEST
58
Antioxidative systems of organism
1. Enzymes (endogenous)
superoxide dismutase, catalase, glutathionperoxidase
2. high molecular weight antioxidants (endogenous)
transferrin, ferritin, ceruloplasmin,… bind free metal ions
3. low molecular weight antioxidants (exogenous,
endogenous)
• reducing compounds with phenol -OH (tocoferol, flavonoids, urate)
• reducing compounds with enolo -OH (ascorbate)
• reducing compounds with -SH group (glutathion, dihydrolipoate)
• compounds with extensive systém of konjugated double bonds
(carotenoides)
12B-biological oxidation
TEST
5912B-biological oxidation
60
Superoxide dismutase
• present in every cell, phylogenetically very old enzyme
• catalyse dismutation of superoxide
2 ·O2
- + 2 H+  O2 + H2O2
• oxidation numbers of oxygen in reaction:
two isoforms: SOD1 (Cu, Zn, cytosol), dimer, Cu = redox center
cytosol, intermitochondrial space, hepatocyte, brain, erytrocyte,
high permeability, catalysis at pH 4,5-9,5
• SOD2 (Mn, mitochondria), tetramer, mitochondrial matrix, lower
stability than Cu, Zn – SOD, phylogenetically younger
(-½)  (0) + (-I)
12B-biological oxidation
61
Elimination of H2O2 in organism
• catalase present in erytrocytes
disproportionation H2O2, H2O2  ½ O2 + H2O, at high levels of H2O2
detoxication of alkylperoxides : H2O2 +ROOH  O2 + H2O+
ROH
• glutathion peroxidase (elimination of interacellular hydroperoxides)
• contains selenocystein, second substrate - glutathion (G-SH) reduces H2O2 and
hydroperoxides of phospholipides (ROOH)
detoxication of hydrogen peroxide, GSSG reduces to GSH using glutathionreductase
2 G-SH + H-O-O-H  G-S-S-G + 2 H2O
2 G-SH + R-O-O-H  G-S-S-G + R-OH + H2O
harmless derivate12B-biological oxidation
Glutathionperoxidases
eliminate intracellular hydroperoxides and H2O2
2 GSH + ROOH  GSSH + H2O + ROH
• cytosol GSH – glutathion peroxidase (EC 1.11.1.9,
cGPx)
• extracellular GSH – glutathion peroxidase
(eGSHPx)
• phospholipidhydroperoxide GSH - peroxidase (EC
1.11.1.12, PHGPx)
6212B-biological oxidation
63
Low molecular weight antioxidants
Lipophilic Hydrophilic
Tocoferol
Carotenes
- Lycopene
- Lutein
Ubiquinola
L-ascorbate
Flavonoids
Dihydrolipoatea
Glutathiona
Urci acid a
a Endogenous compounds.
12B-biological oxidation
64
Tocoferol
• Lipophilic antioxidant of cell membranes and lipoproteins
• Reduces peroxyl radicals of phospholipids to hydroperoxides, which are
further reduced by GSH, tocoferol oxidises to stable radical
• PUFA-O-O· + Toc-OH  PUFA-O-O-H + Toc-O·
• Toc-O· partially reduces to Toc-OH by ascorbate to GSH (phare interface)
• Toc-O· + ascorbate  Toc-OH + semidehydroascorbate
O
HO
CH3
CH3
H3C
R
CH3
O
O
CH3
CH3
H3C
R
CH3
12B-biological oxidation
65
Carotenoides
• Carotenoides are polyisoprenoid carbohydrates (tetraterpens)
• Eliminate peroxyl radicals while changing themselves to
stable carotene radical
• Are able to quench (deexcitate) singlet oxygen
• Sources in food: leaf vegetable, yellow, orange and red
coloured vegetable and fruit
• The most efficient antioxidant is lycopene, present in some
food, mainly tomatos and their products (ketchup, puree) –
je thermally stable
• High intake of lycopene in the Mediterranean
12B-biological oxidation
66
Contain of lycopene in food (mg/100 g)
Tomato puree
Ketchup
Tomato juice/sauce
Melon
Papaya fresh
Tomatos fresh
Apricot compote
Apricot fresh
10-150
10-14
5-12
2-7
2-5
1-4
~ 0,06
~ 0,01
For lycopene effective release and
absorbtion is suitable to cook tomatos
and consume with oil
CH3
CH3
CH3
CH3
CH3
CH3CH3
CH3
CH3
CH3
12B-biological oxidation
67
Zeaxanthine and luteine
OH
CH3 CH3
CH3
CH3 CH3
CH3 CH3
CH3 OH
CH3CH3
CH3
CH3 OH
CH3 CH3
OH
CH3
CH3
CH3
CH3CH3
CH3
zeaxanthine (two chiral centers)
luteine (three chiral centers)
• belong to xanthophyles, oxygenic derivates of carotenoides
• differs in double bond location and number of chiral centers
• present especially in green leaf vegetable
• present in yellow spot (macula lutea) and protects it from degeneration
12B-biological oxidation
68
Ubiquinol (QH2)
• Present in every membrane
• Endogenous synthesis of interstinal mikroflóra from
tyrosine and farnesyl diphosphate (turn in
cholesterol biosynthesis)
• Exogenous sources: sprout oil, liver, meat
• Reduced form of QH2 helps in tocoferol
regeneration
• Toc-O· + QH2  Toc-OH + ·QH
12B-biological oxidation
69
L-Ascorbate (vitamine C)
• Cofactor of proline hydroxylation (colagene synthesis)
• Cofactor (reductant) of dopamine to noradrenaline hydroxylation
• Strong reduction agent (Fe3+ Fe2+, Cu2+  Cu+)
• Facilitates Fe absorbtion from food
• Reduces radicals ·OH, ·O2
-, HO2·, ROO·,...
• Regenerates tocoferol radical
• Eliminates to oxalate !!
• Excessive ascorbate has prooxidative effects:
Fe2+ a Cu+ catalyse formation of hydroxyl radical
ascorbate + O2  ·O2
- + ·monodehydroascorbate
12B-biological oxidation
70
L-Ascorbic is diprotic acid
Two conjugated pairs:
ascorbic acid / hydrogenascorbate
hydrogenascorbate / ascorbate
O
HO O
O
C
CH2OH
H OH
O
O O
O
C
CH2OH
H OH
O
HO OH
O
C
CH2OH
H OH
two enol hydroxyls
pKA1 = 4,2 pKA2 = 11,6
12B-biological oxidation
71
L-Ascorbic acid has reduction effects
O
O O
O
C
CH2OH
H OH
O
HO OH
O
C
CH2OH
H OH
ascorbic acid dehydroascorbic acid
(reduced form) (oxidized form)
12B-biological oxidation
72
Flavonoids and other polyphenols
• Ubiquitary spread in plants, most common reduction
compounds in our food
• Total intake is about 1 g (much higher than vitamines)
• Derivates of chromane (benzopyrane) containe many phenol
hydroxyls
• Main representative is quercetin
• reduce free radicals while are transformed to little reactive
fenoxyl radicals
• Chelatate metal ions (Fe2+, Cu+) preventing them from
participating in Fenton´s reaction
12B-biological oxidation
73
Main sources of flavonoids and other
polyphenols
• vegetable (especially
onion)
• fruit (apples, citruses,
grapes)
• green, black tea
• cocoa, chocolate
• olive oil (Extra Virgin)
• red wine
O
OH
OOH
HO
OH
OH
quercetin
12B-biological oxidation
74
Glutathion (GSH)
• tripeptid
• γ-glutamylcysteinylglycine
• produced in every cell
• reduction agent (-SH)
• reduced H2O2 and ROOH (glutathion peroxidase)
• reduces different oxygen radicals
• regenerates -SH groups of proteins and coenzyme A
• participates in tocoferol and ascorbate regeneration
HOOC
N
N COOH
O
H
CH2
SH
O
H
NH2


g
12B-biological oxidation
75
Regeneration of GSH reduced form
• fluent regeneration of glutathion (GSH) reduced form
must be ensured
• Glutathion reductase, important in erytrocytes
• GSSG + NADPH + H+  2 GSH + NADP+
pentose
cycle
12B-biological oxidation
76
Dihydrolipoate
• cofactor of oxidative decarboxylation of pyruvate and 2-
oxoglutarate
• reduces many radicals (mechanism is unknown)
• participates in tocoferol regeneration
• terapeutic using (acidum thiocticum) – diabetic neuropathy
S S
COOHCOOH
SH SH
dihydrolipoate
(reduced form)
lipoate
(oxidized form)
12B-biological oxidation
77
Uric acid
• Final catabolit of purine bases, diprotic acid
• In tubules resorbes from 90 %
• The most common antioxidant of blood plasma
(150-400 μmol/l)
• Significant reduction effects, reduces RO· radicals
• Binds Fe and Cu cations
12B-biological oxidation
78
Lactim form of uric acid
is diprotic acid
N
N
N
N
H
OH
HO
OH
N
N
N
N
H
OH
HO
O
N
N
N
N
H
OH
O
O
uric acid hydrogenurate urate
pKA1 = 5,4 pKA2 = 10,3
2,6,8-trihydroxypurine
12B-biological oxidation
79
Reduction effects of uric acid
different
transformations
radical (oxidized
form)
hydrogenurate cleaves one electron
R· is for example·OH, superoxide,…
+
N
N
N
N
H
OH
HO
O
N
N
N
N
H
OH
HO
OR H RH+ +
hydrogenurate
(reduced form)
Compare concentrations in blood plasma:
Ascorbate: 10 - 100 μmol/l
Urate: 200 - 420 μmol/l
12B-biological oxidation
• Bind ionts of transition metals, change their
oxidation state and prevent their participation in
radical reactions
• Ferooxidase activity- mobilization of Fe intracellular
reserves
• Transport proteins (transferin, lactoferin,
ceruloplasmin)
• Reserve proteins (feritin, hemosiderine,
neuromelanine), haptoglobine, hemopexine
• Proteins containing large amount of thiol groups
(metalothioneins, albumine)
80
High molecular weight
antioxidants
12B-biological oxidation
Trace elements affecting FR
Selenium
affects vit. E resorption, part of
selenoproteins
 Se = insufficient immune response,
hemolysis of erythrocytes, synthesis of
methemoglobine Zinc
stabilization of cell membranes,
amplification of immune response, Fe
antagonist 8112B-biological oxidation
ROS
Enzymes
Proteins
SOD, CAT, GPX,
GST, MSH,
RSH-PX
1GP, Trf,Alb
ferritin
Metabolits
Vitamins
A, E, C, KoQ
bilirubin, urate
Mg, Mn, Zn, Se
lipoate,
karnosine
NAD(P)H
Radiation
INFLAMMATION
(neutrophils,
macrophages)
Arginine
(NOS)
Autooxidation
Electron
-transport
Polutants
Oxidases
(+)
(-) (-)
DNA, RNA
PROTEINS
EnzymesLIPIDS
Transcription
Translation
errors
Oxid. nucleic
acids
Altered
PROTEINS (enzymes)
Oxid. modific..
oxLP
82
TEST