Reactive Oxygen Species in the Body, Antioxidant...
Transcript of Reactive Oxygen Species in the Body, Antioxidant...
1
Reactive Oxygen Species
in the Body
Antioxidant Defence
MUDr Jan Plaacuteteniacutek PhD
Uacutestav leacutekařskeacute biochemie a laboratorniacute diagnostiky 1LF UK
What is a free radical
- any chemical species (molecule atom ion)
capable of independent existence that contains
at least one unpaired electron
O2middotndash
radical ion
molecule
2
For what do we need oxygen
Transfer of electrons (oxidation) from organic substances tooxygen releases huge amount of energy
Eg Glucose
CC66HH1212OO66 + 6 O+ 6 O22 rarrrarrrarrrarr 6 CO6 CO22 + 6 H+ 6 H22OO
∆∆∆∆∆∆∆∆GGoorsquorsquo = = ndashndash 2820 2820 kJkJ mol mol (180 g (180 g of of glugluccososee))
In addition reactivity of oxygen can be controlled by catalysiswith transition metals (iron copper)
Does sugar burn
3
4
Spin restriction of dioxygen
bull Normal (triplet) O2 is a biradical with high affinity to electrons
bull But gain of an electron requires one of the unpaired electrons changes its spin which is a slow process
bull Thatrsquos why we do not burn in the air
bull Singlet O2 is an excited highly reactive species
Respiratory chain in the
mitochondrial inner
membrane
5
The respiratory chain
Electrons flow lsquodownhillrsquo and terminate at oxygen
Overall reaction
NADH + H+ + 12 O2 rarrrarrrarrrarr NAD+ + H2O
∆∆∆∆∆∆∆∆GGoorsquorsquo = = ndashndash21925 kJ mol21925 kJ mol--11
hellipand resulting proton gradient powers ATP synthesis
6
Reactive oxygen species (ROS lsquooxygen radicalsrsquo) can be derived from intermediates of oxygen reduction to water
O2
2H2O
+4 e-
(+4 H+)
O2middotndash
superoxide
H2O2hydrogen peroxide
OHmiddothydroxyl
radical
+3 e-
(+3 H+)
+2 e-
(+2 H+)
+1 e-
( +H2O )
Superoxidebull Sources in the body
ndash Escape of electrons to oxygen
- Respiratory chain in mitochondria
- Other similar redox carrier systems eg microsomalcytochrome P450 monooxygenase
ndash NAD(P)H Oxidase
- Phagocytes (rdquorespiratory burstrdquo)
- Non-phagocytic cells
ndash Some enzymes
- Xanthine oxidase
- Cyclooxygenase
- Lipoxygenase
ndash Reaction of FeII-hemoglobin with oxygen
ndash Autoxidation (reaction with oxygen) of various compounds (ascorbate glutathione and other thiols catecholamines)
O2middotndash
7
Superoxide production in the respiratory chain
bull 1-2 of total body oxygen consumption (from in vitro experiments in vivo surely much less)
bull Escape of electrons from redox centers of complexes I and III mostly from semiquinone
Origin of superoxide from oxyhemoglobin
Štiacutepek S et al Antioxidanty a volneacute radikaacutely ve zdraviacute a nemoci GradaPublishing Praha 2000
8
Superoxide
bull Properties
- Moderate reactivity both oxidising and reducing agent
- Limited ability to cross biological membranes (only through anion channels or in its protonated form)
- Releases iron from Fe-S clusters
bull Fate
ndash Dismutation
ndash Reaction with nitric oxide
O2middotndash
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
NO + O2middotndash rarr OONOndash (peroxynitrite)
Hydrogen peroxide
bull Formation in the body
ndash Dismutation of superoxide (spontaneous or catalyzed by superoxide dismutase)
ndash Directly by action of some enzymesbull Xanthine oxidase
bull Monoamine oxidase (MAO)
H2O2
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
9
Hydrogen peroxide
bull Properties
- No radical- Freely crosses biological membranes- Fairly unreactive as such- But rapidly reacts with reduced transition metals such
as iron copper (the Fenton reaction)
bull Fate
ndash The Fenton reaction
ndash Safe decomposition by glutathione peroxidase peroxiredoxin or catalase
H2O2
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
hydroxyl radical
Hydroxyl radical
bull Formation in the body
ndash Ionising radiation
ndash The Fenton reaction
bull Properties
- Extremely high reactivity Damages biomolecules near its site of formation
OH middot
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
H2O rarr H + OHmiddot
10
Reactive Oxygen Species (ROS)bull Radicals
ndash Superoxide O2middotndash
ndash Hydroperoxyl radical HO2middot
ndash Hydroxyl radical OHmiddotndash Peroxyl radicals ROOmiddotndash Alkoxyl radicals ROmiddot
bull Non-radicalsndash Hydrogen peroxide H2O2
ndash Hypochlorous acid HClOndash Ozone O3
ndash Singlet oxygen 1O2
Nitric oxide
bull Formation in the body
ndash NO synthase-catalysed reaction
ndash Three different NO synthases- NOS I (neuronal constitutive)- NOS II (phagocytes inducible)- NOS III (endothelial constitutive)
NO middot
L-Arginine + O2 + NADPH rarr L-Citrulline + NADP+ +NO
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
2
For what do we need oxygen
Transfer of electrons (oxidation) from organic substances tooxygen releases huge amount of energy
Eg Glucose
CC66HH1212OO66 + 6 O+ 6 O22 rarrrarrrarrrarr 6 CO6 CO22 + 6 H+ 6 H22OO
∆∆∆∆∆∆∆∆GGoorsquorsquo = = ndashndash 2820 2820 kJkJ mol mol (180 g (180 g of of glugluccososee))
In addition reactivity of oxygen can be controlled by catalysiswith transition metals (iron copper)
Does sugar burn
3
4
Spin restriction of dioxygen
bull Normal (triplet) O2 is a biradical with high affinity to electrons
bull But gain of an electron requires one of the unpaired electrons changes its spin which is a slow process
bull Thatrsquos why we do not burn in the air
bull Singlet O2 is an excited highly reactive species
Respiratory chain in the
mitochondrial inner
membrane
5
The respiratory chain
Electrons flow lsquodownhillrsquo and terminate at oxygen
Overall reaction
NADH + H+ + 12 O2 rarrrarrrarrrarr NAD+ + H2O
∆∆∆∆∆∆∆∆GGoorsquorsquo = = ndashndash21925 kJ mol21925 kJ mol--11
hellipand resulting proton gradient powers ATP synthesis
6
Reactive oxygen species (ROS lsquooxygen radicalsrsquo) can be derived from intermediates of oxygen reduction to water
O2
2H2O
+4 e-
(+4 H+)
O2middotndash
superoxide
H2O2hydrogen peroxide
OHmiddothydroxyl
radical
+3 e-
(+3 H+)
+2 e-
(+2 H+)
+1 e-
( +H2O )
Superoxidebull Sources in the body
ndash Escape of electrons to oxygen
- Respiratory chain in mitochondria
- Other similar redox carrier systems eg microsomalcytochrome P450 monooxygenase
ndash NAD(P)H Oxidase
- Phagocytes (rdquorespiratory burstrdquo)
- Non-phagocytic cells
ndash Some enzymes
- Xanthine oxidase
- Cyclooxygenase
- Lipoxygenase
ndash Reaction of FeII-hemoglobin with oxygen
ndash Autoxidation (reaction with oxygen) of various compounds (ascorbate glutathione and other thiols catecholamines)
O2middotndash
7
Superoxide production in the respiratory chain
bull 1-2 of total body oxygen consumption (from in vitro experiments in vivo surely much less)
bull Escape of electrons from redox centers of complexes I and III mostly from semiquinone
Origin of superoxide from oxyhemoglobin
Štiacutepek S et al Antioxidanty a volneacute radikaacutely ve zdraviacute a nemoci GradaPublishing Praha 2000
8
Superoxide
bull Properties
- Moderate reactivity both oxidising and reducing agent
- Limited ability to cross biological membranes (only through anion channels or in its protonated form)
- Releases iron from Fe-S clusters
bull Fate
ndash Dismutation
ndash Reaction with nitric oxide
O2middotndash
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
NO + O2middotndash rarr OONOndash (peroxynitrite)
Hydrogen peroxide
bull Formation in the body
ndash Dismutation of superoxide (spontaneous or catalyzed by superoxide dismutase)
ndash Directly by action of some enzymesbull Xanthine oxidase
bull Monoamine oxidase (MAO)
H2O2
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
9
Hydrogen peroxide
bull Properties
- No radical- Freely crosses biological membranes- Fairly unreactive as such- But rapidly reacts with reduced transition metals such
as iron copper (the Fenton reaction)
bull Fate
ndash The Fenton reaction
ndash Safe decomposition by glutathione peroxidase peroxiredoxin or catalase
H2O2
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
hydroxyl radical
Hydroxyl radical
bull Formation in the body
ndash Ionising radiation
ndash The Fenton reaction
bull Properties
- Extremely high reactivity Damages biomolecules near its site of formation
OH middot
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
H2O rarr H + OHmiddot
10
Reactive Oxygen Species (ROS)bull Radicals
ndash Superoxide O2middotndash
ndash Hydroperoxyl radical HO2middot
ndash Hydroxyl radical OHmiddotndash Peroxyl radicals ROOmiddotndash Alkoxyl radicals ROmiddot
bull Non-radicalsndash Hydrogen peroxide H2O2
ndash Hypochlorous acid HClOndash Ozone O3
ndash Singlet oxygen 1O2
Nitric oxide
bull Formation in the body
ndash NO synthase-catalysed reaction
ndash Three different NO synthases- NOS I (neuronal constitutive)- NOS II (phagocytes inducible)- NOS III (endothelial constitutive)
NO middot
L-Arginine + O2 + NADPH rarr L-Citrulline + NADP+ +NO
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
3
4
Spin restriction of dioxygen
bull Normal (triplet) O2 is a biradical with high affinity to electrons
bull But gain of an electron requires one of the unpaired electrons changes its spin which is a slow process
bull Thatrsquos why we do not burn in the air
bull Singlet O2 is an excited highly reactive species
Respiratory chain in the
mitochondrial inner
membrane
5
The respiratory chain
Electrons flow lsquodownhillrsquo and terminate at oxygen
Overall reaction
NADH + H+ + 12 O2 rarrrarrrarrrarr NAD+ + H2O
∆∆∆∆∆∆∆∆GGoorsquorsquo = = ndashndash21925 kJ mol21925 kJ mol--11
hellipand resulting proton gradient powers ATP synthesis
6
Reactive oxygen species (ROS lsquooxygen radicalsrsquo) can be derived from intermediates of oxygen reduction to water
O2
2H2O
+4 e-
(+4 H+)
O2middotndash
superoxide
H2O2hydrogen peroxide
OHmiddothydroxyl
radical
+3 e-
(+3 H+)
+2 e-
(+2 H+)
+1 e-
( +H2O )
Superoxidebull Sources in the body
ndash Escape of electrons to oxygen
- Respiratory chain in mitochondria
- Other similar redox carrier systems eg microsomalcytochrome P450 monooxygenase
ndash NAD(P)H Oxidase
- Phagocytes (rdquorespiratory burstrdquo)
- Non-phagocytic cells
ndash Some enzymes
- Xanthine oxidase
- Cyclooxygenase
- Lipoxygenase
ndash Reaction of FeII-hemoglobin with oxygen
ndash Autoxidation (reaction with oxygen) of various compounds (ascorbate glutathione and other thiols catecholamines)
O2middotndash
7
Superoxide production in the respiratory chain
bull 1-2 of total body oxygen consumption (from in vitro experiments in vivo surely much less)
bull Escape of electrons from redox centers of complexes I and III mostly from semiquinone
Origin of superoxide from oxyhemoglobin
Štiacutepek S et al Antioxidanty a volneacute radikaacutely ve zdraviacute a nemoci GradaPublishing Praha 2000
8
Superoxide
bull Properties
- Moderate reactivity both oxidising and reducing agent
- Limited ability to cross biological membranes (only through anion channels or in its protonated form)
- Releases iron from Fe-S clusters
bull Fate
ndash Dismutation
ndash Reaction with nitric oxide
O2middotndash
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
NO + O2middotndash rarr OONOndash (peroxynitrite)
Hydrogen peroxide
bull Formation in the body
ndash Dismutation of superoxide (spontaneous or catalyzed by superoxide dismutase)
ndash Directly by action of some enzymesbull Xanthine oxidase
bull Monoamine oxidase (MAO)
H2O2
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
9
Hydrogen peroxide
bull Properties
- No radical- Freely crosses biological membranes- Fairly unreactive as such- But rapidly reacts with reduced transition metals such
as iron copper (the Fenton reaction)
bull Fate
ndash The Fenton reaction
ndash Safe decomposition by glutathione peroxidase peroxiredoxin or catalase
H2O2
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
hydroxyl radical
Hydroxyl radical
bull Formation in the body
ndash Ionising radiation
ndash The Fenton reaction
bull Properties
- Extremely high reactivity Damages biomolecules near its site of formation
OH middot
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
H2O rarr H + OHmiddot
10
Reactive Oxygen Species (ROS)bull Radicals
ndash Superoxide O2middotndash
ndash Hydroperoxyl radical HO2middot
ndash Hydroxyl radical OHmiddotndash Peroxyl radicals ROOmiddotndash Alkoxyl radicals ROmiddot
bull Non-radicalsndash Hydrogen peroxide H2O2
ndash Hypochlorous acid HClOndash Ozone O3
ndash Singlet oxygen 1O2
Nitric oxide
bull Formation in the body
ndash NO synthase-catalysed reaction
ndash Three different NO synthases- NOS I (neuronal constitutive)- NOS II (phagocytes inducible)- NOS III (endothelial constitutive)
NO middot
L-Arginine + O2 + NADPH rarr L-Citrulline + NADP+ +NO
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
4
Spin restriction of dioxygen
bull Normal (triplet) O2 is a biradical with high affinity to electrons
bull But gain of an electron requires one of the unpaired electrons changes its spin which is a slow process
bull Thatrsquos why we do not burn in the air
bull Singlet O2 is an excited highly reactive species
Respiratory chain in the
mitochondrial inner
membrane
5
The respiratory chain
Electrons flow lsquodownhillrsquo and terminate at oxygen
Overall reaction
NADH + H+ + 12 O2 rarrrarrrarrrarr NAD+ + H2O
∆∆∆∆∆∆∆∆GGoorsquorsquo = = ndashndash21925 kJ mol21925 kJ mol--11
hellipand resulting proton gradient powers ATP synthesis
6
Reactive oxygen species (ROS lsquooxygen radicalsrsquo) can be derived from intermediates of oxygen reduction to water
O2
2H2O
+4 e-
(+4 H+)
O2middotndash
superoxide
H2O2hydrogen peroxide
OHmiddothydroxyl
radical
+3 e-
(+3 H+)
+2 e-
(+2 H+)
+1 e-
( +H2O )
Superoxidebull Sources in the body
ndash Escape of electrons to oxygen
- Respiratory chain in mitochondria
- Other similar redox carrier systems eg microsomalcytochrome P450 monooxygenase
ndash NAD(P)H Oxidase
- Phagocytes (rdquorespiratory burstrdquo)
- Non-phagocytic cells
ndash Some enzymes
- Xanthine oxidase
- Cyclooxygenase
- Lipoxygenase
ndash Reaction of FeII-hemoglobin with oxygen
ndash Autoxidation (reaction with oxygen) of various compounds (ascorbate glutathione and other thiols catecholamines)
O2middotndash
7
Superoxide production in the respiratory chain
bull 1-2 of total body oxygen consumption (from in vitro experiments in vivo surely much less)
bull Escape of electrons from redox centers of complexes I and III mostly from semiquinone
Origin of superoxide from oxyhemoglobin
Štiacutepek S et al Antioxidanty a volneacute radikaacutely ve zdraviacute a nemoci GradaPublishing Praha 2000
8
Superoxide
bull Properties
- Moderate reactivity both oxidising and reducing agent
- Limited ability to cross biological membranes (only through anion channels or in its protonated form)
- Releases iron from Fe-S clusters
bull Fate
ndash Dismutation
ndash Reaction with nitric oxide
O2middotndash
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
NO + O2middotndash rarr OONOndash (peroxynitrite)
Hydrogen peroxide
bull Formation in the body
ndash Dismutation of superoxide (spontaneous or catalyzed by superoxide dismutase)
ndash Directly by action of some enzymesbull Xanthine oxidase
bull Monoamine oxidase (MAO)
H2O2
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
9
Hydrogen peroxide
bull Properties
- No radical- Freely crosses biological membranes- Fairly unreactive as such- But rapidly reacts with reduced transition metals such
as iron copper (the Fenton reaction)
bull Fate
ndash The Fenton reaction
ndash Safe decomposition by glutathione peroxidase peroxiredoxin or catalase
H2O2
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
hydroxyl radical
Hydroxyl radical
bull Formation in the body
ndash Ionising radiation
ndash The Fenton reaction
bull Properties
- Extremely high reactivity Damages biomolecules near its site of formation
OH middot
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
H2O rarr H + OHmiddot
10
Reactive Oxygen Species (ROS)bull Radicals
ndash Superoxide O2middotndash
ndash Hydroperoxyl radical HO2middot
ndash Hydroxyl radical OHmiddotndash Peroxyl radicals ROOmiddotndash Alkoxyl radicals ROmiddot
bull Non-radicalsndash Hydrogen peroxide H2O2
ndash Hypochlorous acid HClOndash Ozone O3
ndash Singlet oxygen 1O2
Nitric oxide
bull Formation in the body
ndash NO synthase-catalysed reaction
ndash Three different NO synthases- NOS I (neuronal constitutive)- NOS II (phagocytes inducible)- NOS III (endothelial constitutive)
NO middot
L-Arginine + O2 + NADPH rarr L-Citrulline + NADP+ +NO
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
5
The respiratory chain
Electrons flow lsquodownhillrsquo and terminate at oxygen
Overall reaction
NADH + H+ + 12 O2 rarrrarrrarrrarr NAD+ + H2O
∆∆∆∆∆∆∆∆GGoorsquorsquo = = ndashndash21925 kJ mol21925 kJ mol--11
hellipand resulting proton gradient powers ATP synthesis
6
Reactive oxygen species (ROS lsquooxygen radicalsrsquo) can be derived from intermediates of oxygen reduction to water
O2
2H2O
+4 e-
(+4 H+)
O2middotndash
superoxide
H2O2hydrogen peroxide
OHmiddothydroxyl
radical
+3 e-
(+3 H+)
+2 e-
(+2 H+)
+1 e-
( +H2O )
Superoxidebull Sources in the body
ndash Escape of electrons to oxygen
- Respiratory chain in mitochondria
- Other similar redox carrier systems eg microsomalcytochrome P450 monooxygenase
ndash NAD(P)H Oxidase
- Phagocytes (rdquorespiratory burstrdquo)
- Non-phagocytic cells
ndash Some enzymes
- Xanthine oxidase
- Cyclooxygenase
- Lipoxygenase
ndash Reaction of FeII-hemoglobin with oxygen
ndash Autoxidation (reaction with oxygen) of various compounds (ascorbate glutathione and other thiols catecholamines)
O2middotndash
7
Superoxide production in the respiratory chain
bull 1-2 of total body oxygen consumption (from in vitro experiments in vivo surely much less)
bull Escape of electrons from redox centers of complexes I and III mostly from semiquinone
Origin of superoxide from oxyhemoglobin
Štiacutepek S et al Antioxidanty a volneacute radikaacutely ve zdraviacute a nemoci GradaPublishing Praha 2000
8
Superoxide
bull Properties
- Moderate reactivity both oxidising and reducing agent
- Limited ability to cross biological membranes (only through anion channels or in its protonated form)
- Releases iron from Fe-S clusters
bull Fate
ndash Dismutation
ndash Reaction with nitric oxide
O2middotndash
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
NO + O2middotndash rarr OONOndash (peroxynitrite)
Hydrogen peroxide
bull Formation in the body
ndash Dismutation of superoxide (spontaneous or catalyzed by superoxide dismutase)
ndash Directly by action of some enzymesbull Xanthine oxidase
bull Monoamine oxidase (MAO)
H2O2
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
9
Hydrogen peroxide
bull Properties
- No radical- Freely crosses biological membranes- Fairly unreactive as such- But rapidly reacts with reduced transition metals such
as iron copper (the Fenton reaction)
bull Fate
ndash The Fenton reaction
ndash Safe decomposition by glutathione peroxidase peroxiredoxin or catalase
H2O2
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
hydroxyl radical
Hydroxyl radical
bull Formation in the body
ndash Ionising radiation
ndash The Fenton reaction
bull Properties
- Extremely high reactivity Damages biomolecules near its site of formation
OH middot
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
H2O rarr H + OHmiddot
10
Reactive Oxygen Species (ROS)bull Radicals
ndash Superoxide O2middotndash
ndash Hydroperoxyl radical HO2middot
ndash Hydroxyl radical OHmiddotndash Peroxyl radicals ROOmiddotndash Alkoxyl radicals ROmiddot
bull Non-radicalsndash Hydrogen peroxide H2O2
ndash Hypochlorous acid HClOndash Ozone O3
ndash Singlet oxygen 1O2
Nitric oxide
bull Formation in the body
ndash NO synthase-catalysed reaction
ndash Three different NO synthases- NOS I (neuronal constitutive)- NOS II (phagocytes inducible)- NOS III (endothelial constitutive)
NO middot
L-Arginine + O2 + NADPH rarr L-Citrulline + NADP+ +NO
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
6
Reactive oxygen species (ROS lsquooxygen radicalsrsquo) can be derived from intermediates of oxygen reduction to water
O2
2H2O
+4 e-
(+4 H+)
O2middotndash
superoxide
H2O2hydrogen peroxide
OHmiddothydroxyl
radical
+3 e-
(+3 H+)
+2 e-
(+2 H+)
+1 e-
( +H2O )
Superoxidebull Sources in the body
ndash Escape of electrons to oxygen
- Respiratory chain in mitochondria
- Other similar redox carrier systems eg microsomalcytochrome P450 monooxygenase
ndash NAD(P)H Oxidase
- Phagocytes (rdquorespiratory burstrdquo)
- Non-phagocytic cells
ndash Some enzymes
- Xanthine oxidase
- Cyclooxygenase
- Lipoxygenase
ndash Reaction of FeII-hemoglobin with oxygen
ndash Autoxidation (reaction with oxygen) of various compounds (ascorbate glutathione and other thiols catecholamines)
O2middotndash
7
Superoxide production in the respiratory chain
bull 1-2 of total body oxygen consumption (from in vitro experiments in vivo surely much less)
bull Escape of electrons from redox centers of complexes I and III mostly from semiquinone
Origin of superoxide from oxyhemoglobin
Štiacutepek S et al Antioxidanty a volneacute radikaacutely ve zdraviacute a nemoci GradaPublishing Praha 2000
8
Superoxide
bull Properties
- Moderate reactivity both oxidising and reducing agent
- Limited ability to cross biological membranes (only through anion channels or in its protonated form)
- Releases iron from Fe-S clusters
bull Fate
ndash Dismutation
ndash Reaction with nitric oxide
O2middotndash
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
NO + O2middotndash rarr OONOndash (peroxynitrite)
Hydrogen peroxide
bull Formation in the body
ndash Dismutation of superoxide (spontaneous or catalyzed by superoxide dismutase)
ndash Directly by action of some enzymesbull Xanthine oxidase
bull Monoamine oxidase (MAO)
H2O2
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
9
Hydrogen peroxide
bull Properties
- No radical- Freely crosses biological membranes- Fairly unreactive as such- But rapidly reacts with reduced transition metals such
as iron copper (the Fenton reaction)
bull Fate
ndash The Fenton reaction
ndash Safe decomposition by glutathione peroxidase peroxiredoxin or catalase
H2O2
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
hydroxyl radical
Hydroxyl radical
bull Formation in the body
ndash Ionising radiation
ndash The Fenton reaction
bull Properties
- Extremely high reactivity Damages biomolecules near its site of formation
OH middot
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
H2O rarr H + OHmiddot
10
Reactive Oxygen Species (ROS)bull Radicals
ndash Superoxide O2middotndash
ndash Hydroperoxyl radical HO2middot
ndash Hydroxyl radical OHmiddotndash Peroxyl radicals ROOmiddotndash Alkoxyl radicals ROmiddot
bull Non-radicalsndash Hydrogen peroxide H2O2
ndash Hypochlorous acid HClOndash Ozone O3
ndash Singlet oxygen 1O2
Nitric oxide
bull Formation in the body
ndash NO synthase-catalysed reaction
ndash Three different NO synthases- NOS I (neuronal constitutive)- NOS II (phagocytes inducible)- NOS III (endothelial constitutive)
NO middot
L-Arginine + O2 + NADPH rarr L-Citrulline + NADP+ +NO
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
7
Superoxide production in the respiratory chain
bull 1-2 of total body oxygen consumption (from in vitro experiments in vivo surely much less)
bull Escape of electrons from redox centers of complexes I and III mostly from semiquinone
Origin of superoxide from oxyhemoglobin
Štiacutepek S et al Antioxidanty a volneacute radikaacutely ve zdraviacute a nemoci GradaPublishing Praha 2000
8
Superoxide
bull Properties
- Moderate reactivity both oxidising and reducing agent
- Limited ability to cross biological membranes (only through anion channels or in its protonated form)
- Releases iron from Fe-S clusters
bull Fate
ndash Dismutation
ndash Reaction with nitric oxide
O2middotndash
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
NO + O2middotndash rarr OONOndash (peroxynitrite)
Hydrogen peroxide
bull Formation in the body
ndash Dismutation of superoxide (spontaneous or catalyzed by superoxide dismutase)
ndash Directly by action of some enzymesbull Xanthine oxidase
bull Monoamine oxidase (MAO)
H2O2
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
9
Hydrogen peroxide
bull Properties
- No radical- Freely crosses biological membranes- Fairly unreactive as such- But rapidly reacts with reduced transition metals such
as iron copper (the Fenton reaction)
bull Fate
ndash The Fenton reaction
ndash Safe decomposition by glutathione peroxidase peroxiredoxin or catalase
H2O2
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
hydroxyl radical
Hydroxyl radical
bull Formation in the body
ndash Ionising radiation
ndash The Fenton reaction
bull Properties
- Extremely high reactivity Damages biomolecules near its site of formation
OH middot
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
H2O rarr H + OHmiddot
10
Reactive Oxygen Species (ROS)bull Radicals
ndash Superoxide O2middotndash
ndash Hydroperoxyl radical HO2middot
ndash Hydroxyl radical OHmiddotndash Peroxyl radicals ROOmiddotndash Alkoxyl radicals ROmiddot
bull Non-radicalsndash Hydrogen peroxide H2O2
ndash Hypochlorous acid HClOndash Ozone O3
ndash Singlet oxygen 1O2
Nitric oxide
bull Formation in the body
ndash NO synthase-catalysed reaction
ndash Three different NO synthases- NOS I (neuronal constitutive)- NOS II (phagocytes inducible)- NOS III (endothelial constitutive)
NO middot
L-Arginine + O2 + NADPH rarr L-Citrulline + NADP+ +NO
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
8
Superoxide
bull Properties
- Moderate reactivity both oxidising and reducing agent
- Limited ability to cross biological membranes (only through anion channels or in its protonated form)
- Releases iron from Fe-S clusters
bull Fate
ndash Dismutation
ndash Reaction with nitric oxide
O2middotndash
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
NO + O2middotndash rarr OONOndash (peroxynitrite)
Hydrogen peroxide
bull Formation in the body
ndash Dismutation of superoxide (spontaneous or catalyzed by superoxide dismutase)
ndash Directly by action of some enzymesbull Xanthine oxidase
bull Monoamine oxidase (MAO)
H2O2
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
9
Hydrogen peroxide
bull Properties
- No radical- Freely crosses biological membranes- Fairly unreactive as such- But rapidly reacts with reduced transition metals such
as iron copper (the Fenton reaction)
bull Fate
ndash The Fenton reaction
ndash Safe decomposition by glutathione peroxidase peroxiredoxin or catalase
H2O2
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
hydroxyl radical
Hydroxyl radical
bull Formation in the body
ndash Ionising radiation
ndash The Fenton reaction
bull Properties
- Extremely high reactivity Damages biomolecules near its site of formation
OH middot
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
H2O rarr H + OHmiddot
10
Reactive Oxygen Species (ROS)bull Radicals
ndash Superoxide O2middotndash
ndash Hydroperoxyl radical HO2middot
ndash Hydroxyl radical OHmiddotndash Peroxyl radicals ROOmiddotndash Alkoxyl radicals ROmiddot
bull Non-radicalsndash Hydrogen peroxide H2O2
ndash Hypochlorous acid HClOndash Ozone O3
ndash Singlet oxygen 1O2
Nitric oxide
bull Formation in the body
ndash NO synthase-catalysed reaction
ndash Three different NO synthases- NOS I (neuronal constitutive)- NOS II (phagocytes inducible)- NOS III (endothelial constitutive)
NO middot
L-Arginine + O2 + NADPH rarr L-Citrulline + NADP+ +NO
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
9
Hydrogen peroxide
bull Properties
- No radical- Freely crosses biological membranes- Fairly unreactive as such- But rapidly reacts with reduced transition metals such
as iron copper (the Fenton reaction)
bull Fate
ndash The Fenton reaction
ndash Safe decomposition by glutathione peroxidase peroxiredoxin or catalase
H2O2
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
hydroxyl radical
Hydroxyl radical
bull Formation in the body
ndash Ionising radiation
ndash The Fenton reaction
bull Properties
- Extremely high reactivity Damages biomolecules near its site of formation
OH middot
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
H2O rarr H + OHmiddot
10
Reactive Oxygen Species (ROS)bull Radicals
ndash Superoxide O2middotndash
ndash Hydroperoxyl radical HO2middot
ndash Hydroxyl radical OHmiddotndash Peroxyl radicals ROOmiddotndash Alkoxyl radicals ROmiddot
bull Non-radicalsndash Hydrogen peroxide H2O2
ndash Hypochlorous acid HClOndash Ozone O3
ndash Singlet oxygen 1O2
Nitric oxide
bull Formation in the body
ndash NO synthase-catalysed reaction
ndash Three different NO synthases- NOS I (neuronal constitutive)- NOS II (phagocytes inducible)- NOS III (endothelial constitutive)
NO middot
L-Arginine + O2 + NADPH rarr L-Citrulline + NADP+ +NO
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
10
Reactive Oxygen Species (ROS)bull Radicals
ndash Superoxide O2middotndash
ndash Hydroperoxyl radical HO2middot
ndash Hydroxyl radical OHmiddotndash Peroxyl radicals ROOmiddotndash Alkoxyl radicals ROmiddot
bull Non-radicalsndash Hydrogen peroxide H2O2
ndash Hypochlorous acid HClOndash Ozone O3
ndash Singlet oxygen 1O2
Nitric oxide
bull Formation in the body
ndash NO synthase-catalysed reaction
ndash Three different NO synthases- NOS I (neuronal constitutive)- NOS II (phagocytes inducible)- NOS III (endothelial constitutive)
NO middot
L-Arginine + O2 + NADPH rarr L-Citrulline + NADP+ +NO
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
11
Nitric oxidebull Properties- Gaseous radical- Reaction with heme iron of guanylate cyclase
(hellipphysiological effects)- Reaction with heme iron of hemoglobin (hellipinactivation)
- Reaction with sulfhydryl groups on glutathione etc to nitrosothiol (helliptransportstorage)
- Reaction with superoxide to peroxynitrite and finally hydroxyl radical (helliptoxicity)
NO middot
NO + O2middotndash rarr OONOndash peroxynitrite
OONOndash + H+ rarr HOONO rarr OHmiddot + NO2
peroxynitrous acid hydroxyl radical
NO causes relaxation of smooth muscles in blood
vessel wall
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
12
Reactive Nitrogen Species (RNS)bull Radicals
ndash Nitric oxide NOndash Nitric dioxide NO2
bull Non-radicalsndash Nitrosonium NO+
ndash Nitroxyl NOndash
ndash Nitrous acid HNO2
ndash Dinitrogen trioxide N2O3
ndash Dinitrogen tetraoxide N2O4
ndash Nitronium NO2+
ndash Peroxynitrite ONOO ndash
ndash Alkylperoxynitrite ROONO
Oxidative damage to biomolecules
bull Lipids peroxidation of polyunsaturated fatty acids in membranes
bull Proteins oxidation of -SH carbonylation -NH2 hydroxylationnitrosylation of aromatic AA cross-linking degradation
bull Nucleic acids strand breaks hydroxylation of bases mutation cancerogenesis
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
13
Lipid peroxidationLipid peroxidation((rancidityrancidity))
Further fate of lipid peroxides
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
14
Ionising radiationHydroxyl radical originates from ionisation of water
H2O + hν rarr H + OHmiddot
Reactive oxygen species in the body
One-electron reduction of oxygen (mitochondria NADPH oxidase) forms superoxide O2
middotndash
Dismutation of superoxide produces hydrogen peroxideO2
middotndash + O2middotndash + 2 H+ rarr O2 + H2O2
Fenton reaction with Fe or Cu converts peroxide tohydroxyl radicalH2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
bull Ribonucleotide reductase (hellipDNA synthesis)
bull Synthesis of prostanoids (enzymatic lipoperoxidation)
bull Useful heme peroxidasesndash Thyreoid peroxidase
ndash Myeloperoxidase of neutrophils
ndash Lactoperoxidase in milk respiratory tract mucus and saliva
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
15
Where RONS are beneficial
I Active centers of some enzymes
bull Monooxygenases (cytochromes P450)
ndash Detoxication of drugs synthesis of steroid hormones andbile acids NO synthases
Mechanism in essence controlled Fenton reaction
Fe3+ + SH + 2 e- + O2 + H+ rarr FeOOH SH
FeOOH SH rarr FeO3+ SH + H2O
FeO3+ SH rarr FeOH3+Sbull rarr Fe3+SOH rarr Fe3+ + SOH
Where RONS are beneficial
II Signaling
bull Eg nitric oxide NOndash Smooth muscle relaxation in blood vessels
gastrointestinal tract corpus cavernosum penis
ndash neurotransmitter neuromodulator in CNS function in synaptic plasticity learning andmemory
ndash Inhibition of adhesion and aggregation of platelets
ndash Inhibition of adhesion of leukocytes
ndash (etc)
Superoxide as well with effects opposing NO
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
16
NITRIC OXIDEProduced by NO synthases
Vasodilation inhibition of leukocyte activation inhibition of platelet
aggregation etc
SUPEROXIDEPEROXIDEProduced by NADPH oxidasesVasoconstriction activation of
leukocytes aggregation of platelets etcAnd if meet destroy each other
NO + O2middotndash rarr OONOndash
Concept of redox signaling
bull Redox environment inside the cell ndashdetermined mostly by ratios GSHGSSG and NADHNAD
bull Redox sensors on proteinsndash critical -SH groups
ndash Fe-S centers
bull Targets of redox signalingndash transcription factors
ndash protein kinases and phosphatases
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
17
Redox signaling
bull Oxidative stress activatescertain protein kinases andtranscription factors
bull Effect of ROSantioxidant on the cell can be veryspecific eg stimulationof proliferationdifferentiation cellularsenescence (ageing) orapoptosis
(T Finkel amp NJ Holbrook Nature 408 (2000) 239-247)
Where RONS are beneficial
III Phagocytosis
bull Neutrophils eosinophils monocytes macrophages microglia
bull Often the target particle needsto be marked (coated withopsonins)
bull Phagocyte tasksndash Recognizendash Engulfndash Killndash Digest
copy 1998 Garland Publishing
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
18
Fig httpwalchecklabumnedu_rsrc1417821135388research-interestsneutrophil-recruitment
Activation of a phagocytic cell Respiratory burst
bull Dramatic increase in consumption of oxygen (for superoxide production not respiration)
bull Dramatic increase in consumption of glucose(for the pentose cycle ndash produces NADPH forNADPH oxidase)
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
19
NADPH oxidase
bull Membrane enzyme producing a superoxide fluxoutwardly or into a phagosome
bull Catalyzes reactionNADPH + 2O2 rarr NADP+ + H+ + 2O2middotndash
bull Latent in resting cell assembles on the cell membrane from 6 subunits during phagocyteactivation
bull Necessary for an ability to kill some kinds of engulfed bacteria (hereditary deficit chronicgranulomatous disease)
bull Similar enzymes are present also on theendothelial and many other non-phagocytic cells
Myeloperoxidase
bull 2 ndash 5 of total protein in neutrophils
bull Hemoprotein that gives pus its green tinge
bull Non-specific peroxidase in the body catalyzes reaction
Clminus + H2O2 + H+ rarr HClO + H2O
bull Deficit in humans is common (1 in 2000-4000) and is not severe
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
20
Further neutrophil weaponsbull Powerful proteolytic enzymes elastase collagenase
cathepsin G gelatinase etcbull Lysozyme cleaves polysaccharide of Gram+ bacterial
cell wallbull Lactoferrin binds iron to make unavailable for bacteriabull Defensins cationic proteins forming channels in
bacterial cell membranebull NETs (neutrophil extracellular traps) fibers of DNA
and serine proteases catch bacteria extracellularly
RONS generators and other weapons can synergizeeg HClO inhibits α1-antitrypsin
RONS as phagocyte weapons
iNOS is important in macrophage but rather insignificant in human neutrophils In contrast macrophages do not possessmyeloperoxidase
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
21
Oxidative stress
bull Levels of reactive oxygen species are kept within certain limits by mechanisms of antioxidant defence
bull Oxidative stress occurs if the oxidantantioxidant balance shifts to oxidation
Antioxidant defence
bull prevention of ROSRNS formation (regulation of producing enzymes sequestration of metals)
bull scavenging trapping and quenching of radicals
bull reparation systems (phospholipases proteasome DNA repair)
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
22
Antioxidant in food chemistry
bull Reducing agent capableof terminating chainreaction of lipid peroxidation hellipldquochain-breakingrdquo
bull Egndash Butylated
hydroxytoluene (BHT)
ndash Butylated hydroxyanisol(BHA)
ndash Tocopherol (Vitamin E)
Antioxidant defence of human body
bull Anatomy of the body limiting tissue oxygen
bull Antioxidant enzymes
bull Sequestration of redox active metals
bull Antioxidant substrates (scavengers)
bull Stress response
bull (Repair systems)
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
23
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
24
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 mmHg O2
Lung capillaries 100 mmHg O2
Arterial blood 85 mmHg O2
Arterioles 70 mmHg O2
Capillaries 50 mmHg O2
Cells 1-10 mmHg O2
Mitochondria lt 05 mmHg O2
Fig Wikipedia
Antioxidant defence II
Antioxidant enzymes
bull Superoxide dismutase
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Catalase
2 H2O2 rarr 2 H2O + O2
bull Glutathione peroxidase peroxiredoxin H2O2 + 2 R-SHrarr 2 H2O + RS-SR
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
25
Superoxide dismutase (SOD)bull Catalyses dismutation of superoxide
O2middotndash + O2
middotndash + 2 H+ rarr O2 + H2O2
bull Absolutely required for life in oxygen
bull SOD1 Cu+Zn (cytosol of eukaryotic cells)
bull SOD2ndash Mn (mitochondrial matrix)
ndash Fe (bacteria)
bull EC-SOD (SOD3) extracelullar Cu+Zn ndash MW 135000 binds to heparane sulfate on the
inner surface of blood vessels
Glutathione peroxidases (GPX)bull Reduction of peroxides coupled to oxidation of
glutathione2 GSH + H2O2 rarr GS-SG + 2 H2O
(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenocysteine
bull Cytosolic glutathione peroxidasendash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidasendash reduces LOOH even in membranes
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
26
Glutathione (GSHGSSG)bull tripeptide in every cell
1-10 mM
bull keeps ICT reduced
bull substrate for GPX etc
bull also non-enzymatic reactions with ROS and mixed disulfides with proteins products of GSH oxidation are toxic for cell
bull in oxidative stress the cell exports GSSG out
Catalasebull Tetramer every subunit contains heme with
Fe
bull Dismutation of hydrogen peroxide
2 H2O2 rarr 2 H2O + O2
bull Red blood cells peroxisomes
bull Also peroxidase activity
H2O2 + ROOH rarr H2O + ROH + O2
(in comparison to GPX less significant)
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
27
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
Glutathioneperoxidase
H2O2
H2O
Glutathionereductase
GS-SG
GSHTranshydrogenase
NADPH+H+
NADP+
NADH+H+
NAD+
Pentose cycle
O2middotndash
Superoxide dismutase
Reduced glutathione(GSH)
Oxidised glutathione(GS-SG)
ATPATP
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
28
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
H2O2
GSH
O2middotndash
Superoxide dismutase
H2O
Thioredoxin
reductase RED
Thioredoxin
reductase OX
NADPH+H+
NADP+
SH HS
S S
Peroxiredoxin
RED
Peroxiredoxin
OX
SH HS
S S
Thioredoxin
RED
Thioredoxin
OX
FADH2 (Se)
FAD (Se)
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
29
Antioxidant defence III
Sequestration of metalsbull Redox-active transition metals (Fe Cu)
acceptdonate one electron easilyndash alleviation of spin restriction of dioxygenndash metals are in active centers of all oxygen handling-
enzymes
bull But the same properties of Fe Cu are deleterious if uncontrolledndash the Fenton oxidant
H2O2 + Fe2+ rarr OHndash + OHmiddot + Fe3+
oxidative damage to biomolecules
Antioxidant defence III
Sequestration of metalsbull Ironcopper handling proteins
ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leucocytes ndash ferritin H and L subunits H is ferroxidase Fe
storage (up to 4500 atoms Fe3+) ndash haptoglobin binds hemoglobin in circulationndash hemopexin binds heme in circulationndash ceruloplasmin contains Cu function
ferroxidase (export Fe from the cells)ndash albumin transport of Cu
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
30
ICT ECT
Superoxide
Peroxide FeCu
Superoxide
Peroxide FeCu
Superoxide dismutaseGlutathione peroxidaseCatalase
GlutathioneTocopherolAscorbate
Antioxidant enzymes amp
glutathione levels very low
TocopherolAscorbateCarotenoids uric acid albumin
glucose bilirubin
Labile iron pool (LIP) present Sequestration of iron and copper- transferrin lactoferrin- hemopexin- haptoglobin- ceruloplasmin (ferroxidase)- Cu bound to albumin
bull THIOLS ndash Glutathionendash Thioredoxin
bull OTHER ENDOGENOUS METABOLITESndash Bilirubin ndash Uric acidndash Lipoic acid
bull DIETARYndash Ascorbate (Vitamin C)ndash α-Tocopherol (Vitamin E)ndash Carotenoidsndash Plant phenols
Antioxidant defence IVLow-molecular-weight antioxidant substrates
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
31
Tocopherols (Vitamin E)
bull group of 8 isomers α-tocopherol most effective
bull antioxidant ofmembranes(lipophilic)
bull ldquochain-breakingrdquo terminates thechain reaction of lipid peroxidation
Ascorbate (Vitamin C)
bull Redox-active saccharide
bull In most animals synthesized from glucuronic acid
bull Vitamin for humans other primates bats and guinea pigs
bull Deficit causes scurvy (scorbut)
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
32
Ascorbate in the bodybull Main function is pro-oxidant cofactor of hydroxylases
ndash Hydroxylation of Pro and Lys in collagen synthesisndash Synthesis of noradrenaline from dopaminendash Synthesis of carnitine (hellip role in oxidation of fat)ndash Activation of hypothalamic peptidic hormones by
amidation (CRH GRH oxytocin vasopressin substance P)
bull Reductant for iron promotes its intestinal absorptionbull Potentially dangerous pro-oxidant if iron sequestration
impaired (hemochromatosis) ()bull Daily need 70-100 mg high doses po excreted by
urine (renal threshold cca 200 mg24 hours)
membrane compartment hydrophilic compartment
LH
L
Tocopherol
Tocopheryl
radical
LOOH
LOO
chain reaction of
lipid peroxidation
Ascorbate
Semidehydro-
ascorbate
Dehydroascorbate
+e-
-e-
dehydroascorbatereductase
2GSH
GSSG
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
33
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
bull Selenium
ndash Trace element (daily need 55 microg) possibility of deficiency as well as intoxication
ndash Component of several antioxidant enzymes(glutathione peroxidase thioredoxin reductase) and also eg 5lsquo-dejodase
bull ββββ-carotene (provitamin A) is precursor forsynthesis ofndash Retinal hellip vision
ndash Retinoic acid hellipregulator of gene expression cellular growth and differentiation
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence
34
Antioxidant defence V
Stress responseOxidation or nitrosylation of sensor -SH
Transcription factors (NFκB Nrf-2hellip)activation nuclear translocation
Induction of gene expressionbull chaperones (heat shock proteins)bull antioxidant enzymes
bull metallothioneinbull hemoxygenase 1
helliprarr more resistant to further
oxidative stress
Apoptosis as theultimate antioxidant defence