1
Antioxidant Defence
Ageing
Dietary Antioxidants
Jan Plaacuteteniacutek MD PhD
Uacutestav leacutekařskeacute biochemie a laboratorniacute diagnostiky 1LF UK
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+
2
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)
3
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)
4
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
5
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 160 mmHgmmHg OO22
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 Olt 05 mmHg O22
Fig Wikipedia
Mitochondria originated from
phagocytedparasitic bacteria
6
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
Superoxide dismutase (SOD)
bull Catalyses dismutation of superoxideO2
middotndash + O2middotndash + 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
7
Glutathione peroxidases (GPX)
bull Reduction of peroxides coupled to oxidation of glutathione
2 GSH + H2O2 rarr GS-SG + 2 H2O(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenium as selenocysteine
bull Cytosolic glutathione peroxidase (GPX1)ndash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidase(GPX4)ndash reduces LOOH even in membranes
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
8
Catalase
bull 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)
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
9
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
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
10
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)
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
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
2
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)
3
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)
4
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
5
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 160 mmHgmmHg OO22
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 Olt 05 mmHg O22
Fig Wikipedia
Mitochondria originated from
phagocytedparasitic bacteria
6
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
Superoxide dismutase (SOD)
bull Catalyses dismutation of superoxideO2
middotndash + O2middotndash + 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
7
Glutathione peroxidases (GPX)
bull Reduction of peroxides coupled to oxidation of glutathione
2 GSH + H2O2 rarr GS-SG + 2 H2O(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenium as selenocysteine
bull Cytosolic glutathione peroxidase (GPX1)ndash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidase(GPX4)ndash reduces LOOH even in membranes
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
8
Catalase
bull 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)
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
9
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
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
10
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)
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
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
3
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)
4
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
5
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 160 mmHgmmHg OO22
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 Olt 05 mmHg O22
Fig Wikipedia
Mitochondria originated from
phagocytedparasitic bacteria
6
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
Superoxide dismutase (SOD)
bull Catalyses dismutation of superoxideO2
middotndash + O2middotndash + 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
7
Glutathione peroxidases (GPX)
bull Reduction of peroxides coupled to oxidation of glutathione
2 GSH + H2O2 rarr GS-SG + 2 H2O(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenium as selenocysteine
bull Cytosolic glutathione peroxidase (GPX1)ndash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidase(GPX4)ndash reduces LOOH even in membranes
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
8
Catalase
bull 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)
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
9
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
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
10
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)
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
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
4
O2
First organism
(anaerobic)
Developantioxidantdefence
Die out
Resort to anaerobic
condition
O2Clump together
O2
5
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 160 mmHgmmHg OO22
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 Olt 05 mmHg O22
Fig Wikipedia
Mitochondria originated from
phagocytedparasitic bacteria
6
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
Superoxide dismutase (SOD)
bull Catalyses dismutation of superoxideO2
middotndash + O2middotndash + 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
7
Glutathione peroxidases (GPX)
bull Reduction of peroxides coupled to oxidation of glutathione
2 GSH + H2O2 rarr GS-SG + 2 H2O(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenium as selenocysteine
bull Cytosolic glutathione peroxidase (GPX1)ndash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidase(GPX4)ndash reduces LOOH even in membranes
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
8
Catalase
bull 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)
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
9
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
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
10
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)
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
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
5
Antioxidant defence I
Regulation of tissue O2
Inhaled air 160 160 mmHgmmHg OO22
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 Olt 05 mmHg O22
Fig Wikipedia
Mitochondria originated from
phagocytedparasitic bacteria
6
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
Superoxide dismutase (SOD)
bull Catalyses dismutation of superoxideO2
middotndash + O2middotndash + 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
7
Glutathione peroxidases (GPX)
bull Reduction of peroxides coupled to oxidation of glutathione
2 GSH + H2O2 rarr GS-SG + 2 H2O(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenium as selenocysteine
bull Cytosolic glutathione peroxidase (GPX1)ndash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidase(GPX4)ndash reduces LOOH even in membranes
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
8
Catalase
bull 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)
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
9
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
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
10
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)
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
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
6
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
Superoxide dismutase (SOD)
bull Catalyses dismutation of superoxideO2
middotndash + O2middotndash + 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
7
Glutathione peroxidases (GPX)
bull Reduction of peroxides coupled to oxidation of glutathione
2 GSH + H2O2 rarr GS-SG + 2 H2O(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenium as selenocysteine
bull Cytosolic glutathione peroxidase (GPX1)ndash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidase(GPX4)ndash reduces LOOH even in membranes
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
8
Catalase
bull 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)
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
9
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
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
10
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)
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
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
7
Glutathione peroxidases (GPX)
bull Reduction of peroxides coupled to oxidation of glutathione
2 GSH + H2O2 rarr GS-SG + 2 H2O(glutathione is subsequently regenerated by glutathione reductases)
bull Active site contains selenium as selenocysteine
bull Cytosolic glutathione peroxidase (GPX1)ndash reduces H2O2 and LOOH after release from
phospholipids
bull Phospholipide hydroperoxide-GSH-peroxidase(GPX4)ndash reduces LOOH even in membranes
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
8
Catalase
bull 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)
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
9
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
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
10
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)
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
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
8
Catalase
bull 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)
OxidaOxidationtion of very longof very long chain fattychain fatty acids in peracids in peroxisomeoxisomess
9
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
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
10
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)
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
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
9
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
PeroxiredoxinThioredoxin
bull Recently discovered antioxidant system more important for removal of hydrogen peroxide than GPX
10
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)
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
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
10
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)
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
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
11
Antioxidant defence III
Sequestration of metals
bull Ironcopper handling proteins ndash transferrin binds 2 atoms Fe3+ (transport)ndash lactoferrin analogous to transferrin but no Fe
release ( only sequestration) leukocytesndash 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
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
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
12
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
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
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
13
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)
Ascorbate in the body
bull Main function is pro-oxidant cofactor of hydroxylasesndash 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)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
14
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
Activated neutrophiles
accumulate
dehydroascorbate (DHA)GLUT1
DHA
Ascorbate
Protects membrane of the neutrophilefrom its own ROS
GSH
GSSG
Glutaredoxin
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
15
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 (T4rarrT3)
bull Carotenoids
ndash ββββ-carotene (provitamin A) is precursor forsynthesis of
bull Retinal hellip vision
bull Retinoic acid hellipregulator of gene expression cellular growth and differentiation
ndash Antioxidants in the skin and in the eye
Plant (poly)phenolsbull Thousands of substances
(quercetin resveratrol curcumin catechinshellip)
bull Fruits vegetables tea redwine soy sauce coffee chocolate herbs spiceshellip
bull Excellent antioxidants(reductants) in vitro
bull In vivo more complexndash Absorption in digestive tract
ndash Conversion to otherderivatives
ndash Other specific biologicaleffects
Fighttpwwwjustaboutskincom
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
16
Antioxidant defence V
Stress response
Oxidation 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
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
17
Free radicals in pathogenesis of human diseases
bull Cause of disease egbull cancerogenesis due to exposition to ionising radiationbull retinopathy of the newborn (fibroplasia retrolentalis)
bull Contribute to pathogenesis egbull atherosclerosisbull diabetes mellitusbull hypertensionbull some kinds of cancerbull brain traumahemorrhagebull ischemiareperfusion injury of heart and other organsbull Parkinson diseasebull Alzheimer diseasebull ageing
bull Merely an epiphenomenon (general consequence of tissue damage)
What is ageing
bull Looks similar in various animals butproceeds with variable speed hellip must beuniversal process
bull Stochastic process unlike eg embryogenesis is not directly programmedby genome (but genes do play a role )
bull On molecular level inability to keep fidelity of biomolecules
indefinitely
bdquosystemic molecular disorderldquo (Hayflick)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
18
Mitochondrial
genome
Mitochondrial DNA
mutates 10 times faster
than nuclear DNA
Exposed to oxygen radicals Not covered by histonesRepair insufficient
Vicious circle of mitochondrial oxidativedamage
Production of oxygen radicals in mitochondria
Accumulation of mtDNA mutations with age
Respiratory chain deficiency
Heart failure muscle weakness diabetes mellitus dementia neurodegeneration hellip
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
19
Free-radicalmitochondrial theory of ageing
bull Accumulation of oxidative damage with age (DenhamHarman 1956)
bull Later refined to mitochondrial theory
ndash hellipmitochondria are the main source of ROS in the body
bull But Difficult to prove that human mitoDNA is more damagedby ROS or that significantly accumulates mutations with age
bull Model of Kirkwood and Kovald
ndash Certain amount of ROS always escapes mitochondria anddamages other cellular structures
ndash Prevention of ROS formation and repair systems are never100 effective
ndash Slightly damaged mitochondria produce less energy thanthe cell would need
How the cells get rid of worn-out proteins and organelles
bull Calpains proteasome (short-lived proteins)bull Autophagy (long-lived proteins organelles)
ndash Macroautophagy (whole organelles)ndash Microautophagy (macromolecules small organelles)ndash Chaperone-mediated autophagy (KFERQ proteins)
Fig httpcpmcnetcolumbiaedudeptgsasanatomyFacultyKessinautophagyhtml
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
20
Ageing as a catabolic insufficiencyIncomplete digestion in lysosomes release of Fe from mito
ROS lipid peroxidation cross-linking aggregation andpolymeration of oxidised proteins and lipids
uarr LIPOFUSCIN (in lysosomes)In cytosol defective mito and
indigestible protein aggregates
Loss of hydrolases delivered to lipofuscin-loaded lysosomesDamaged and hypertrophic (giant) mito not degradable
Less ATP more ROS damaged mito amp lysosomes caninitiate apoptosishellip
+
Fig httpwwwuni-mainzdeFBMedizinAnatomieworkshopEMEMtLysohtml
Stress response becomes permanent in ageing
Nick Lane Oxygen The Molecule that made the World Oxford University Press 2002
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
21
Antioxidants as elixirs of youth
bull Vitamin E (tocopherol)
bull Vitamin C (ascorbate)
bull β-carotene
bull Selenium
Fig httpwwwoselcz
Antioxidant dietary supplements caneven be harmful
bull Recent meta-analysis of total mortality in 68 studies on administration of antioxidant supplements (232 606 participants 385 publications)
ndash β-carotene vitamin A and vitamin E significantly increase mortality
ndash Vitamin C and selenium have no effect
(Bjelakovic G et al JAMA 2007 297 842-857)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
22
Why the antioxidants do not help or even harm
bull High doses are ineffectivebull Suppress the beneficial oxidations
ndash Inhibition of the stress responsendash Impair defence against infection cancer
physiologic apoptosis
bull Have other effects in addition to antioxidant ndash tocopherols anti-inflammatoryndash β-carotene co-carcinogen (together with
smoking or environmental toxins)
httpwwwcalpolyedu~lcimarelknowhtm
Diet rich in fruit and vegetables (optim 5x 80 g daily) is associated with lower risk of
cardiovascular diseases diabetes and certainkinds of cancer (lung oropharynx pancreas
stomach prostate)
(but we do not know whyhellip)
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