Transcript Zdroje volných radikál* ROS

Biochemistry of aging – Free
radicals and antioxidants
Petr Tůma and Eva Samcová
Oxygen
• Origin of O2 – photosynthesis
• 6CO2 + 12H2O → C6H12O6 + 6H2O
+ 6O2
• Cyanobacteria produce oxygen
first 2 billion years ago
• aerobic metabolism
Two basic equilibrium
• Acid-base – proton transfer
– base + H+ ↔ acid
• Oxidation-reduction – electron transfer
– oxidation form + e- ↔ reducing form
Reactive oxygen species (ROS)
• Reactive oxygen species are involved in
releasing and conversion of energy
necessary for life processes,
• are part of enzyme mechanisms,
• and some are also signaling molecules.
• Damages of organisms only when there is
a loss of control.
Reactive oxygen species - ROS
• Gradual 4 electrons reduction of O2 to water
• superoxide (radical)
O2 + e- → O2·• Hydrogen peroxide
O2·- + e- + 2H+ → H2O2
• Hydroxyl radical
H2O2 + e- → OH- + HO·
• water
HO· + e- → OH-
Source of superoxide
1. Respiration chain in mitochondrion
– 1-4% O2 is reduced incompletely to ROS
– complex I (NADH – dehydrogenase)
– complex III (ubiquinol: cytochrome c- reductase)
2. Cytochrome P-450 in endoplasmic reticulum
– ROS bound to enzyme – biotransformation, ethanol oxidation
3. Specialized cells – leukocytes, macrophages
– NADPH – oxidase in cytoplasmic membrane – baktericidal
prophylactic system
– myeloperoxidase – production of HClO
4. Oxidation of hemoglobin to methemoglobin
Source of H2O2
• Dismutation of superoxide:
2 O2·- + 2H+ = O2 + H2O2
– spontaneous
– Enzyme Superoxide Dismutase
• Direct reduction of O2 action of
oxidases
– Monoamine oxidase, Glutathione oxidase,
Xanthine oxidase
• Peroxisomes
– Equipped with several enzymes, which are used
for oxidation of diferent organic substrates
(ethanol, phenols, formaldehyde)→ H2O2
– Oxidation of long and side-chain fatty acids
Nonenzymic sources of ROS
Besides enzymes, the oxygen is reduced in cells by small
endogenous and exogenous molecules (they transfer
electron to O2 from different reductases (e.g. from
NADPH-cytochrom-P450 reductase and others)
1. Quinone antibiotics
• adriamycine, daunomycine, streptonigrin, cardiotoxic...
2.
Pyridine herbicides
• paraquat, diquat – lung injury
3.
Low-molecular complexes of Fe with phosphates,
nucleotides (other toxic carrier of electrons)
• Complexes with ATP, ADP
Reactive nitrogen species – nitric oxide
Nitric oxide
•important second messenger
•antimicrobial effects – macrophages
•vasodilator
•Nitric oxide synthases – NOS
– NOS I – neuronal(brain)
– NOS II – macrophage
– NOS III – endothelial
H2N
NH
O
C
C
NH
NH
CH2
H2N
H2N
O2 + NADPH
CH2
CH2
CH2
CH2
CH2
CH
COOH
H2N
arginin
Peroxynitrite
•NO· + O2·- = OONO•Important powerful oxidant – oxidation of amino acids in proteins
•Antimicrobial effects – macrophages
CH
NO·
COOH
citrulin
Physiological functions of free radicals
Free radicals are a tool of oxidases and
oxygenases
1. Respiratory chain
•
•
Inner mitochondrial membrane
Aerobic phosphorylation
2. Biotransformation of xenobiotics
•
•
Mitochondrial cytochrome oxidase – P450
Superoxide and peroxide bound to enzyme
3. Synthesis in cells
•
•
Monooxygenases in endoplasmic reticulum of
hepatocyte or in mitochondria of adrenal gland
Hydroxylation of xenobiotics, synthesis of cholesterol
and bile acids
ROS and RNS as an effective weapon of
phagocytes against germs
Neutrophils and macrophages
– Bactericidal prophylactic system (removes dead
cells and kills bacteria)
– NADPH-oxidase (enzyme membrane complex)
• Activated after absorption of foreign particle
→reduction of oxygen to superoxide→H2O2
• Fenton reaction
– Myeloperoxidase
•
Synthesis of HClO from H2O2 and Cl-
– Synthetase of NO (NOS II)
•
•
NO concentration increases by several orders of
magnitude
Synthesis of peroxynitrite - NO + superoxide – OONO-
ROS and RNS as signal molecules
• Information net
– Primary messenger, secondary messenger
– Protein kinases – influencing activities of enzyme,
transcription factors → gene expression
• Sensitivity of information net depends on redox
state of cell (influencing of protein kinases)
• Redox state
– Capacity of antioxidant system (accessibility of
reducing equivalents)
– Intensity of oxidation load (RONS)
• Nitric oxide NO (nitrogen oxide)
– Secondary messenger
– Neurotransmitter in CNS and autonomic nervous
system (vegetative)
– Vasodilatation of vessels
Antioxidant protective system
1. Restriction of excessive formation of ROS
and RNS
–
–
Regulation of enzyme activity
Trap of transition elements from reactive sites
2. Trap and elimination of radicals
–
–
scavengers, trappers, quenching
enzymes, substances which form with radicals more
stable products
3. General reparative mechanisms of injured
macromolecules
–
–
–
phospholipases
reparative enzymes of DNA
proteolysis of proteins injured by oxidative stress
Enzyme antioxidant systems
catalase
O2
·-
SOD
H2O2
NADPH+H+
+ Fe2+
2 GSH
H2O + ½ O2
·OH
+ Fe3+ + OH-
GSHPx
2 H 2O
NADP+
GSSG
Superoxide dismutase – SOD
•
accelerates the dismutation of superoxide by 4 orders
•
present in most of aerobic cells and in extracellular fluid
•
several isoenzymes with different cofactors: Cu, Zn, Mn, Fe
Types of superoxide dismutases :
mitochondrial (SOD2 = Mn-SOD, Fe-SOD)
– tetramer in prokaryotes and in mitochondria matrix
cytoplasmic (SOD1 =CuZn-SOD)
– dimer, atom Cu and Zn in each subunit(also intermembrane space)
– elimination of SOD1 decreases life time, and causes the
development of degenerative disease associated with old age –
carcinogenesis
extracellular (SOD3 = EC-SOD)
– elimination has only minimal effect
Glutathione peroxidase
• Removal of intracellular hydroperoxides
• Proteins with selen – selenocystein in active
center
• 2 GSH + ROOH = GSSH + H2O + ROH
• cytosol GSH – glutathione peroxidase (cGPx)
– decomposes hydroperoxides of fatty acids after
releasing from lipids by phospholipase A2, H2O2
• Phospholipid hydro peroxide-GSH-peroxidase
(PHGPx)
– reduces phospholipid hydroperoxides directly in
plasmatic membrane without releasing of fatty acids
from phospholipids
Catalase
• Two-electron dismutation of hydrogen
peroxide
• 2H2O2 = 2 H2O + O2
• Inactivation of H2O2 – peroxisomes and
mitochondria of hepatocyte, cytoplasm of
erythrocyte
High-molecular endogenic antioxidants
Proteins which bind transition elements Fe and Cu =
inactivation of these elements for catalysis
• transferrin – binds Fe3+ in blood
• lactoferrin – binds Fe3+ in leukocytes
• ferritin – intracellular, storage of Fe in the cell
• haptoglobin – uptake of extracellular hemoglobin
• ceruloplazmin – binds Cu in blood plasma
• albumins – bind on its –SH groups Cu2+oxidation to Cu3+
and damage the surrounding structures of albumin
• metalothioneins– proteins with many cysteins and via sulfur
atoms form chelates with metal ions in the nucleus
Low-molecular endogenic antioxidants
Soluble in water
• ascorbic acid – vitamin C
• glutathione
• uric acid
• lipoic acid
Soluble in fat
• carotenoids and vitamin A
• α-tocopherol – vitamin E
• ubiquinol – coenzym Q
Ascorbic acid – vitamin C
• Derivative of monosaccharides occuring in animals and plants
• Essential for synthesis of collagen, hydroxylation of proline and
lysine and in conversion of dopamine to norepinephrine
• Reduces radicals – O2·-, HOO·, HO·, ROO·, NO2
• Transfer to hydroascorbate (ascorbyl radical)
• Regeneration by NADH
• In combination with Fe – prooxidative effect
– reduces Fe3+ to Fe2+ (absorption of Fe in intestine)
HO·
+ H 2O
a – tocopherol and vitamin E
Group of 8 isomers –most significant a-tocopherol
Most important lipophilic antioxidant
Antioxidant of biological membranes
Reduces alkylperoxyl radicals LOO· of lipids to hydrogen
peroxides, which after are reduced by glutathione
peroxidase
• From tocopherol arises slightly reactive tocopheryl radical
• Regeneration by ascorbate
•
•
•
•
CH3
CH3
HO
O
R
R-O-O·
H3C
O
R
CH3
H3C
O
CH3
a-tokoferol
CH3
tokoferylový radikál
CH3
+ R-O-O-H
Ubiquinone/ubiquinol – coenzyme Q10
• Transfer of reducing equivalents in respiratory chain in
the mitochondria
• It serves as an antioxidant in mitochondria and
membranes (together with tocopherol)
• Partly is synthesized, partly accepted by diet
• Its level decreases in the mitochondria with increased age
(old age). Then
– Heart failure
– Myocardial infarction
– Atherosclerosis
Carotenoids, b-carotene and
vitamin A
• chemically isoprenoids
• Remove radicals centered on carbon and
alkylperoxyl radicals ROO· in lipids
CH3
H3C
CH3
CH3
CH3
CH3
CH3
b - karoten
CH3
CH3
CH3
Glutathione GSH
• Glutathione (tripeptide- g-glutamylcysteinylglycine) is
synthesized in the body
• The most significant intracellular redox buffer
• 2 GSH = GSSG + 2H+ + 2e• Nonenzymatic removal of ROS – HO·, RO·, ROO·
• Keeps in reduced form –SH groups of proteins, cysteine,
CoA, regenerates ascorbate, Its regeneration is
catalyzed by glutathione reductase
• Necessary substrate of glutathione peroxidase
O
HOOC
CH
CH2
CH2
C
O
NH
CH
CH2
NH2
glutathion
SH
C
NH
CH2
COOH
Uric acid (urate)
• End product of purine catabolism in human
and primates
• Most plentiful antioxidant in blood plasma
• Traps RO·, HClO, binds Fe a Cu
• Hyperuricemia – gout
Lipoic acid (lipoate)
• cofactor pyruvate dehydrogenase and aoxoglutarate dehydrogenase multienzyme
complex (cytric acid cycle)
• antioxidant ROO·, ascorbyl radical, HO·, NO·,
O2 · -
Uric acid
Breaking antioxidant protection
• Oxidative stress – unbalancing between formation and
removal of ROS a RNS
– excessive production of radicals
– inadequate antioxidant protection
• Causes of excessive production of ROS and RNS
– reoxygenation of tissue after ischemia
– after receiving redox active xenobiotics
– release of chemical bonds of Fe a Cu from bonds to
storage proteins
– excessive production of NO and congestion load of
SOD
NO + O2·- = peroxynitrite - strong oxidant
Key role of Fe in oxidative damage to the
body
• Fenton reaction
Fe2+ + H2O2 = Fe3+ + HO· + OH• Catalytic ability of Fe in active enzyme centers
(minute amount)– reactivity of Fe is rectified in
favor of life events
• Fe reacts as in the case of nonspecific protein,
lipid, and NA binding, and also after release
from transferrin and ferritin – Damage to
biomolecules
• Human body – 4 grams of Fe
– in oxidoreductases only tiny part
– 70% in hemoglobin, 10% in myoglobinu
Lipid peroxidation (LPO)
•
•
in vitro – rancidity of oils – auto-oxidation
radical reaction
in vivo – lipid peroxidation – polyunsaturated
fatty acids (PUFA)
1. nonenzymatic
– Caused by non-specific pathological factors
– FFA cleavage on hydrocarbons - ethane, pentane
and aldehydes→decrease of membrane fluidity
2. Enzymatic
- takes place at the active centers of
hydroperoxidases and endoperoxidases →
prostaglandins and leukotrienes
H
H
R
H
lipid
HO· - H2O
R
alkyl radical
R
H
O2
R
O
O
H
Fe
R
O
peroxyl radical
alkoxyl radical
H
H
R
C 2H 6
alkane
O
alkenal
DNA damage
• Reaction with HO· radical
• removal of deoxyribose H
atom - interrupts chain
• addition of HO· to bases hydroxy and oxo derivatives
NH2
OH
N
N
OH
N
N
OH
N
N
H
8-hydroxyadenin
OH
N
OH
H2 N
N
N
H
8-hydroxyguanin
HO
N
5-hydroxyuracil
Protein damage
• Oxidation of amino acid residues
– methionine – methionine sulfoxide
– cysteine – cysteic acid
– arginine – aldehyde of glutamic acid
– proline – glutamic acid
• Hydroxylation of amino acids
– Aromatic amino acids
• Products of lipid peroxidation
- ROS and RNS react with membrane proteins and
proteins lipoprotein particles
- other products LPO (reactive aldehydes MDA…) is
covalently linked to the ɛ- lysine group → aggregation,
networking
Non-enzymatic glycation of proteins
and Diabetes mellitus
• Hyperglycemia is a major symptom of diabetes- high concentration
of glucose – reactive molecule
• Covalent bond of aldehyde group glucose to amine group of proteins
= glycation (Shiff base)
• Non-enzymatic glycation
• -early stage-hours-Shiff´s bases – ketomines
• -transitional stage-days-Amadori products –
fructosamines
- advanced stage –weeks, months – linking chains(transversal
covalent bonds) – Advanced glycation end products (AGE =
Maillard´s products)
Glycation is accompanied glykooxidation (AGE and glucose are
oxidized → ROS
Diabetes mellitus
• Glycated hemoglobin
– long-term blood glucose control in diabetics
– the percentage of glycated hemoglobin formed is
directly proportional to the glucose concentration and
the time that the red cells have been exposed to
glucose.Measurement of it gives an integrated picture
of the mean blood glucose during preceding 60 days.
• Physiological level – less than 4%
• Controlled diabetes ( DM) – 5%
• Impulse to change therapy – more than 6%