biochemistry of aging – free radicals and antioxidants petr tůma and eva samcová
TRANSCRIPT
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 ROSBesides 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
Peroxynitrite •NO· + O2
·- = OONO-
•Important powerful oxidant – oxidation of amino acids in proteins•Antimicrobial effects – macrophages
H2N CH COOH
CH2
CH2
CH2
NH
C
H2N NH
arginin
H2N CH COOH
CH2
CH2
CH2
NH
C
H2N O
citrulin
O2 + NADPHNO·
Physiological functions of free radicalsFree radicals are a tool of oxidases and
oxygenases1. 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 system1. 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
O2·- H2O2
H2O + ½ O2
·OH + Fe3+ + OH-
2 H2O
SOD
catalase
GSHPx
+ Fe2+
2 GSH
GSSG
NADPH+H+
NADP+
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 antioxidantsProteins 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·
+ H2O
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
O
CH3
CH3
O
H3C CH3
R
tokoferylový radikál
R-O-O·
+ R-O-O-HO
CH3
CH3
HO
H3C CH3
R
-tokoferol
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
CH3
CH3
CH3 CH3
H3C
CH3
CH3CH3CH3
- karoten
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
NH2
CHHOOC CH2 CH2 C NH
O
CH C
CH2
SH
NH
O
CH2 COOH
glutathion
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 a-
oxoglutarate dehydrogenase multienzyme complex (cytric acid cycle)
• antioxidant ROO·, ascorbyl radical, HO·, NO·, O2·-
N
NN
NH
OH
HO
OH
kyselina močováUric 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 fluidity2. Enzymatic- takes place at the active centers of
hydroperoxidases and endoperoxidases → prostaglandins and leukotrienes
R
H H
R
H
R
H
R
HOO
R
HO
R
O
H
HO· - H2O
O2
Fe
C2H6
lipid
alkyl radical
peroxyl radical
alkoxyl radical
alkenalalkane
DNA damage
• Reaction with HO· radical• removal of deoxyribose H
atom - interrupts chain
• addition of HO· to bases - hydroxy and oxo derivatives
N
NNH
N
NH2
OH
8-hydroxyadenin
N
NNH
N
OH
OH
H2N
8-hydroxyguanin
N
N
OH
HO
OH
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%