Biochemistry of aging – Free radicals and antioxidantsPetr 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 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%