1. Biochemistry of aging – Free radicals and antioxidants
Petr Tůma and Eva Samcová

2. 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

3. 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.

4. 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-

5. 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

6. 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

7. 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)
Quinone antibiotics
adriamycine, daunomycine, streptonigrin, - cardiotoxic...
Pyridine herbicides
paraquat, diquat – lung injury
Low-molecular complexes of Fe with phosphates, nucleotides (other toxic carrier of electrons)
Complexes with ATP, ADP

8. Reactive nitrogen species – 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
O2 + NADPH
NO·

9. Physiological functions of free radicals
Free radicals are a tool of oxidases and oxygenases
Respiratory chain
Inner mitochondrial membrane
Aerobic phosphorylation
Biotransformation of xenobiotics
Mitochondrial cytochrome oxidase – P450
Superoxide and peroxide bound to enzyme
Synthesis in cells
Monooxygenases in endoplasmic reticulum of hepatocyte or in mitochondria of adrenal gland
Hydroxylation of xenobiotics, synthesis of cholesterol and bile acids

10. 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-

11. 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

12. Antioxidant protective system
Restriction of excessive formation of ROS and RNS
Regulation of enzyme activity
Trap of transition elements from reactive sites
Trap and elimination of radicals
scavengers, trappers, quenching
enzymes, substances which form with radicals more stable products
General reparative mechanisms of injured macromolecules
phospholipases
reparative enzymes of DNA
proteolysis of proteins injured by oxidative stress

13. Enzyme antioxidant systems
H2O + ½ O2
catalase
SOD
+ Fe2+
O2·-
H2O2
·OH + Fe3+ + OH-
2 GSH
NADPH+H+
GSHPx
2 H2O
GSSG
NADP+

14. 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

15. 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

16. Catalase Two-electron dismutation of hydrogen peroxide
2H2O2 = 2 H2O + O2
Inactivation of H2O2 – peroxisomes and mitochondria of hepatocyte, cytoplasm of erythrocyte

17. 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

18. 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

19. 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

20. 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
R-O-O·
+ R-O-O-H

21. 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

22. Carotenoids, b-carotene and vitamin A
chemically isoprenoids
Remove radicals centered on carbon and alkylperoxyl radicals ROO· in lipids

23. 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

24. Uric acid (urate) Lipoic acid (lipoate)
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·-
Uric acid

25. 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

26. 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

27. Lipid peroxidation (LPO)
in vitro – rancidity of oils – auto-oxidation radical reaction
in vivo – lipid peroxidation – polyunsaturated fatty acids (PUFA)
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

28. lipid
HO·
- H2O
alkyl radical O2
peroxyl radical Fe
alkoxyl radical
C2H6
alkane
alkenal

29. DNA damage Reaction with HO· radical
removal of deoxyribose H atom - interrupts chain
addition of HO· to bases - hydroxy and oxo derivatives

30. 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

31. 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

32. 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%