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OXIDANTS, ANTI-OXIDANTS

AND FREE RADICALS


OXIDANTS (1)
1. Oxidants are implicated in several disease processes such as :
• Cardiovascular diseases
• Respiratory diseases
• Reperfusion injury
• Diabetes mellitus
• Impairments of the immune system
• Carcinogenesis
• Aging
2. Oxidants may affect cell's integrity because they react with
and destroy cell's components either structural (e.g. cell
membrane & cytoskeleton) or functional (e.g. enzymes & DNA)
3. Oxidants maybe produced inside our body (endogenous) or
may come from sources outside the body (exogenous)
OXIDANTS (2)
ENDOGENOUS OXIDANTS
 Are produced as a by product of normal physiological processes such
as:
 ATP production (oxidative phosphorylation) in mitochondria
 Oxygenation of Hb in erythrocytes
 Secreted by inflammatory cells
 Formed by the action of ionizing radiation (e.g. x-rays)

EXOGENOUS OXIDANTS
May come from several sources such as :
 Pollutants
 Drugs
 Food additives
 Chemicals used in industry

etc
OXIDANTS & FREE RADICALS (1)
Oxidants are electron acceptors
Example: Fe+++

Fe 3+ + e-  Fe 2+

Free radicals are atoms or molecules possessing one


or more unpaired electron
Example : homolytic cleavage of water due to ionizing radiation

H : 0 : H ( H – 0 – H ) .H + .OH

H atom Hydroxyl radical


(Free Radical) (Free Radical)
OXIDANTS & FREE RADICALS (2)
Free radicals show a great tendency to attract electrons
( e– ) due to the presence of unpaired electron.

a. Electron donor is another radical:


R1· + R2·  R1 : R2 (R1 – R2)
(non radical)

b. Electron donor is a non radical


R1 · + R2 : H (R2 – H)  R1 : H (R1 - H) + R2·

radical non radical non radical new radical

The newly formed radical can then attack other molecules


OXIDANTS & FREE RADICALS (3)
1. Oxidants are electron acceptors, i.e. they attract
electrons:
Fe3+ + e–  Fe2+

2. Since free radicals also attract electrons, free radicals


can also be considered oxidants

3. Free radicals are all oxidants but not all oxidants are
free radicals.
Example :
Hydrogen peroxide (H2O2): oxidant, non radical
Hydoxyl radical (.OH) : oxidant, radical
FREE RADICALS CAN TRIGGER
CHAIN REACTIONS
1. When a free radical react with a nonradical the result will
be the formation of a new radical:
R1. + R2 – H  R1 – H + R2.

2. The newly formed radical can again react with a


nonradical giving rise to another radical.
R2. + R3 – H  R2 – H + R3.
This process can be repeated again and again resulting in
a chain reaction

3. Such a chain reaction will only stop when 2 radicals meet


R1. + R1.  R1 – R1
R1. + R2.  R1 – R2 etc.
THE 3 STAGES OF A CHAIN REACTION
1. Initiation
2. Propagation
3. Termination
Example:
Initiation : Fe2+ + H2O2  Fe3+ + OH- + • OH
R1 – H + .OHR1. + H2O
Propagation : R1. + R2 – H  R1 – H + R2.
R2. + R3 – H  R2 – H + R3.
etc.
Termination : R1. + R1. R1 – R1
R1. + R2. R1 – R2
R1. + R3. R1 – R3
etc.
FREE RADICALS :
Are more damaging than nonradical oxidants
because of their :
• Higher reactivity
• Tendency to trigger chain reactions
REACTIVE OXYGEN SPECIES (1)
(ROS)
1. Reactive oxygen species (ROS) are endogenous oxidants
derived from oxygen
These includes :
• Superoxide (an) ion : O 2.-
• Peroxyl radical : . OOH
• Hydrogen peroxide : H 2O 2
• Hydroxyl radical : . OH
• Hypochlorite (an) ion : CIO-
• Singlet oxygen : 1O 2
As can be seen, some are free radicals (O2. -, . OOH, .OH )
others are not
2. Of all the ROS, .OH is the most dangerous because of its high reactivity
3. 1O2 is a special form of oxygen which is more reactive than
"ordinary" oxygen (O2) due to its different electron configuration.
(O2 itself is a relativery weak oxidant)
REACTIVE OXYGEN SPECIES (2)
(ROS)
1. The reduction of O2 to H2O involves the transfer of
4 electrons:

O2 + 4 H+ + 4e-  2 H2O

2. All ROS except CIO- and 1O2, can be considered as being


the result of incomplete oxygen reduction :
1 e-transfer : O2 + e-  O2.-
O2 + e- + H+  .OOH
2 e-transfer : O2 + 2e- + H+  H2O2
3 e-transfer : O2 + 3e- + 3H+  H2O + .OH
4 e-transfer : O2 + 4e- + 4H+  2H2O
GENESIS & RELATIONSHIP BETWEEN ROS
A summary
CIO-

H2O2 1
O2
Fe /CU
++ +

O2 .OOH .OH
+
Fe+ /
++ CU
+
/CU Fe
+

O2.- & H2O2 are primary ROS from which all the
others are derived
FORMATION OF PRIMARY R.O.S.
H2 O 2
OXIDASES (ENDOPLAS MIC RETICULUM, PEROXISOME)
RH2 + O2 R + H 2O 2

H2 O 2 :

1. Oksidan kuat  dpt mengoksidasi berbagai senyawa dlm sel,


misal :
2GSH + H2O2  GSSG + 2 H2O

2. Reaksi Fenton membentuk radikal hidroksil :


1. H2O2 + Fe++ / Cu+  Fe+++ / Cu++ + OH- + .OH

3. Menghasilkan ion hipoklorit (E.myeloperoksidase di sel radang


spt granulosit, monosit, makrofag menkatalisis H2O + Cl- 
H2O + ClO-) yang dapat mengoksidasi berbagai senyawa,
misal :
R + ClO-  RO + Cl-
FORMATION OF PRIMARY R.O.S.
•O 2 ¯
TRANSITION METALS :
O2 + Fe++ (Cu+) •O2¯ + Fe+++ (Cu++ )

NADPH OXIDASE (INFLAMMATORY CELLS)


2 O2 + NADPH 2 •O2¯ + NADP+ + H+

XANTHINE OXIDASE (REPERFUSION INJURY)


2 O2 + XH + H2O 2 •O2¯ + 2 H+ + X – OH
(XANTHINE) (URIC ACID)

BY PRODUCTS OF :
1. NORMAL PROCESSES OXIDATIVE PHOSPHORYLATION
2. HEMOGLOBIN OXYGENATION (FORMATION OF Hb O2)
3. Hydroxylation by E.Monoaxygenation (sit.P450 dan sit b4)
XANTHINE OXIDASE
 Tak ada pada sel mamalia
 Terbentuk dari xantin dehidrogenase
dalam keadaan hipoksia / iskhemia
 XD  XO + peptida
 Reaksi ini tak reversibel sehingga jika pasokan
oksigen menjadi normal dapat terbentuk ion
superoksid yang dapat merusak jaringan (re
perfusion injur
ION SUPEROKSID
Sebenarnya tak terlalu reaktif
•O2¯ + H+  .OOH (radikal peroksil yg reaktif)
.OOH + XH  .X + H2O2
 Ion superoksid berbahaya jika ada bersama dg
H2O2 (reaksi Haber Weiss)
 •O2¯ + H2O2  O2 + OH- + .OH
 Radikal hidroksil :
 berbahaya karena paling reaktif
 Bukan produk primer proses biologi
 Berasal dari H2O2 dan •O2¯
FORMATION OF OTHER R.O.S.
•OOH
•O2¯ + H+ •OOH

•OH
•O2¯ + H2O2 Fe+ + +
O2 + OH¯ + •OH
(Cu++ ) HABER-WEISS REACTION

H2O2 + Fe++ /Cu+ Fe+++ /Cu++ + OH¯ + •OH


(FENTON REACTION)
ClO¯
H2O2 + Cl¯ H2O + ClO¯
1 (MYELOPEROXIDASE)

O2 1
H2O2 + ClO¯ (MYELOPEROXIDASE) H2O + Cl¯ + O2
EFFECT OF ROS
1. ROS can react with many substances either simple
compounds such as amino acids, fatty acids,
cholesterol etc or more complex substances such as
DNA & proteins

2. The most damaging effect of ROS, however, is on


3 important components of cells :
• Membrane lipids
• Proteins
• DNA
EFFECT OF ROS ON MEMBRANE LIPIDS
1. The cell membrane is a lipid bilayer formed mostly by phospholipids and
cholesterol.
2. Phospholipids contain polyunsaturated fatty adds (PUFA) which is prone
to attack by ROS esp. .OH causing a chain reaction called Lipid
Peroxidation :
a. Initiation
LH + .OH  L. + H 2O

Lipid radical
b. Propagation
L. + O2  LOO.
Peroxylipid radical
LH + LOO.  L. + LOOH

Lipid peroxide
c. Termination
L. + L.  L – L
3. LOOH undergo further reactions leading to clevage of the F.A. chain
4. Termination causes cross-linking between 2 FA Chain
EFFECT OF ROS ON MEMBRANE LIPIDS
EFFECT OF ROS ON DNA(1)
EFFECT OF ROS ON DNA(2)
EFFECT OF ROS ON DNA(3)
EFFECT OF ROS ON PROTEINS
ANTIOXIDANTS (1)
1. In its original definition in chemistry antioxidants
are electron donors
Example: Cu +  Cu2+ + e-
2. In Biology (and Medicine) however, antioxidants
have a much broader meaning, it is:
any substance that prevent the formation or
activity of oxidants
3. This broader definition in Biology includes not only
electron donors but other sustances such as :
• Metal binding proteins
• Enzymes
ANTIOXIDANTS (2)
ANTIOXIDANTS CAN BE CLASSIFIED ACCORDING :
A. Its mode of action
1. Preventive antioxidants prevent undue accumulation of oxidants
2. Chain breaking antioxidants prevent propagation of chain reactions
initiated by free radicals

B. Its solubility
1. Lipophilic antioxidants
Hydrophobic, fat solluble molecules, act in cell membranes :
Tocopherols (vitamin E)
 carotene (provitamin A)
2. Hydrophilic antioxidants
Hydrophilic, water solluble molecules act in cytosol and E.C.F.:
Ascorbic acid (vitamin C)
Glutathione
Cysteine
Others (e.g.: uric acid)
PREVENTIVE ANTIOXIDANTS
Preventive antioxidants prevents accumulation of: O 2, H2O2 and
Transition metal ions Fe2+ and Cu+

1. O2.- & H2O2 are primary ROS from which all others ROS are
derived Thus preventing the accumulation of O 2. and H2O2
will prevent accumulation of all the other ROS

2. Among all the ROS, .OH is the most reactive


Thus it is most important to prevent generation of .OH
3. .OH is mainly generated through 2 reactions :
a. Fenton reaction
Fe2+ (Cu+) + H2O2  Fe3+ (Cu2+) + OH- + .OH

b. Haber – Weiss reaction


H2O2 +O2.-  O2 – OH- + .OH

This reaction requires Fe3+ or Cu2+ and is believed


to occur in 2 steps, the 2nd step of which is the
Fenton reaction
Fe3+ (Cu2+) + O2.-  Fe2+ (Cu+) + O2
Fe2+ (Cu+) + H2O2  Fe3+ (CU2+) + OH- + .OH

Thus preventing accumulation of free Fe 2+ and Cu+ ions is


important to prevent generation of .OH even if O 2.- and H2O2
are present
4. Accumulation of free Fe2+ & Cu+ ions are prevented by
transition metal binding proteins:

Fe2+ : Transferrin & Ferritin


Cu+ : Coeruloplasmin & Albumin

5. Accumulation of O2.- is prevented by a reaction catalyzed


by superoxide dismutase (SOD):

2O2.- + 2 H+  O2 + H2O2

Mammalian cells contain 2 species of SOD one containing


Cu & Zn (CuZnSOD) and another containing Mn (Mn SOD)
6. Accumulation of H2O2 is prevented by the actions of enzymes called Catalase
and Peroxidases

a. Catalase : 2H2O2  2H2O + O2


b. Peroxidases are enzymes catalyzing the general reaction
A + H2O2  AO + H2O
Among the peroxidases. the most important of which is
Glutathione peroxidase (GsPx or GPX) a Se containing
enzyme catalyzing the reaction

2 GSH + 2H2O2  GSSG + 2H2O


(Glutathione) (Oxidized glutathione)

GSH is restored by the action of glutathione reductase :


GSSG + NADPH + H+  2 GSH + NADP+

7. OH once generated can still be inactivated by glutathione (GSH) or


cysteine (Cys SH)
GSH : GSH + .OH  GS. + H2O Cys SH : Cys Sh + .OH  Cys. + H2O
2 GS.  GSSG 2Cys.  Cys SS Cys (Cystine)
CHAIN - BREAKING ANTIOXIDANTS
1. Lipid peroxidation is quantitatively the most important chain reaction
occuring in cells.
Thus only lipophilic antioxidants can stop this reaction from progressing

2. Tocopherols are major lipophilic antioxidant present in cell membranes


(and also in lipoproteins)
Although tocopherols (TocH) can react with lipid radicals (L.):

L. + TocH  LH + Toc.
(Tocopheryl radical)

Its main action is probably on peroxylipid radicals (LOO.):

LOO. + TocH  LOOH + Toc. (radikal Vit.E)

3. Although Toc. Is relatively stable because of electron delocalisation, it still


remains to be inactivated
4. Inactvation of Toc. Can occur by several ways :
a. Intramolecular rearrangement can give rise to a nonradical
called tocoquinone (ToqQ)
b. Moving to the cell membrane surface, it reacts with ascorbic acid
(Assc H2)

ToC. + AscH2  TocH + Asc.- + H+


Ascorbyl radical
The ascorbyl radical is then spontaneously inactivated by
a dismutation reaction

2 Asc.- + 2H+  AscH2 + DHAA


(dehydro-ascorbic acid)

5. Alternatively, Toc. Can also react with cysteine (Cys SH) or glutathione
(GSH), generating cystine (Cys SS Cys) or oxidised glutathione (GSSG)

6. Tocopherols can only react at a relatively high PO 2. At low PO2, the role
of tocopherols is replaced by R-carotene, whose radical ( -carotenyl
radical) is also relatively stable due to electron delocalisation