MEASUREMENT THE EFFECT

OF OXIDANTS, AND FREE

RADICALS
 Prof. Soetjipto, dr., M.S., Ph.D.
• Jabatan: Staf Pengajar Departemen Biokimia FK UNAIR
Staf Pengajar Program Pascasarjana UNAIR
Wakil Rektor III UNAIR

• Pendidikan :
– S-1/Dokter : FK UNAIR
– S-2/ Magister Sains : Program Pascasarjana UNAIR
– S-3/ Ph.D. : Kobe University, Japan

E-mail address : warek3@unair.ac.id

soetjiptojtbr@sby.centrin.net.id
OXIDANTS & FREE RADICALS (1)
Oxidants are electron acceptors
Example : Fe+++
Fe 3+ + e -  Fe 2+
Free radicals are atoms or molecules possessing one
Ore more unpaired electron
Example : homolytic cleavage of water due to ionizing
radiation
H : O : H (H – O – H)  .H + .OH

H atom Hydroxyl radical

(Free Radical) (Free Radical)
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 & cytosceleton) 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 (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. it also 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) : oxidants, non radical
Hydoxyl radical (.OH) : oxidant, radical
OXIDANTS & FREE RADICALS (4)

1. The tendency to attract electrons among free

radicals may vary, some are highly reactive, very
unstable and have a short half-life others are less
reactive, relatively stable and have a longer half-
life.
2. The relative stability of certain radicals is due to a
physico-chemical phenomenon called; electron
delocalisation (“wandering electron”).
3. Nonradical oxidants, on the other hand, are stable
compounds and their oxidative reactivity are thus
less than the most stable free 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 . + R 3 .  R 1 – R 3
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 : O2.-
 Peroxyl radical : . OOH
 Hydrogen peroxide : H2O2
 Hydroxyl radical : .OH
 Hypochlorite (an) ion : CIO-
 Singlet oxygen : 1O2
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. 1
O2 is a special form of oxygen which is
more reactive than “ordinary” oxygen (O2)
due to its different electron configuration.
(O2 itself is a relatively 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 CI- 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 + e- + H +  H2O2
3 e-transfer : O2 + 3e- + 3H+  H2O + .OH
4 e-transfer : O2 + 4e + 4H+  2H2O
GENESIS & RELATIONSHIP BETWFEN ROS
A summary
CIO-

H2O2 1
O2
Fe /CU
++ +

O2 .OOH .OH

+
Fe+ ++ /CU
+
/CU+ Fe
O2 .

O2. - & H2O2 are primary ROS from which all the
Others are derived
EFFECT OF ROS
1. ROS can react 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:
• Lipid Membranes
• Proteins
• DNA
EFFECT OF ROS ON LIPID MEMBRANES (1)

1. The cell membrane is a lipid bilayer formed mostly by

phospholipids and cholesterol.

2. Phospholipids contain polyunsaturated fatty acids

(PUFA) which is prone to attack by ROS esp.
.OH causing a chain reaction called Lipid
Peroxidation:
a. Initation
LH + . OH  L. + H2O

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 cleavage of the Fatty acid
chain

4. Termination causes cross-linking

between 2 Fatty acid Chain.
 EFFECT OF ROS ON LIPID MEMBRANES (2)
Membrane lipids
ROS
Lipid peroxidation

Cross linking FA Cleavage FA w3 & w6

chain PUFA chain

Toxic products
• MDA
• 8 – OH non-enal
• Ethane (C2 H6)
• Pentane (C5 H10)

Membrane cell damage

Disturbed osmotic balance

Water enters cell

PUFA : Polyunsaturated
fatty acid Cell swelling
MDA : Malondialdehyde
Cell lysis
EFFECT ROS ON CHOLESTEROL
• ROS react with cholesterol giving a mixture of
compounds, an example :
EFFECT OF ROS DNA (2)
EFFECT OF ROS ON DNA (3)
EFFECT OF ROS ON DNA
DNA

1. Hydroxylation of Cleavage of
Pu & Py bases phosphodiester
2. Ring opening of backbone
Pu & Py bases

Not repaired Repaired Not repaired

Mutations No effect

Mutation on Chromosome If severe

Proto – or aberrations
antioncogenes

Pu : Purine Cancer Other Effects Cell dies

Py : Pyrimidine
EFFECT OF ROS ON AMINO ACIDS

Example :
1. Cysteine
-
OOC – CH – CH2 – SH

NH3+ Cys – SH + .OH  Cys.S. + H2O

Cysteine (Cys – SH) 2 Cys – S.  Cys-S-S-Cys
Cystine
2. Histidine
CH – COO- CH – COO-

N NH NH3+ N NH NH3+
.OH H2O

Histidine  .OH

CH – COO-
CH – COO-

N NH NH3+
HN NH NH3+

OH
O 2-oxo-Histidine
 EFFECT OF ROS ON PROTEINS
Proteins

Cysteine residues Other amino acids

residues

Formation of Disulfide Modified side chain

(S – S) bonds

Intra- or
interchain
Cross-lilnking

LOSS OF BIOLOGICAL FUNCTION

(e.g. enzymes, peptide hormones, receptors, Channel
proteins etc)
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 substances such as :
 Metal binding proteins
 Enzymes
ANTIOXIDANT (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 sollubility
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 Extracellular fluid.:
Ascorbic acid (vitamin C)
Glutathione
Cysteine
Others (e.g.; uric acid)
PREVENTIVE ANTIOXIDANTS (1)

Preventive antioxidants prevents accumulation of: O2¯,

H2O2 and transition metal ions Fe2+ and Cu+
1. O2.- & H2O2 are primary ROS from which all others
ROS are derived This preventing the accumulation
of O2.- and H2O2 will prevent accumulation of all the
other ROS
2. Among all the ROS, .OH is the most reactive
This 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

This preventing accumulation of free Fe2+ and Cu+ ions

is important to prevent generation of .OH even if O2.-
and H2O2 are present
PREVENTIVE ANTIOXIDANTS (2)

1. Accumulation of free Fe2+ & Cu+ ions are prevented by

transition metal binding proteins :
Fe2+ : Transferrin & Ferritin
Cu+ : Ceruloplasmin & Albumin
• Accumulation of O2.- is prevented by a reaction catalyzed
by superoxide dismutase (SOD) :
2O2.- + 2 H+  O2 + H2O
Mammalian cells contain & species of SOD one
containing Cu & Zn (CuZnSOD) and another containing
Mn (Mn SOD)
3. Accumulation of H2O2 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+
4. .OH once generated can still be inactivated
by glutathione (GSH) or cysteine (Cys SH)

GSH : GSH + .OH  GS. + H2O

2 GS.  GSSG

Cys SH : Cys SH + .OH  Cys. + H2O

2Cys.  Cys SS Cys (Cystine)
CHAIN – BREAKING ANTIOXIDANTS
1. Lipid peroxidation is quantitatively the most important
chain reaction occuring in cells.
This only lipophilic antioxidants can stop this reaction from
progressing
2. Tocopherols is major lipophilic antioxidants 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.
3. Although ToC. Is relatively stable because of electron
delocalisation, it still remains to be inactivated
4. Inactivation 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.- + N+
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 oxidized glutathione (GSSG)

6. Tocopherols can only react at a relatively high Po2.

at low PO2, the role of tocopherols is replaced by
-carotene, whose radical (-carotenyl radical) is
also relatively stable due to electron
delocalisation
RESONANCE STRUCTURAL OF VITAMIN E
RADICAL (ELECTRON DELOCALISATION)
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