Oxidant and Antioxidant

Dr. Citrawati Dyah Kencono Wungu, dr., M.Si

citrawati.dyah@fk.unair.ac.id

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 Outlines
• Oxidants
• Free radicals (FR)
• Oxidative Stress
• Radical damage on DNA, Lipids, and Protein
• Antioxidants

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OXIDANT

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 What are Oxidants?
Oxidants: atoms or molecules that oxidize other atoms or molecules by pulling
electrons, hydrogen or by adding oxygen: reduction processà electron acceptor

Ferric ion (Fe3+) à Ferrous ion (Fe2+)

Oxidant

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 What are Free Radicals?
• Free Radicals:
– Particles with an unpaired electron spinning around the nucleus
– Tend to reach equilibrium, plucks an electron from the nearest intact molecule
à highly reactive
– Have ability to change molecules into free radicals

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 Oxidant/Free Radical
Electron donors can be either:
– Between free radicals reaction:
R1٠ + R2٠ à R1- R2 or (R2٠٠R2)
– Free radical and Non-Free Radical reaction:
٠R1 + R2=H à R1-H + ٠R2 à……..

Oxidant is electron acceptor and has tendency to pull electrons.

Free radicals have a tendency to pull out electrons from other compounds.

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 Oxidant/ Free Radical

All free radicals are oxidants,

but not all oxidants are free radicals

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 Free radicals Non-radical oxidants
superoxide, O2 · - hydrogen peroxide, H2O2
hydroxyl radical, OH · (Fenton´s reaction)
peroxyl, ROO · hypochlorous acid, HClO
alkoxyl, RO · ozone, O3
hydroperoxyl, HO2 · singlet oxygen, 1O2

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 Free Radical
Higher reactivity
Tendency to trigger chain reactions

Free radical are more damaging than

non-radical oxidant

Structural Component Functional Component

Damage Damage

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 Characteristics of Oxidants & Free Radicals
FRs Oxidants
• Have short half life à hardly difficult • Longer half-life
to measure their levels in the body • Stable
• Unstable • Less reactivity
• Reactivity >>

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Free radicals have a tendency to trigger
chain reactions

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 Higher reactivity
Tendency to trigger Chain reaction
• Electron donor :
– Other free radicals :
·R1 + ·R2 → R1 : R2 (R1-R2) (non radical)
– Non radical compounds :
·R1 + R2-H → R1:H + ·R2 (new FR)
this new free radical (·R2) reacts with other compounds
→ new radical may formed à chain reaction.
– Chain reaction is terminated when 2 radicals react
·R1 + ·R1 → R1-R1
·R1 + ·R2 → R1-R2, and etc.
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 The 3 Stages of Chain Reaction
1. Initiation
2. Propagation
3. Termination

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 Source of Oxidants
• Endogenous
• Exogenous

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 Endogenous Oxidants
• This process is a natural phenomenon called the energy
formation system, but can also provide oxidants or free
radicals that can damage the body.

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 Endogenous sources:
Oxidative Phosphorylation

4H+ + O2 → 2H2O (~95% of the time)

O2 → *O2•- → H2O2 *Only 1-5% generate free radicals 16
 Endogenous sources:
Oxidative Phosphorylation
• During oxidative phosphorylation process,
coenzyme Q (CoQ) is reduced twice (through 2 steps):
Q + H+ + e à .QH (CoQ semiquinone)
.QH + H + + e à QH2 (reduced CoQ)

• The formation of superoxide anions (.O2-):

.QH + O2 à Q + .O2- + H +
• Superoxide anion is very reactive
à à à the formation of H2O2 and hydroxyl radical (.OH):
2.O2- + 2H+ à H2O2
.O2- + H2O2 à .OH
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Free radicals formation

From incomplete
reduction of O2 à H2O

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Formation of ROS (Reactive Oxygen Species)

Superoxide and hydrogen

peroxide are the first ROS to
be formed
à from both, other ROS will
be produced

O2· & H2O2 : primary ROS

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 Endogenous sources:
Secreted by inflammatory cells

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 Endogenous sources:
Macrophage

Oxidants secreted by immune cells during infection by microorganismsà initial purpose is to eradicate
these microorganisms 22
 Endogenous sources:
Produced by endothelial cells

For smooth muscle cells relaxation

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 Exogenous Oxidants
• Radiation
UV light, X-rays, gamma-rays
• Chemicals that react to form peroxides
ozone and singlet oxygen
• Chemicals that promote superoxide formation
quinones, nitroaromatics, bypyrimidiulium herbicides
• Chemicals that are metabolized to radicals
polyhalogenated alkanes, phenols, aminophenols
• Chemicals that release iron
ferritin
• Certain drugs
Paracetamol, some antibiotics, chemotherapy drugs, anti malaria
• Others
cigarette, smoke, junk food, processed food
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 ROS (Reactive Oxygen Species) and
RNS (Reactive Nitrogen Species)
All living organisms (aerobic) need oxygen to
produce energy efficiently.

ROS and RNS are the result of normal cell

metabolismà can be beneficial or destructive

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 ROS
Free radicals Not free radicals
superoxide, O2 · - hydrogen peroxide, H2O2
hydroxyl radical, OH · (Fenton´s reaction)
peroxyl, ROO · hypochlorous acid, HClO
alkoxyl, RO · ozone, O3
hydroperoxyl, HO2 · singlet oxygen, 1O2

Most dangerous, because of its reactivity

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 RNS
Free radicals Not free radicals
nitrogen(II) oxide, NO . nitrosyl, NO+
nitrogen(IV) oxide, NO2 . nitrous acid, HONO
nitrogen(III) oxide, N2O3
peroxynitrite, ONOO -
alkylperoxinitrite, ROONO

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 ROS Formation
• ROS is formed when the reduction is not perfect.
• One electron transfer reaction :
O2 + e à .O2-
O2 + e + H+ à .OOH
• Two electron transfer reaction:
O2 + 2e + H+ à H2O2
• Three electron transfer reaction:
O2 + 3e + 3H+ à H2O + .OH
• Four electron transfer reaction:
O2 + 4e + 4H+ à H2O

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Connection between ROS-RNS (Reactive Nitrogen Species) and
among ROS

OOH.
H+ Myeloperoxidase (MPO)
H+

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 Biomedical Importance
• Oxidant may affect cell’s integrity: damage lipid in membrane cell, plasma lipoprotein,
protein, and nucleic acid
• Oxidant is involved in several disease:
– Cardiovascular
– Respiratory
– Impairment of the immune system
– Reproduction
– Aging

Patologic
Physiologic

Antioxidant

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 High concentration :
free radicals and oxidants
have destructive effects
to the body
Low / moderate concentration :
free radicals and oxidants have
beneficial effects / physiological
function to the body
• cellular response
• immune system
Free radicals physiological function
What is Oxidative Stress?

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 Impact due to ROS

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medical.net%2Fimage.axd%3Fpicture%3D2018%252F4%252Fshutterstock_sakurra.jpg&imgrefurl=https%3A%2F%2

Oxidative stress (OS) is included in various pathological

conditions related to disease, heart disease, cancer, neurologic
disease, diabetes mellitus (DM), hypertension (HT), aging etc..
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 ROS Related to Disease

ROS effect to Oxidative conditions

Mitochondria cause inflammation
and elevation of
oxidase activity
Mitochondria are involved in the production of reactive oxygen
species àone-electron carriers in the respiratory chain
Atherosclerosis
Mitochondrial structures are also very susceptible to oxidative
stress as evidenced by massive information on lipid Chronic inflammation
peroxidation, protein oxidation, and mitochondrial DNA Ischemic
(mtDNA) Reperfusion injury
• Ex: cancer and DM

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 ROS Related to Disease
Convincing evidence of the link between oxidative stress and acute and
chronic disease lies in the biomarkers of oxidative stress.
Defense system is the enzyme
system, including superoxide
dismutase, glutathione peroxidase,
and catalase.
Some important minerals including
selenium, manganese, copper, and
zinc are needed for the formation
or activity of these enzymes.
Disease
These biomarkers must
be measured objectively
and evaluated .
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 ROS Impact
ROS can react with many substances, either simple
compounds such as amino acids, fatty acids, cholesterol, etc
or with more complex substances such as proteins and DNA

The most damaging effect of ROS is on 3 (three) important

components of the cells: Lipid membranes, DNA, and Proteins

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These radicals can bind
covalently to the
macromolecules and
induce peroxidative
degradation of the
membrane lipids rich in
polyunsaturated fatty
acids, leads to the
formation of lipid
peroxides followed by
multiple pathological
changes
Impact ROS to Lipid Membran Cell
Cell membrane stucture is a lipid bilayer which is
composed mainly by phospholipid and
cholesterol.

ROS

Lipid peroxidation reactions

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 Lipid Peroxidation

LH +.LOO à LH + .OH à .L (lipid radikal) + H2O

.L + LOOH (lipid
peroksida)

.L + O2 à .LOO (radikal peroksilipid)

.L + .L à L – L

https://www.google.com/url?sa=i&s TERMINATION 44
ource=images&cd
Fatty Acid Cross Linking

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 Consequences of Lipid Peroxidation
• Structural changes in membranes
– Alter fluidity and ion channels
– Alter membrane-bound signaling proteins
– Increase membrane permeability
• Form adducts/crosslinking with non-lipids
– E.g., protein and DNA
• Cause direct toxicity
– E.g., 4-hydroxynonenal, MDA
• DNA damage and mutagenesis
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ROS Impact

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Impact ROS to Cholesterol Membran Cell
• Cholesterol is also present in cell structures.

The structure
is different
from original
cholesterol

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Impact ROS to DNA

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Effect of ROS on DNA

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 Impact ROS to Protein
Proteins are composed of a series of amino acids

Histidine.
Cysteine

2-oxo-histidine radical
Cysteine Radical

Cystine 51
Impact ROS to Protein

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So, is there any way to get rid of these free radicals?
What is antioxidant?
Definition in chemistry: electron donor.
Example:
Cu+ à Cu2+ + e-
Cupro Cupri
Antioxidant

Definition in biology (Medicine):

1. Electron donor
2. Any substance that prevent the
formation or suppress the activity of
oxidants
example: Mental binding protein and
enzyme
 How do antioxidants work?

Antioxidants donor their electronà

Antioxidants undergo oxidation process

Antioxidant resources must be continuously restored in the body

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Antioxidant donor their electronà Antioxidants run into
oxidation process

To get rid of the free radicals

Changed into pro-oxidant

Produce ROS and RNS

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 Source of Protection Against
Oxidative Stress
1. Producing antioxidants (endogenous
antioxidants)
2. Supplying from externally (exogenous
antioxidants).

To neutralize excess free radicalsà protect cells

from the effects of free radical toxins and
contribute to the prevention of disease
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 Antioxidant Classification

How it works its solubility

Prevention Chain breakers Hydrophilic Lipophilic

Cytoplasm: Vitamin Cell membrane:

C, Glutathione, Vitamin D, E, A
Cysteine, Uric acid
 Antioxidant Classification
Based on the activity

Non-enzymatic
Enzymatic Antioxydant
Antioxydant

by breaking down and by interrupting free

removing free radicals radical chain reactions

convert dangerous oxidative

products to hydrogen peroxide
(H2O2) and then to water. vitamin C, vitamin E, plant
polyphenol, carotenoids, and
Presence of cofactors such as glutathione.7
copper, zinc, manganese, and
iron.
 ANTIOXIDANT MECHANISM
Preventive Antioxidants
Preventing the buildup of primary ROS (superoxide (O2 ·) and
hydrogen peroxide (H2O2)) and transition metal ions (ferrous
ion (Fe2 +)) and cuprous ion (Cu +)).
 MECHANISM OF ANTIOXIDANTS
Preventive Antioxidants
1. Preventing the buildup of primary ROS → secondary ROS are not formed
2. Preventing the formation of hydroxyl radical (OH ·): very reactive
3. Preventing the formation of primary OH and ROS by preventing the
buildup of transition metal ions through the reaction of Fenton and
Haber-Weiss:
This reaction requires the presence of Ferrous ions (Fe2 +) or Cuprous ions
(Cu +)
• Fenton Reaction:
Fe2+ (Cu+) cuprous + H2O2 à Fe3+ (Cu2+) cupric + OH- + .OH
• Haber-Weiss Reaction:
H2O2 +.O2- à O2 + OH- + .OH

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 MECHANISM OF ANTIOXIDANTS
Preventive Antioxidants
4. Preventing the buildup of free Ferrous ion and free Cuprous
ions à transition metal binding proteins,
example: Transferrin and Ferritin (function of binding to Ferro ions)
and Ceruloplasmin and Albumin (binding to Cuprous ions)
5. Preventing the buildup of superoxide anions à superoxide
dismutase (SOD) enzymes:
2.O2- + 2H+ à O2 + H2O
6. Preventing the accumulation of H2O2 à catalase and
peroxidase enzymes
 Enzyme Classification
REMINDER!!!

Oxidoreductase-Hydroperoxydase
III. HYDROPEROXYDASE
Enzyme that uses H2O2 as a substrate

Two types:
1. PEROXYDASE, requires other substance as the electron acceptor.
H2O2 + AH2 2 H2O + A

2. CATALASE, can use H2O2 as both electron donor and acceptor.

H2O2 + H2O2 2H2O + O2
 ANTIOXIDANT
Chain Breaking Antioxidants

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Chain Breaking Antioxidants: Lipid Peroxidation
• Break lipid peroxidation chain reaction
• Work especially in cell membranes
• Only lipophilic antioxidants

• Vitamin E (Tocopherol) is a lipophilic antioxidant found in the

cell membranes (and also in lipoproteins).

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 Chain Breaking Antioxidants: Lipid Peroxidation
Vitamin E
Tocopherol (ToCH) can react with lipid radical (.L) in the following
way:
.L (Lipid radical) + ToCH à LH + .ToC (Tocopheryl radical)

.LOO (peroxylipid radical) + ToCH à LOOH + .ToC

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 Inactivation of Tocopheryl Radical
• Intramolecular rearrangement occurs à Tocopheryl radical
turns into Tocoquinone (TocQ)
• Tocopheryl radicals subsequently move to the surface of the
cell membrane, and then react with vitamin C (Ascorbic Acid)
in the following way:
– ToC + AscH2 à TocH + .Asc + H+ (Ascorbyl radical)
This ascorbiyl radical will then be spontaneously inactivated through a
dismutation reaction, as follows:
– 2.Asc + 2H+ à AscH2 + DHAA (Dehydroascorbic acid)
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 Vitamin E and C
Interaction between antioxidants in the lipid phase (cell membranes) and
the aqueous phase (cytosol)

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 Chain Breaking Antioxidants– β carotene

• It should be understood that tocopherol can only react at a

relatively high oxygen partial pressure
• In relatively low partial pressure of oxygen, the role of
tocopherol is replaced by beta-carotene
• β-carotene (CarH)+ free radicals à β–carotenyl radical (Car.)
• β-carotenyl radical has relatively stable properties

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Endogenous Antioxidant Enzymes

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 Main Antioxidant Enzymes
Main antioxidant enzymes directly involved in the neutralization
of ROS and RNS are:
• superoxide dismutase (SOD): frontliner
• catalase (CAT)
• glutathione peroxidase (GPX)
• glutathione reductase (GR)

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Main Antioxidant Enzymes Mechanisms

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 Glutathione Peroxidase
• Among peroxidases, the most important one is glutathione
peroksidase.
• Contains Selenium.
• Catalyzes the following reaction:
2 GSH + 2H2O2 à GSSG + 2 H2O
(Reduced Glutathione) (Oxidized Glutathione)

GSH is returned through a reaction catalyzed by glutathione reductase :

GSSG + NADPH + H+ à 2GSH + NADP+
 Glutathione
Reductase

Pentose Phosphate Pathway/

HMP Shunt

The figure is found at http://www.med.unibs.it/~marchesi/ppp.html (March 2007)

Glucose 6-Phosphate Dehydrogenase Deficiency in Erythrocytes
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