Antioxidants (MBBS Lecture)

FREE RADICALS
AND
ANTI-OXIDANTS
Dr. Seema Lekhwani

Assistant Professor
Biochemistry Department
Pt. B. D. S. PGIMS, Rohtak (Haryana).
The outermost orbital in an
atom or molecule contains
two electrons, each
spinning in opposite
directions.
The chemical covalent

bond consists of a pair of
electrons, each component
of the bond donating one
electron each.
Definition:
A free radical is a molecule or molecular fragment that
contains one or more unpaired electrons in its outer-
most orbital.
Free radical is generally represented by a superscript
dot, (R°).
Oxidation reactions ensure that molecular oxygen is completely reduced to water.
The products of partial reduction of oxygen are highly reactive and create havoc
in the living systems.
Hence, they are also called Reactive Oxygen Species (ROS).
The sequential univalent reduction steps of oxygen:
The following are the members of this group i.e. ROS:
i) Superoxide anion radical (O2‒º)
ii) Hydroperoxyl radical (HOOº)
iii) Hydrogen peroxide (H2O2)
iv) Hydroxyl radical (OHº)
v) Lipid peroxide radical (ROOº)
vi) Singlet oxygen (1O2)
vii) Nitric oxide (NOº)
viii) Peroxy nitrite (ONOO ‒ ‒ º)
Though Hydrogen peroxide (H2O2) and Singlet oxygen (O2) are not free
radicals, due to their extreme reactivity, are included in ROS group. Also
compare oxygen with superoxide anion.
Important characteristics of the
ROS are:
a) Extreme reactivity
b) Short life span
c) Generation of new ROS by
chain reaction
d) Damage to various tissues
Generation
of
Free Radicals
i) They are constantly produced during the normal oxidation of
foodstuffs, due to leaks in the electron transport chain in
mitochondria.
About 1-4% of oxygen taken up in the body is converted
to free radicals.
Mitochondrial ROS production is modulated largely by
the rate of electron flow through respiratory chain
complexes.
ii) Some enzymes such as xanthine oxidase and aldehyde oxidase
form super-oxide anion radical or hydrogen peroxide.

iii) NADPH oxidase in the
inflammatory cells (neutrophils,
eosinophils, monocytes and
macrophages) produce superoxide
anion by a process of respiratory burst.
The superoxide is converted to
hydrogen peroxide and then to
hypochlorous acid (HClO) with
the help of superoxide dismutase
(SOD) and myeloperoxidase
(MPO).
The superoxide and hypochlorous ions
are the final effectors of bactericidal
action. This is a deliberate production of
free radicals by the body.
Along with the activation of

macrophages, the consumption of oxygen
by the cell is increased drastically; this is
called respiratory burst.
In chronic granulomatous disease (CGD), the NADPH oxidase is
absent in macrophages and neutrophils.
In this condition, macrophages ingest bacteria normally, but cannot

destroy them.
However, streptococci and pneumococci themselves produce H 2O2.
Therefore, they are destroyed by the myeloperoxidase system of the

macrophages.
But staphylococci being catalase positive can detoxify H 2O2 in
the macrophages and therefore are not destroyed in such
persons. Hence, recurrent pyogenic infection by staphylococci
are common in chronic granulomatous disease.
iv) Macrophages also produce NO from
arginine by the enzyme nitric oxide
synthase. This is also an important
anti-bacterial mechanism.
v) Peroxidation is catalyzed by lipo-oxygenase in platelets and
leukocytes.
Under hypoxic conditions, the mitochondrial respiratory chain
also produces nitric oxide (NO), which can generate other
reactive nitrogen species (RNS).
Excess ROS and RNS can lead to oxidative and nitrosative stress.
Low levels of both species function in hypoxic signaling

pathways, which have important implications for cancer,
inflammation and a variety of other diseases.
Free Radical
Scavenger Systems
1) Superoxide dismutase (SOD)
The mitochondrial SOD is Mn dependent;
cytoplasmic enzyme is
Cu-Zn dependent.
SOD is non-heme protein.

A defect in SOD gene is seen in patients with
amyotrophic lateral sclerosis
(Lou Gehrig’s disease; named after the American baseball captain who
succumbed to the illness.)
2) Catalase
When is H2O2 generated in large quantities, the enzyme catalase is

also used for its removal.
2 H2O2 Catalase
O2 + H2O
3) Glutathione peroxidase
In the next step, H2O2 is removed by glutathione peroxidase

(POD), a Se dependent enzyme.
4) Glutathione reductase (GR)
The oxidized glutathione, in turn, is reduced by the glutathione reductase,

in presence of NADPH.
This NADPH is generated with the help of glucose-6-phosphate

dehydrogenase (GPD) in HMP shunt pathway.
Therefore, in GPD deficiency, the RBCs are liable to lysis, especially

when oxidizing agents are administered (drug induced hemolytic anemia).
5) Polyphenols
Consumption of polyphenol-rich fruits, vegetables and beverages is

beneficial to human health.
Dietary polyphenols represent a wide variety of compounds that occur

in fruits, vegetables, wine, tea and chocolate.
They contain flavones, isoflavones, flavonols, catechins and phenolic

acids.
Polyphenols act as agents having antioxidant, antiapoptosis, anti-aging,
anti-carcinogenic, anti-inflammatory, anti-atherosclerotic effect.
They are protective against cardiovascular diseases.
Grape polyphenols can prevent damage due to alcohol.
Oral administration of grape polyphenol extract ameliorates cerebral

ischemia induced neuronal damage.
Grape-seed procyanidins prevent low grade inflammation by modulating

cytokine expression in rats.
Damage Produced
by
Reactive Oxygen Species
Free radicals are extremely reactive.
Their mean effective radius of action is only 30 Aº.
Their half-life is only a few milliseconds.
When a free radical reacts with a normal compound,

other free radicals are generated.
This chain reaction leads to thousands of events.

Peroxidation of PUFA (poly unsaturated fatty acids) in
plasma membrane leads to loss of membrane functions.
Lipid peroxidation and consequent degradation products

such as malondialdehyde (-CHO-CH2-CHO-) are seen in
biological fluids.
Almost all biological macromolecules are damaged by the

free radicals.
Thus,
oxidation of sulfhydryl group containing enzymes,
loss of function and
fragmentation of proteins are noted.
Polysaccharides undergo degradation.
DNA is damaged by strand breaks.

The DNA damage may
directly cause inhibition of protein and enzyme

synthesis and
indirectly cause cell death or mutation and

carcinogenesis.
Clinical Significance
1) Chronic inflammation
Chronic inflammatory diseases such as rheumatoid

arthritis are self perpetuated by the free radicals released
by neutrophils.
ROS induced tissue damage appears to be involved in

pathogenesis of chronic ulcerative colitis, chronic
glomerulonephritis etc.
2) Acute inflammation
At the inflammatory site, activated macrophages

produce free radicals.
Respiratory burst and increased activity of NADPH

oxidase are seen in macrophages and neutrophils.
3) Respiratory diseases
Breathing of 100% oxygen for more than 24 hrs produces

destruction of endothelium and lung edema.
This is due to the release of free radicals by activated

neutrophils.
Respiratory diseases (continued..)
In premature newborn infants, prolonged exposure to high

oxygen concentration is responsible for bronchopulmonary
dysplasia.
Adult respiratory distress syndrome (ARDS) is

characterized by pulmonary edema. It is produced when
neutrophils are recruited to lungs which subsequently release
free radicals.
Respiratory diseases (continued..)
Cigarette smoke contains free radicals.
Soot attracts neutrophils to the site which releases more

free radicals, leading to lung damage.
4) Diseases of the Eye
Retrolental fibroplasia (retinopathy of prematurity) is a

condition seen in premature infants treated with pure
oxygen for a long time.
It is caused by free radicals, causing thromboxane

release, sustained vascular contracture and cellular injury.
Diseases of the Eye (continued..)
Cataract formation is related with aging process.
Cataract is partly due to photochemical generation of

free radicals.
Tissues of the eye, including the lens, has high

concentration of free radical scavenging enzymes.
5) Reperfusion injury
Reperfusion injury after myocardial ischemia is caused by

free radicals.
During ischemia, the activity of xanthine oxidase is

increased.
When reperfused, this causes conversion of hypoxanthine

to xanthine and superoxide anion.
Reperfusion injury (continued..)
At the same time, the availability of scavenging

enzymes is decreased, leading to aggravation of
myocardial injury.
Allopurinol, a xanthine oxidase inhibitor, reduces the

severity of reperfusion injury.
6) Atherosclerosis and Myocardial infarction
Low density lipoproteins (LDL) are deposited under

the endothelial cells, which undergo oxidation by free
radicals.
This attracts macrophages.
Macrophages are then converted into foam cells.

Atherosclerosis and Myocardial infarction
(continued…)
This initiates the atherosclerotic plaque formation.
Anti-oxidants offer some protective effect..

7) Shock related injury
Release of free radicals from phagocytes damage membranes

by lipid peroxidation.
They release leukotrienes from platelets and proteases from

macrophages.
All these factors cause increased vascular permeability,

resulting in tissue edema.
Anti-oxidants have a protective effect.

8) Skin diseases
Certain plant products, called psoalens are administered

in the treatment of psoriasis and leukoderma.
When the drug is applied over the affected skin and then
irradiated by UV light, singlet oxygen is produced with
clinical benefit.
9) Carcinogenesis and treatment
Free radicals produce DNA damage, and accumulated

damages lead to somatic mutations and malignancy.
Cancer is treated by radiotherapy.
Irradiation produces reactive oxygen species in the cells

which trigger the cell death.
10) Aging process
Reactive oxygen metabolites (ROM) play a pivotal

role in the degenerative brain disorders such as
Parkinsonism, Alzheimer’s dementia and multiple
sclerosis.
Cumulative effects of free radical injury cause gradual

deterioration in aging process.
Free radical toxicity
Lipid Peroxidation
1. Initiation Phase
Polyunsaturated fatty acids (PUFA) present in cell

membranes are easily destroyed by peroxidation.
During the initiation phase, the primary event is the
production of Rº (carbon centered radical) (PUFA
radical) or ROOº (lipid peroxide radical) by the
interaction of a PUFA molecule with free radicals
Peroxidation of polyunsaturated fatty acids (PUFA).
RH + OHº --------® Rº + H2O
metal ion
ROOH ----------® ROOº + H+
The Rº and ROOº , in turn, are degraded to malondialdehyde (3

carbon).
It is estimated as an indicator of fatty acid breakdown by

free radicals.
2. Propagation Phase
The carbon centered radical (R' ) rapidly reacts with

molecular oxygen forming a peroxyl radical (ROO' )
which can attack another polyunsaturated lipid
molecule.
Rº + O2 ® ROOº
ROOº + RH ® ROOH + Rº
This would lead to continuous production of
hydroperoxide
with consumption of PUFA.
One free radical generates another free radical in the

neighboring molecule; a "chain reaction" or
"propagation" is initiated.
This is called "death kiss" by free radicals.

3. Termination Phase
The reaction would proceed unchecked till a peroxyl radical

reacts with another peroxyl radical to form inactive
products.
ROOº + ROOº ® RO--OR + O2
Rº + Rº ® R--R
ROOº + Rº ® RO--OR
Preventive anti-oxidants:
They will inhibit the initial production of free radicals.
They are catalase, glutathione peroxidase, and ethylene

diamine tetra acetate (EDTA).
Chain breaking anti-oxidants:
They can inhibit propagative phase.
They include:
 superoxide dismutase,
 uric acid and
 vitamin E.
Alpha tocopherol (T-OH) (vitamin E)
would intercept the peroxyl free radical and inactivate it

before a PUFA can be attacked.
T-OH + ROOº ® TOº + ROOH
The tocoperoxyl radical can react with another peroxyl

radical getting converted to inactive products.
TOº + ROOº ® inactive products

Vitamin E (Alpha tocopherol) acts as the most
effective naturally occurring chain breaking anti-
oxidant in tissues.
Only traces of tocopherol is required to protect
considerable amounts of polyunsaturated fat
(1 tocopherol molecule per 1000 lipid molecules).
While acting as anti-oxidant, alpha tocopherol is

consumed.
Hence it has to be replenished by daily dietary supply.

Biomarkers of Oxidative Stress
1. Lipid peroxidation generates

Malondialdehyde (MDA) O O
Hydroxy nonenal (HNE)

Acrolein (2-propenal) O
2. Protein cleavage, crosslinking inhibition of

enzyme activity altered immunogenicity
3. 8-iso-PGF2a or F2-isoP It is most reliable

assessment for oxidative stress status.
New risk factor for coronary disease
4. Glutathione
Reduced glutathione (GSH)
Glutathione disulphide(GSSG)
5. 3-nitrotyrosine (NO2-Tyr)
Nitrated proteins in diseases
6. Carbonylated proteins (Protein carbonyls) oxidation

of amino acid side chains Lys, Arg, Pro, Thr
7. DNA breaks
8-hydroxy-deoxy-guanosine (8OHdG)
Anti-oxidants
1. Vitamin E is the lipid phase antioxidant.
2. Vitamin C is the aqueous phase antioxidant.
3. Ceruloplasmin can act as an antioxidant in extracellular fluid.
4. Caffeine
5. Cysteine, glutathione
6. vitamin A , Beta carotene

Antioxidants (MBBS Lecture)

Uploaded by

Copyright:

Available Formats

Antioxidants (MBBS Lecture)

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Antioxidants (MBBS Lecture)

Uploaded by

Copyright:

Available Formats

FREE RADICALS

Dr. Seema Lekhwani

The chemical covalent

About 1-4% of oxygen taken up in the body is converted

Mitochondrial ROS production is modulated largely by

the rate of electron flow through respiratory chain

form super-oxide anion radical or hydrogen peroxide.

Along with the activation of

In this condition, macrophages ingest bacteria normally, but cannot

However, streptococci and pneumococci themselves produce H 2O2.

Therefore, they are destroyed by the myeloperoxidase system of the

Low levels of both species function in hypoxic signaling

The mitochondrial SOD is Mn dependent;

SOD is non-heme protein.

amyotrophic lateral sclerosis

When is H2O2 generated in large quantities, the enzyme catalase is

In the next step, H2O2 is removed by glutathione peroxidase

The oxidized glutathione, in turn, is reduced by the glutathione reductase,

This NADPH is generated with the help of glucose-6-phosphate

Therefore, in GPD deficiency, the RBCs are liable to lysis, especially

Consumption of polyphenol-rich fruits, vegetables and beverages is

Dietary polyphenols represent a wide variety of compounds that occur

They contain flavones, isoflavones, flavonols, catechins and phenolic

They are protective against cardiovascular diseases.

Grape polyphenols can prevent damage due to alcohol.

Oral administration of grape polyphenol extract ameliorates cerebral

Grape-seed procyanidins prevent low grade inflammation by modulating

Their mean effective radius of action is only 30 Aº.

Their half-life is only a few milliseconds.

When a free radical reacts with a normal compound,

This chain reaction leads to thousands of events.

Lipid peroxidation and consequent degradation products

Almost all biological macromolecules are damaged by the

oxidation of sulfhydryl group containing enzymes,

loss of function and

fragmentation of proteins are noted.

Polysaccharides undergo degradation.

DNA is damaged by strand breaks.

directly cause inhibition of protein and enzyme

indirectly cause cell death or mutation and

Chronic inflammatory diseases such as rheumatoid

ROS induced tissue damage appears to be involved in

At the inflammatory site, activated macrophages

Respiratory burst and increased activity of NADPH

Breathing of 100% oxygen for more than 24 hrs produces

This is due to the release of free radicals by activated

In premature newborn infants, prolonged exposure to high

Adult respiratory distress syndrome (ARDS) is

Cigarette smoke contains free radicals.

Soot attracts neutrophils to the site which releases more

Retrolental fibroplasia (retinopathy of prematurity) is a

It is caused by free radicals, causing thromboxane

Cataract formation is related with aging process.

Cataract is partly due to photochemical generation of

Tissues of the eye, including the lens, has high

Reperfusion injury after myocardial ischemia is caused by

During ischemia, the activity of xanthine oxidase is

When reperfused, this causes conversion of hypoxanthine

At the same time, the availability of scavenging

Allopurinol, a xanthine oxidase inhibitor, reduces the