(1) Free radicals are highly reactive molecules with unpaired electrons that can cause oxidative damage. They are produced through normal metabolic processes and from environmental sources. (2) Antioxidants protect against free radical damage by neutralizing free radicals through enzymatic and non-enzymatic mechanisms. Key antioxidant enzymes include superoxide dismutase and catalase. Vitamins C and E are important non-enzymatic antioxidants. (3) Oxidative stress occurs when there is an imbalance between free radicals and antioxidants in favor of free radicals, potentially leading to cell and tissue damage associated with various diseases if left unchecked.

1. Free Radicals and
Antioxidants
DR. MANOJ ACHARYA
JUNIOR RESIDENT (1st YEAR)
DEPARTMENT OF BIOCHEMISTRY
BPKIHS
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2. OUTLINES
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ROLES OF ANTIOXIDANTS
OXIDATIVE STRESS
INTRODUCTION TO FREE RADICAL
LIPID PERIOXIDATION
SOURCES
DISEASE ASSOCIATIONS

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INTRODUCTION
A free radical can be defined as any molecular species
capable of independent existence that contains an
unpaired electron in an atomic orbital.
They can either donate an electron to or accept an electron
from other molecules, therefore behaving as oxidants or
reductants.
Are highly reactive due to unpaired electrons.
Causes generation of new radical.
Have very short lifespan
Causes damage to various tissues.
INTRODUCTION

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• A radical is an atom with one
unpaired electron.
• When a free radical is
formed it hunts other atoms
to take an electron from
them.
• This causes the creation of
another free radical, as
shown on the left and chain
of event continues often
causing destruction of parts
of the cell

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https://www.researchgate.net/figure/Involvement-of-mitochondria-in-oxidative-stress-and-
diseases_fig2_332556819

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Free radical is generally represented by a superscript dot, (R .)
The products of partial reduction of oxygen are highly reactive
and also called as reactive oxygen species.
The most important are
i. Superoxide anion radical (O2
–•)
ii. Hydroperoxyl radical (HOO• )
iii. Hydrogen peroxide (H2O2 )
iv. Hydroxyl radical (OH• )
v. Lipid peroxide radical (ROO• )
vi. Singlet oxygen ( 1O2 )
vii. Nitric oxide (NO• ) Reactive nitrogen species
viii. Peroxy nitrite (ONOO–•).

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Hydrogen peroxide and oxygen are listed to compare
with free radicals.
Superoxide anion is the precursor of all oxygen related
reactive oxygen species.

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• Sequential univalent reduction steps of oxygen is
represented as
• Superoxide radical
• Hydrogen peroxide
• Hydroxyl radical
• Water

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Common sources of free radicals in the body
Endogenous sources
Produced by body with various metabolic reactions
1.During oxidation of food stuffs, there is leakage of
electron from electron transport chain, contributing free
radicals.
2.NADPH oxidase reaction.
Explained with respiratory burst.

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Respiratory burst (RB) is a
rapid increase in the
production of reactive
oxygen species (ROS)
during the phagocytosis of
microbes.
Requires oxygen.
Note: In respiratory burst
there is synthesis of
superoxide anion

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3. Xanthine oxidase reaction
In purine degradation hypoxanthine, xanthine are
converted to uric acid with help of xanthine oxidase and in
this reaction there is production of ROS like form
superoxide anion radical or hydrogen peroxide.
4. Prostaglandin synthesis in platelets and leucocyte
during lipooxygenase pathway is also a source of
synthesis of ROS.
5. Synthesis of nitric oxide from arginine
6. Autooxidation of metals (eg metal ions,)

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7. Lipid perioxidation
Polyunsaturated fatty acids (PUFA) present in cell
membranes are easily destroyed by peroxidation.
1.Initiation phase
RH + OH• ------→ R• + H2O
PUFA reacts with hydroxyl radical and forms R•
(carbon centered radical) or PUFA radical.

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2. Propagation phase
R• + O2 → ROO•
carbon centered radical (R• ) rapidly reacts with
molecular oxygen forming a peroxide radical (ROO• )
which can attack another polyunsaturated lipid molecule
Has tendency to combine with another PUFA.
ROO• + RH → ROOH + R•
The net result of reactions is the conversion to ROOH (a
hydroperoxide)
This propagation phase do not get stopped until
PUFA gets exhausted.

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3. Termination phase
The reaction would proceed unchecked till a peroxyl radical
reacts with another peroxyl radical to form inactive products.
ROO• + ROO• → RO--OR + O2
These are the process from where free radicals are
produced from lipid perioxidation

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Iron produces free radicals

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• Exogenous sources

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Free radicals and diseases
• Cardiovascular disease
Oxidized low density lipoproteins, formed by the action of free
radicals, promotes atherosclerosis and CAD
Increased LPL is converted to oxidized LDL by free
radical attracts the macrophages which are
converted into foam cells leading to formation of
plaques.
• Aging process and neurological involvement
Overproduction of free radicals can cause oxidative damage
to biomolecules (lipids, proteins, DNA), eventually leading to
neurodegeneration.
Free radicals play vital role in normal aging process
Diseases like Parkinson’s disease, alzheimer’s and multiple
sclerosis.

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Cancer
Free radicals can damage DNA and nucleic acid cause
mutagenicity and cytotoxicity,
ROS can induce mutations, and inhibit DNA repair process,
resulting in inactivation of certain tumor suppressor genes
leading cancer.
Free radicals also promote biochemical and molecular
changes for rapid growth of tumor cells.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698794/

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Respiratory Distress
Direct exposure to lungs with 100% O2 for longer period, known
to destroy lungs endothelium which ultimately causes lung
edema.
This is mediated by ROS
Cigeratte smoke, contain free radicals also promotes the
generation of free radical
Diabetes Mellitus
Free radicals affects or destroys islets of pancreas after it
accumulates.
Responsible for IDDM
https://pubmed.ncbi.nlm.nih.gov/15111505/

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Antioxidants
Antioxidants are the compounds that protect the body
against the toxic effect of free radicals.
The protective mechanisms of antioxidants serve to
scavenge (remove) the free radicals.

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Action of antioxidants
Different antioxidants acts at different level
They may prevent initiation of chain reactions by removing
free radicals. (Preventive antioxidants)
They may scavenge free radicals generated in chain
reactions, thereby interrupting the chain sequence (chain
breaking)

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Enzymatic antioxidant system
Following antioxidant enzyme destroy the superoxide
radical and H2O2
The protective action of these enzymes need not be
independent.
They may function cooperatively.
Superoxide dismutase (chain breaking Antioxidant)
Catalase
Preventive antioxidant
Glutathione peroxidase

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Vitamins as antioxidants
Few vitamins have antioxidant property and help in
detoxification of free radical. These vitamins are:
Tocopherol (vitamin E) – most powerful chain breaking
antioxidant
Beta carotenes (vitamin A)
Ascorbic acid (Vitamin C)

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Vitamin E
Biological system contains alfa, beta, gamma and sigma tocopherol
where alfa is most potent antioxidant and acts as a chain breaking
antioxidant.
Hydroxyl group is attached to alfa tocopherol whose hydrogen is easy
to remove,
So when peroxyl radicals are generated in lipid perioxidation they will
combine to antioxidant rather to adjacent fatty acids.
Reduced form of vitamin E (EH) can break the chain process by
reacting with lipid peroxide radical and itself forming a free radical ,
tocopheroxyl radical E.

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Vitamin C is able to generate vitamin E from E. permitting
the vitamin E once more to act as an antioxidant.
Alfa tocopherol radical can migrate to the membrane surface
and be reoxidized to alfa tocopherol by dehydroascorbic
acid.
Vitamin C is also able to reduce and detoxify oxygen
intermediates in cells.

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Minerals as antioxidants
The activity of the antioxidants enzymes depends on
supply of minerals.
Manganese
Copper
Zinc
Selenium
Manganese, copper and zinc are required for the
activity of superoxide dismutase
Selenium is required for the activity of glutathione
peroxidase.

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Interrelationship between antioxidant system.
The role of metabolism in the antioxidant activity of vitamin E, and its
synergism with vitamin C, reduced glutathione, NADPH, and cellular
electron transport proteins

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Ergothioneine Histidine-Derived, Biologically Significant
antioxidants
- Robert D. Short, Jr. and Steven /. Baskin
Ergothioneine (ET) is the trimethylbetaine of 2-thiolhistidine (2-thiol-L-
histidine betaine) and it exists in a variety of biological systems
was initially isolated from the ergot fungus Claviceps purpurea in 1909.
oldest of the sulfur-containing alkaloids is ergothioneine
The most important role is regarded as an antioxidant , controlling reactive
oxygen species (ROS) and reactive nitrogen species (RNS), such as peroxy,
hydroxyl, and peroxynitrite
Because can pass through the blood–brain barrier (BBB), the antioxidant
activity may serve to provide protection from neurodegeneration

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The Reduction of Ferryl Myoglobin by Ergothioneine: A Novel
Function for Ergothioneine
Arduino Arduini, Lynne Eddy, and Paul Hochstein’ Institute for Toxicology, University of Southern
California, Los Angeles, California 90033
The oxidative state of myoglobin may be a critical event in the tissue
damage associated with cardiac ischemia/reperfusion states
In this paper, demonstrated that ergothioneine (ET), a naturally
occurring thiolhistidine, reduces ferrylmyoglobin. The reduction of
ferrylmyoglobin by ET yields the disulflde of ET which the addition of
GSH promptly reduces back to ET. The further addition of ET in the
perfusion buffer of Langendorff rat heart preparations exposed to a
brief period of ischemia prevents the myocardial damage (lactate
dehydrogenase release) which accompanies reperfusion. The results of
these experiments support a view that ET and its redox couple GSH
might function in a Mb redox cycle.
https://pubmed.ncbi.nlm.nih.gov/2383023/

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Antioxidants can be prooxidants called as
antioxidant paradox
Ascorbate, can also be a source of superoxide radicals by
reaction with oxygen, and hydroxyl radicals by reaction
with Cu2+ ions.
However, these pro-oxidant actions require relatively high
concentrations of ascorbate, which are unlikely to be
reached in tissues.

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Beta carotene is indeed a radical trapping antioxidant
under conditions of low partial pressure of oxygen, as in
most tissues,
at high partial pressure of oxygen (as in lungs)
and especially in high concentrations, beta carotene
is an autocatalytic prooxidant and hence can
damage to lipid and protein

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Other nutrient antioxidants
Coenzyme Q10 of ubiquinone
Curcuminoids of turmeric
Proanthocyanidins of grape seeds
Catechins in tea
Quercetin of onions.

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Other metabolic antioxidants
Uric acid – a powerful scavenger of singlet oxygen 1O2 and
OH- radicals.
Ceruloplasmin – inhibits iron and copper dependent lipid
perioxidation
Transferrin - binds to iron and prevents iron catalyzed
free radical formation.
Albumin - can scavenge the free radicals formed on its
surface
Bilirubin – protects the albumin bound free fatty acids from
peroxidation

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Oxidative Stress
Oxidative stress reflects an
- imbalance between the systemic manifestation of
reactive oxygen species and a biological system's
ability to readily detoxify the reactive intermediates or
to repair the resulting damage.

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FRs as tipping the balance toward disease, so that as FRs
increase along the up slope as a function of progressive
disease, antioxidants (AOXs) decrease along the down slope
as they are being consumed due to oxidative stress, so that
homeostasis tips toward disease

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Biomarkers of oxidative stress
Biomarkers of oxidative stress can be analyzed in cells,
tissues, blood, urine, CSF, synovial fluid, saliva, tears etc.
8 hydroxy deoxyguanosine – major product of DNA oxidation.
Polyethyl glycol conjugated SOD - is effective than SOD in scavenging free
radicals in injuries
4-Hydroxy-2-nonenal (HNE) - products of phospholipid peroxidation, owing to
its reactivity and cytotoxicity. It can be formed by several radical-dependent
oxidative processes
Thiobarbituric acid reactive substances (TBARS) byproduct of lipid peroxidation
CUPRAC (CUPric Reducing Antioxidant Capacity) method of antioxidant
measurement

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Detection of free radicals
ELECTRON spin resonance (ESR) spectroscopy provides
the best readily available method for detecting free radicals
in reacting systems
Detects unpaired electron
Xylenol orange assay
TBARS assay:
Malondialdehyde end product of lipid peroxidation react
with thiobarbituric acid under acid pH and elevated temp to
form red fluorescent
Drawback: Malondialdehyde is not only end aldehyde
product of lipid peroxidation and TBARS is not specific for
malondialdehyde.

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Are antioxidants helpful for disease prevention?
V. Hajhashemi,1,* G. Vaseghi,1 M. Pourfarzam,2 and A.
Abdollahi3
2010 Jan-Jun PMCID: PMC3093095 PMID: 21589762
At moderate concentration free radicals and radical-derived
ROS play an important role as regulatory mediators in
signaling processes
Many of the ROS-mediated responses in fact protect the cells
against oxidative stress and reestablish “redox homeostasis”.
At high concentrations however, free radicals and radical-
derived, non-radical reactive species are hazardous for living
organisms and harm all major cellular constituents.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093095/

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Study was done in diseases with atherosclerosis,
Alzheimer's disease, cancer, ocular disease, diabetes,
rheumatoid arthritis and motor neuron disease
Among the antioxidant dietary supplements such as
vitamin E, vitamin C and beta-carotene are widely used.
However, the results of clinical trials was inconsistent

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Findings and conclusion of study
Findings of latest large scale randomized clinical trials
indicate that neither vitamin E nor vitamin C supplementation
reduces risk of major cardiovascular events in middle-aged
and older men.
Similarly antioxidant supplementation, particularly with
vitamin E, vitamin C and beta-carotene does not reduce
primary cancer incidence or cancer mortality.
Beta-carotene supplementation might even increase the risk
of smoking-related cancers, as well as cancer mortality, and
should be avoided by smokers
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6636175/

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The data also indicate that neither Vitamin E nor beta-
carotene supplementation affects overall incidence of
cataract or cataract extraction
Accordingly, it is difficult to assess how antioxidant
supplements may affect disease prevention or mortality in
populations with specific needs or insufficiency in
micronutrients.

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References
• Marshall_Clinical Biochemistry Metabolic and Clinical
Aspects
• Oxidants, antioxidants, and free radicals/edited by
Steven I. Baskin and Harry Salem,
• Harpers Illustrated Biochemistry - McGraw Hill
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6636175/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3093095/
• https://pubmed.ncbi.nlm.nih.gov/15111505/
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698794/
• https://www.researchgate.net/figure/Involvement-of-
mitochondria-in-oxidative-stress-and-diseases_fig2_332556819
• Textbook of Biochemistry for Medical Students
(D. M. V, S.S )

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