Free Radicals and Antioxidants

Free Radical Generation and

Defense Mechanism
 Questions asked
• What are free radicals?
• Types of free radicals
• Sources of free radicals
• Oxygen metabolism
 What are free
radicals?
• Any molecule containing
one or more unpaired
electrons.
• These unpaired electrons
readily form free radical
molecules which are
chemically reactive and
highly unstable.
Types of free radicals
1. Superoxide, O2-
2. Hydrogen peroxide, H2O2
3. Hydroxyl radical, OH-
4. Singlet oxygen, 1O2
5. Hydroperoxy radical, HOO-
6. Lipid peroxide radical, ROO-
7. Nitric oxide, NO-
8. peroxynitrite, ONOO-
 e- e-, 2H+ e-, H+ e-, H+
O2 O2- H2O2 OH- H2O
Superoxide Hydrogen H2O Hydroxyl
peroxide radical
 Properties of free radicals
1. Highly reactive
2. Very short half-life
3. Generate new radicals by chain reaction
4. Cause damage to biomolecules, cells and tissues

Most free radicals in biological systems are

derivatives of oxygen (Reactive Oxygen Species,
ROS), but there are also derivatives of nitrogen
(Reactive Nitrogen Species, RNS), Reactive
Metabolites or Intermediates.
Reactive Oxygen Species (ROS)

• Superoxide (O2.-)
• Hydrogen Peroxide (H2O2)
• Hydroxyl Radical (OH.)
• Singlet oxygen, 1O2

• Reactive Oxygen Species is used in a broad

sense to collectively free radicals (O2.-, OH.)
and non-free radicals (H2O2, 1O2, which are
extremely reactive) of the biological system.
 Generation of Free Radical

1. Cellular metabolism
• About 1-4% of oxygen taken up in the body is converted to
free radicals. They are constantly produced during the normal
oxidation of foodstuffs.
a) due to leaks in the electron transport chain in mitochondria.
b) Some enzymes such as xanthine oxidase and aldehyde
oxidase form superoxide anion radical or hydrogen peroxide.
c) Macrophage also produces NO from arginine by the enzyme
nitric oxide synthase. This is also an important anti-bacterial
mechanism.
 Sources of oxygen free radicals
• In mitochondria:
- generation of energy - ATP
- glucose, fatty acids, amino acids
- O2 2H2O
4e-+4H+
- leakage of O2-. (superoxide)
H2O2 (hydrogen peroxide)
 Generation of Free Radical

2. Environmental effects:
a) due to drug metabolism.
b) due to damages caused by UV or X-rays
c) cigarette or alcohol.
 FREE RADICAL FREE RADICALS : THE CAUSE
OF VIRTUALLY ALL

FORMATION DISEASES
Pesticides and herbicides
Industrial pollution

Excessive
Alcohol &
smoking

Environmental pollution

High fat
foods
 Harmful effects of free radicals
A. Free Radical and biomolecules
1. Proteins
• Cause oxidation of sulfhydryl groups, and modification
of AA. ROS may damage protein by fragmentation,
aggregation results in the loss of biological activity of
proteins.
2. Lipids
• The polyunsaturated lipid molecules of cell
membranes are particularly susceptible to damaging
free radicals process and contribute to the
uncontrolled chain reaction (lipid peroxidation).
Lipid peroxidation
• Lipid peroxidation refers
to the oxidative
degradation of lipids.
• It is the process whereby
free radicals "steal"
electrons from the lipids in
cell membranes, resulting
in cell damage.
• This process proceeds by
a free radical chain
reaction mechanism. It
most often affects
polyunsaturated fatty
acids(PUFA).
• In addition, end products
of lipid peroxidation may
be mutagenic and
carcinogenic
 Harmful effects of free radicals
A. Free Radical and biomolecules

3. Carbohydrates
• Glycation increases the susceptibility of
carbohydrates to the attack by free radicals.

4. Nucleic acid
• cause DNA strand breaks, fragmentation of bases
and deoxyribose results in cytotoxicity and
mutations.
 Oxidative Damage

Free Radicals

Proteins Lipids DNA/RNA

(-SH) (R-OO.) (-OH.)
 Harmful effects of free radicals
B. Diseases
1. Cardiovascular diseases (CHD): ox-LDL, formed
by the action of free radicals, promote CHD and
atherosclerosis (AS).
2. Cancers: damage DNA and cause mutation and
cytotoxicity, play a key role in carcinogenesis.
3. Inflammatory diseases: damage on the extracellular
components such as collagen and hyaluronic acid, promote
glomerulonephritis and ulcerative colitis.
4. Respiratory diseases: destroy endothelium and
cause lung edema. Cigarette smoke contains free radicals
and promotes the production of more free radicals.
• Macrophage take up oxidized LDL, when
overload with lipid, become “foam cells”.
• Conglomerate of foam cells form fatty
streaks or yellow patches visible in the
arterial wall.
• Dying foam cells release lipid that form
lipid pool within the arterial wall.
 Harmful effects of free radicals
B. Free Radical and diseases
5. Diabetes mellitus: Destruction of islets results in
pathogenesis.
6. Cataract
7. Male infertility: reduce sperm motility and
viability.
8. Aging process
9. Others: such as Parkingson’s disease, Alzheimer’s
disease, multiple sclerosis, liver cirrhosis, muscular
dystrophy.
 Antioxidant
• The substance present in low concentrations
relative to the oxidizable substrate that
significantly delays or reduces oxidation of the
substrate.
• They reduce the effect of dangerous oxidants by
binding together with these harmful molecules,
decreasing their destructive power.
• They can also help repair damage already sustained
by cells.
• They may be considered as the scavengers of free
radicals.
• During this reaction the antioxidant sacrifices
itself by becoming oxidized.
• However, antioxidant supply is not unlimited as
one antioxidant molecule can only react with a
single free radical. Therefore, there is a constant
need to replenish antioxidant resources.
 Antioxidants

• Prevents the transfer of electron

from O2 to organic molecules
• Stabilizes free radicals
• Terminates free radical reactions
 Classification of antioxidant
Ⅰ. According to their location
a) Plasma antioxidants:
– ascorbic acid (Vitamin C), bilirubin, uric
acid, transferrin, ceruloplasmin, β-
carotene;
b) Cell membrane antioxidants:
– α-tocopherol (Vitamin E)
c) Intracellular antioxidants:
– superoxide dismutase (SOD), catalase,
glutathione peroxidase (GPx)
 Classification of antioxidant
Ⅱ. According to their nature and action
a) Enzymatic antioxidants:
– SOD, catalase, GPx, glutathione reductase
b) Non-enzymatic antioxidants:
– Nutrient antioxidants:
• β-carotene, α-tocopherol, ascorbic acid,
– Metabolic antioxidants:
• bilirubin, uric acid, ceruloplasmin, ferritin,
transferrin, albumin, glutathione
 Enzymatic antioxidant
1. superoxide dismutase (SOD)
SOD
2O2·⁻+ 2H+
H2O2 + O2

SOD is present in essentially every cell in the body

which actually represented by a group of metalloenzymes
with various prosthetic groups. SOD appears in three
forms:
a) Cu-Zn SOD: in the cytoplasm with two subunits
b) Mn-SOD: in the mitochondrion
c) Cu-SOD: extracellular SOD
This is the first line of defence to protect cells
from the injurious effects of superoxide.
 Enzymatic antioxidant
2. catalase, CAT

2H2O2 catalase 2H2O + O2 

Catalase, iron dependent

enzyme, is present in all body
organs being especially
concentrated in the liver and
erythrocytes. The brain, heart
and skeletal muscle contains
only low amounts.
 Enzymatic antioxidant
3. glutathione peroxidase, GPx

GPx is a selenium-dependent enzyme.

The entire process is driven by energy production at
the cellular level, which involves proper thyroid hormone
levels, healthy mitochondrial function, and an active
pentose-phosphate metabolic pathway.
 Nutrient antioxidant
1. α-tocopherol (vitamin E)

•The most important lipid-soluble antioxidant

•Present in all cellular membranes.
•Protect against lipid peroxidation.
1. Vitamin E was shown to be stored in adipose
tissue.
2. Vitamin E prevents the peroxidation of
membrane phospholipids and avoids cell
membrane damage through its antioxidant
action.
 Nutrient antioxidant
2. ascorbic acid (vitamin C)

+ 2O2·⁻ + 2H+ H2O2 +

Dehydroascorbate, DHA
It is a water-soluble, antioxidant present in citrus
fruits, potatoes, tomatoes and green leafy vegetables.
It is a chain breaking antioxidant as a reducing agent
or electron donor. It scavenges free radicals and inhibits
lipid peroxidation. It also promotes the regeneration of α-
tocopherol.
 Nutrient antioxidant
3. carotenoids

Carotenoids consist of C40 chains with conjugated

double bonds, they show strong light absorption and often
are brightly colored (red, orange). They occur as pigments
in bacteria, algae and higher plants.
 β-carotene is the most important.
• It is composed of two molecules of vitamin A
(retinol) joined together.
• Dietary β-carotene is converted to retinol at
the level of the intestinal mucosa. It can quench
singlet oxygen.
– Quenching of singlet oxygen is the basis for
it's well known therapeutic efficacy in
erythropoietic protoporphyria (a
photosensitivity disorder).
Lycopene is responsible for color of certain
fruits and vegetables like tomato. It also
possesses antioxidant property.
 Nutrient antioxidant
4. α-lipoic acids

• It is vitamin-like compound, produced in the

body, besides the supply from plant and animal
sources.
• It plays a key role in recycling other
important antioxidants such as ascorbic acid, α-
tocopherol and glutathione.
 • Other important nutrient antioxidants

Antioxidant Source
1.Coenzyme Q10 organ meats (best heart), beef, chicken

2. Selenium sea foods, meats, whole grains

3. Proanthocyanidins grape seeds
4. Catechins green tea
5. Quercetin onions, red wine, green tea
6. Ellagic acid berries, walnuts, pomegranates
 Metabolic antioxidant
1. glutathione, GSH

In addition to its role as a substrate in GSH

redox cycle, GSH is also a scavenger of hydroxyl
radicals and singlet oxygen. GSH also has an
important role in xenobiotic metabolism.
 Metabolic antioxidant

1. Uric acid scavenge singlet oxygen and hydroxy radical

2. Ceruloplasmin inhibit iron and copper dependent lipid peroxidation
3. Transferrin prevents iron-catalyzed radical formation
4. Albumin scavenge radicals on its surface
5. Bilirubin protects albumin bound FFA from peroxidation
6. Haptoglobin bind to free Hb and prevent the acceleration
of lipid peroxidation
  

Normally, cellular homeostasis is a delicate balance

between the production of free radicals and our
antioxidant defenses. 
GREEN TEA – A WONDERFUL
ANTIOXIDANT
Primarily consumed in China, Japan,
Middle East, North Africa & North America
- Rich in polyphenol – an antioxidant

• 200 times more powerful than Vitamin E

• Scavenges free radicals, high rate
• Reduce the risk of heart diseases
• Lowers LDL oxidation
• Prevents Red blood Cell breakdown
• Protects against digestive & respiratory
infections
• Prevents cancers of colon, pancreas &
stomach
 Notice: Countering the Harmful
Effects of Free Radicals
• Don’t smoke – if you do then make it a point to quit
• Don't overdo your exposure to the sun
• Don’t over consume alcohol
• Don’t consume foods containing trans fats or
hydrogenated oils
• Get your cardio exercise from sprinting or interval
training
• Do consume antioxidant rich foods and use a good
antioxidant formula
• Keep your stress levels down
 Points
• Free radicals
– Definition, types and Properties
– ROS, RNS
– Generation of Free Radical
– Harmful effects of free radicals
• Antioxidant
– Enzymatic antioxidants
• SOD, catalase, GPx, glutathione reductase
– Non-enzymatic antioxidants
• Nutrient antioxidants: β-carotene, α-tocopherol,
ascorbic acid,
• Metabolic antioxidants: glutathione (GSH)