Free Radicals

@
Antioxidants
 FREE RADICALS

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• Contain single unpaired electron in an outer orbit

• Oxygen free radicals (=Reactive Oxygen Species = (ROS);
Partially reduced, reactive forms of Oxygen
• ROS;Superoxide radical, Hydroperoxyl radical, Hydroxyl
radical, Hydrogen peroxide,Hypochlorous
• Reactive Nitrogen Species; Nitric Oxide (= NO), Peroxynitrite
• The most damaging radicals in biological systems

• Cause Oxidative tissue damage

 ANTIOXIDANTS

• Protect tissues against free radical damage

 www.pubmed.com
FREE RADICAL @ DISEASE ; 71821 Results
• Free radicals cause damage to Nucleic acids, Proteins,
Membrane lipids, Plasma lipoproteins

• This damage can cause Cancer, Atherosclerosis, Coronary

artery disease, Autoimmune diseases
 www.pubmed.com
FREE RADICAL @ ANTIOXIDANTS ; 182790

• Antioxidant nutrients: Selenium, Vitamins C and E,

β-Carotene, Polyphenolic plant compounds
• Many people take one or more antioxidant supplements
• However, intervention trials show little benefit of
antioxidant supplements except among people who were
initially deficient
• Many trials of β-carotene and Vitamin E have shown
increased mortality among those taking the supplements
• About 90% of our O usage is committed to ETC
2

• Enzymes that use O for hydroxylation and oxygenation

2

reactions consume another 10%

• <1%, is converted to ROS
• Mitochondria is considered the major source of ROS where
most oxidative metabolism occurs
 ROS

Medical Biochemistry, Baynes JW, Dominiczak MH

• Reactive, strongly oxidizing forms of oxygen so;

• Source of chronic damage to tissue biomolecules
 IN PEROXISOMES

• Some enzymes use H2O2 as a substrate so are important in

normal metabolism
• During Oxidation of Very long chain fatty acids
H2O2 is produced
Hydroxyl Radicals Formation from Hydrogen peroxide by
Fenton and Haber–Weiss reactions

• Hydroxyl radical (OH•) is the most reactive and

damaging ROS
• Iron and Copper ions catalyze the reaction
• Under physiologic conditions,Haber–Weiss reaction is
catalyzed by redox-active metal ions such as Fe or Cu
 The most reactive and damaging ROS
‘’Hydroxyl radicals’’
• Hydroxyl radicals reacts with biomolecules primarily by
Hydrogen abstraction and addition reactions
• Cell membranes are rich in Polyunsaturated fatty acids
(PUFA) so;
• One of the most sensitive sites of free radical damage
 Peroxidative damage to plasma membrane affects

• Integrity and function of the membrane

• Damage ion gradients and membrane phospholipid
structure
When OH• abstracts a Hydrogen atom from PUFA

• Initiates Lipid peroxidation

reaction
• Produces Secondary
oxidation products such as;
• Lipid peroxides, and lipid
peroxyl radicals
When OH• abstracts a Hydrogen atom from PUFA

• Lipid oxidation products

degrade
• Form reactive carbonyl
compounds, such as;
• Malondialdehyde (MDA) and
Hydroxynonenal (HNE)
When OH• abstracts a Hydrogen atom from PUFA

• MDA and HNE react with

proteins
• Form adducts and crosslinks,
known as Advanced
Lipoxidation End products
(ALE)
• ALE are biomarkers of
Oxidative stress
• This reaction continues until
PUFA supply is exhausted,
unless a termination reaction
occurs
• Vitamin E is the major chain-
terminating antioxidant in
membranes
• Reduces both conjugated
Dienyl and Hydroperoxyl
radicals, quenching chain or
cycle of lipid peroxidation
reactions
• Lipid peroxides may also be
reduced by Glutathione
peroxidase (GPx), forming inert
lipid alcohols
MDA and HNE adducts to Lysine residues are
Icreased in

• Lipoproteins in plasma and vascular wall in atherosclerosis

• Amyloid plaque in Alzheimer’s disease, implicating oxidative
stress and damage in the pathogenesis of these diseases
 ROS also react with Carbohydrates

• Form Reactive Carbonyl compounds that react with Proteins

• Form Advanced Glycation End products (AGE)
• AGE and ALE increase in tissue proteins in Diabetes as a
result of Hyperglycemia and Oxidative stress
• The increase in chemical modification of proteins by AGE
and ALE is implicated in development of Diabetic vascular,
renal, and retinal complications
Oxidized LDL Atherosclerosis
• Nitric Oxide Synthases (NOS) catalyze production of
Nitric Oxide free radical (NO ) from L-Arginine
•
ISOENYMES OF NITRIC OXIDE SYNTHASE

• nNOS in neuronal tissue, where NO• serves as

Neurotransmitter
• iNOS in immune system, where NO• is involved in regulation
of immune response
• eNOS in endothelial cells, where NO•, known as
Endothelium Derived Relaxation Factor (EDRF), has a role in
regulation of vascular tone
In a side reaction at inflammation sites
• NO reacts with Super oxide radical to form Peroxynitrite
•

(ONOO )
−

• Peroxynitrite produce Nitrosative stress by reaction with

biomolecules
• ONOOH has many of the strong oxidizing properties of OH •

but has a longer biological half-life

• It is also a potent nitrating agent
• Produces Nitrotyrosine in proteins, Nitrated phospholipids
in membranes, and nucleotides in DNA
MARKERS OF FREE RADICAL DAMAGE
ANTIOXIDANT DEFENSES
 There are several levels of
protection against oxidative damage
• ROS damage to lipids and proteins is repaired largely by
degradation and resynthesis
• Oxidized proteins are preferred targets for proteasomal
degradation
• Damaged DNA is repaired by a number of excision-repair
mechanisms but
• The process is not perfect
• Some proteins, such as Collagens and Crystallins
turn over slowly,
• So damage accumulates
• Function may be impaired
• Age dependent browning and precipitation of lens
proteins leads to cataracts
• Crosslinking of Collagen and Elastin cause loss of
elasticity
• Cause changes in permeability of vascular wall and
renal basement membrane
• The association between Chronic inflammation and Cancer
indicates that
• Chronic exposure to ROS causes cumulative damage to the
genome in the form of nonlethal mutations in DNA
 First line of defense against ROS damage
‘’Sequestration or chelation of redox-active metal ions’’
• A number of metal-binding proteins that sequester Fe and
Cu in inactive form
• Transferrin transports Fe
• Ferritin stores Fe
• Plasma protein Haptoglobin binds to hemoglobin from
ruptured red blood cells and delivers hemoglobin to liver
• Plasma Hemopexin binds Heme and delivers it to liver
• Albumin has a strong binding site for Cu and effectively
inhibits copper-catalyzed oxidation reactions in plasma
 Carnosine (β-Alanyl-L-Histidine)

• Present in Muscle and Brain

• Potent copper chelators
• May have a role in intracellular antioxidant protection
• Despite potent metal chelation systems, ROS are formed
continuously in body, both by enzymes and by spontaneous
metal-catalyzed reactions
 ANTIOXIDANT ENZYMES

• Provides enzymatic defence against ROS

• SOD= Super Oxide Dismutase
• CAT=Catalase
• GPx= Glutathione Peroxidase
• GR= Glutathione Reductase
• GSH= Reduced Glutathione
• SOD converts Superoxide radical to less toxic H2O2
• Mn SOD isozyme is found in Mitochondria
• Cu,Zn SOD isozyme is found in Cytosol
• EC SOD (Extra Cellular) isoenzyme contains Cu and Zn and
binds to proteoglycans in vascular wall and is thought to
protect against O and ONOO injury
2• −
 CATALASE

• Inactivates H O
2 2

• Found largely in Peroxisomes, the major site of H O

2 2

generation in the cell

 GLUTATHIONE

• Important antioxidant tripeptide

• Present in all cells
 GLUTATHIONE PEROXIDASE

• Widely distributed in cell

• Contains Selenium
• Reduces H2O2 and lipid hydroperoxides to water and a lipid
alcohol, respectively, using Reduced Glutathione (GSH)
• GSH is recycled by Glutathione Reductase (GR) using NADPH
from Pentose phosphate pathway
 The third defense line
Against Oxidative damage

• Provided by Antioxidant Vitamins, A, C, and E,

• Vitamin C (=Ascorbate) in the aqueous phase and Vitamin E
(= α- and γ-Tocopherol) in the lipid phase, act as Lipid
Peroxidation Chain breaking antioxidants
• During Antioxidant action of Vitamin E , radical form of
Vitamin (= Tocopheryl radical) is produced
• Vitamin E is recycled by Vitamin C (= Ascorbate )
• Oxidized Vitamin C (= Dehydro ascorbate ) is recycled by
Glutathione reductase
• Vitamin A (=Carotene) is also a lipophilic antioxidant and
protects against damage from sunlight in retina and skin
• Antioxidants work together to inhibit lipid peroxidation
reactions in plasma lipoproteins and membranes
 S- Glutathionylation of Proteins
(Reaction of Glutathione with SH group of Proteins)
Provides protection against ROS

• When Proteins are exposed to ROS

• Sulfhydryl groups of Proteins are oxidized to Sulfenic acid
• Sulfenic acid reacts with another Sulphydryl group of protein
• Forms a crosslinked protein
• Crosslinking reactions may be reversed by S-
Glutathionylation of proteins
• Native protein with free sulfhydryl groups is regenerated
• During Oxidative stress, there is a significant increase in S-
Glutathionylated proteins in the cell
• S-Glutathionylation is reversed by nonenzymatic reduction
by GSH or by enzymes using thiol protein cofactors
(Thioredoxin, Glutaredoxin)
• This pathway inhibits the formation of crosslinked protein
aggregates, such as Heinz bodies
 Heinz Bodies

• Hemoglobin precipitates that develop in RBC in Glucose- 6-

phosphate dehydrogenase deficiency
• Characterized by decreased levels of GSH
BENEFICIAL EFFECTS OF ROS
 ROS are essential for
Many metabolic and Signaling pathways
• Regulatory functions of NO ( Vasodilator effect )
• Role of ROS in activation of Antioxidant Response Element (=
Enhancer sequence that mediates transcriptional activation of
genes in cells exposed to oxidative stress)
• Requirement for ROS in bactericidal activity of macrophages
• Use of ROS as substrates for enzymes ( H O for Hemeperoxidases
2 2

involved in iodination of Thyroid hormone)

• There is also increasing evidence that ROS, particularly H2O2, are
important signaling molecules involved in the regulation of
metabolism
 Generation and release of ROS
During Phagocytosis

• A cascade of reactions generating ROS is initiated during

phagocytosis to kill invading organisms
• Hydrolytic enzymes are also released from lysosomes to
assist in degradation of microbial debris
 RESPIRATORY BURST IN MACROPHAGES

• Consumption of O2 by NADPH oxidase is responsible for

“Respiratory burst”
• NADPH oxidase is activated to produce Superoxide radical
• Superoxide radical is then converted to Hydrogen peroxide
by SOD
• Hydrogen peroxide is used by Myeloperoxidase (MPO), to
oxidize Chloride ion to Hypochlorous acid (HOCl)
• H O and HOCl mediate Bactericidal activity by Oxidation and
2 2

Chlorination of microbial lipids, proteins, and DNA

• HOCl, is also active oxidizer in Chlorine containing laundry
bleaches
• i.v. infusion of dilute HOCl solutions was used for the
treatment of bacterial sepsis in battlefield hospitals during
World War I, before the advent of penicillin and other
antibiotics
• Chronic granulomatous disease is an inherited disease
resulting from a genetic defect in NADPH oxidase
• Inability to produce Superoxide radicals leads to Chronic life-
threatening bacterial and fungal infections
 ANTIOXIDANT DEFENSES IN
Red Blood Cells
• RBC does not use Oxygen for metabolism, nor is it involved
in phagocytosis
• Because of the high Oxygentension in arterial blood and
heme iron content of RBC;
• ROS are formed continuously in RBC
• Hemoglobin spontaneously produces superoxide radical in a
minor side reaction associated with binding of Oxygen
• Occasional reduction of Oxygen to Superoxide radical is
accompanied by oxidation of normal (Ferro) Hb to
Methemoglobin (Ferrihemoglobin)
• Methemoglobin is a rust-brown protein that does not bind
or transport Oxygen
• Normally, less than 1% of Hb is present as Methemoglobin
 ANTIOXIDANT DEFENSES IN
Red Blood Cells
• Methemoglobin may release Heme, which reacts with O2•
and H2O2 in Fenton-type reactions
• Produces Hydroxyl radical and reactive iron-oxo species
• These ROS initiate Lipid peroxidation reactions
• Lead to loss of membrane integrity and cell death
 Congenital Methemoglobinemia

• Results from Methemoglobin reductase deficiency

• Typically have a dark and cyanotic appearance
• Chocolate blood color, Chocolate cyanosis
• Treatment; Large doses of Vitamin C is used to reduce
Methemoglobin to Hemoglobin
 RBC is rich in Antioxidant molecules

• Catalase
• Superoxide dismutase
• Glutathione peroxidase
• Methemoglobin reductase( Reduces Methemoglobin back
to normal Hemoglobin)
• Reduced Glutathione
 ISCHEMIA/REPERFUSION INJURY
• Ischemia, meaning limited blood flow, is a condition
in which a tissue is deprived of oxygen and
nutrients
• Damage to heart tissue during a myocardial
infarction occurs not during Hypoxic or Ischemic
phase but during Reoxygenation of heart tissue
• This type of damage also occurs after
transplantation and cardiovascular surgery
 ROS are thought to play a major role in
Reperfusion injury

• When cells are deprived of oxygen

• Must rely on Anaerobic glycolysis
and Glycogen stores for ATP
synthesis
• NADH and Lactic acid accumulate
• All components of ETC are
saturated with electrons
• Electrons cannot be transferred to
Oxygen
• Mitochondrial membrane
potential is increased
(Hyperpolarized)
 ROS are thought to play a major role in
Reperfusion injury

• When oxygen is reintroduced

• Great quantities of ROS are rapidly
produced
• Overwhelms antioxidant defenses
• ROS flood throughout the cell
• Damage membrane lipids, DNA,
and other vital cellular
constituents
• Lead to necrosis