View
4.958
Download
5
Category
Preview:
DESCRIPTION
Lecture on Free Radicals
Citation preview
Free radicals and
antioxidants in health and disease
• Electrons in an atom or molecule occupy orbits
• The outer most orbital 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
• A free radical is a molecule or molecular fragment that
contains one or more unpaired electrons in its outer orbital
• Free radical is conventionally represented by a superscript
dot (R●)
Introduction
• Aerobic organisms produce a number of reactive free radicals continuously in cells during respiration, metabolism and phagocytosis.
• Out of these, the most important source of free radicals being the respiratory chain where ~ 1 to 2% oxygen is converted into superoxide radicals (O2•−).
• Oxidation reactions ensure that molecular oxygen is completely reduced to water
• Products of partial reduction of oxygen are highly reactive and make havoc in the living systems
• Hence they are also called Reactive oxygen species (ROS)
Introduction
Members of ROS group
• Superoxide anion radical (O2-)
• Hydroperoxyl radical (HOO●)
• Hydrogen peroxide (H2O2)
• Hydroxyl radical (OH●)
• Lipid peroxide radical (ROO●)
• Singlet oxygen (1O2)
• Nitric oxide (NO●)
• Peroxy nitrite (ONOO--●)
Univalent reduction steps of oxygen
Important characteristics
• Extreme reactivity
• Short life span
• Generation of new ROS by chain reaction
• Damage to various tissues
Generation of free radicals
• Constantly produced during the normal oxidation of foodstuffs,
due to leaks in the electron transport chain in mitochondria
• Some enzymes such as xanthine oxidase and aldehyde oxidase
form superoxide anion radical or hydrogen peroxide
• NADPH oxidase in the inflammatory cells produces superoxide
anion by a process of respiratory burst during phagocytosis
• Macrophages also produce NO from arginine by the enzyme
nitric oxide synthase
• Peroxidation is also catalysed by lipo-oxygenase in platelets and
leukocytes
Generation of free radicals
• Ionising radiation damages tissues by producing hydroxyl radicals, hydrogen peroxide and superoxide anion
• Light of appropriate wavelengths can cause photolysis of oxygen to produce singlet oxygen
• In the presence of free iron, H2O2 can generate OH● which is
highly reactive
• Cigarette smoke contains high concentrations of various free radicals
• Toxic compounds such as carbon tetrachloride, drugs and inhalation of air pollutants will increase the production of free radicals
Respiratory burst
• NADPH oxidase in the inflammatory cells produces superoxide anion
• 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
• About 10% of the oxygen uptake by macrophage is used for free radical generation
• Along with the activation of macrophages, the consumption of oxygen is increased drastically; this is called respiratory burst
Damage produced by ROS
• Free radicals are extremely reactive
• Their mean effective radius of action is only 30Å
• Their half life is only a few milliseconds
• When a free radical reacts with a normal compound, other free radicals are generated
• Peroxidation of PUFA in plasma membrane leads to loss of membrane functions
• Lipid peroxidation and consequent degradation products such as malondialdehyde are seen in biological fluids
• Their estimation in serum is often employed to assess the oxidative stress
Damage produced by ROS
• Almost all biological macromolecules are damaged by the
free radicals
• Oxidation of sulfhydryl containing enzymes, modification
of amino acids, loss of function and fragmentation of
proteins are noticed
• 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 is self-perpetuated by the free radicals released
by neutrophills
• Both corticosteroids and non-steroid anti-inflammatory
drugs interfere with formation of free radicals and
interrupt the disease process
• ROS induced tissue damage appears to be involved in
pathogenesis of chronic ulcerative colitis, chronic
glomerulonephritis etc.
Clinical significance
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
Clinical significance
3. Respiratory diseases
• Breathing of 100% oxygen for more than 24 hrs produces destruction of endothelium and lung edema
• This is due to release of free radicals by activated neutrophils
• In premature newborn infants, prolonged exposure to high oxygen concentration is responsible for broncho-pulmonary dysplasia
• ARDS is produced when neutrophils are recruited to lungs which subsequently release free radicals
• Cigarette smoke contains free radicals. Soot attracts neutrophils to the site which releases free radicals
Clinical significance
4. Diseases of the eye
• Retrolental fibroplasia or 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
• 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
Clinical significance
5. Reperfusion injury
• Reperfusion injury after myocardial ischemia is caused by free
radicals
• During ischemia, the concentration of xanthine oxidase is
increased and when reperfused this causes conversion of
hypoxanthine to xanthine and superoxide anion
• 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
Clinical significance
6. Shock related injury
• Release of free radicals from phagocytes damage membranes
by lipid peroxidation
• They release leucotrienes from platelets and proteases from
macrophages
• All these factors cause increased vascular permeability,
resulting in tissue edema
• Antioxidants have a protective effect
Clinical significance
7. Atherosclerosis and myocardial infarction
• Low density lipoproteins (LDL) promote atherosclerosis
• They are deposited under the endothelial cell, which undergo
oxidation by free radicals released from endothelial cells
• This attracts macrophages which are then converted to foam
cells
• This initiates the atherosclerotic plaque formation
• Alpha tocopherol offers some protective effect
Clinical significance
8. Peptic ulcer
• Peptic ulcer is produced by erosion of gastric mucosa by
HCl
• It is shown that superoxide anions are involved in the
formation of ulcer
• Helicobacter pylori infection perpetuates the disease
• This infection potentiates the macrophage oxidative burst
leading to tissue destruction
Clinical significance
9. Skin diseases
• Due to inborn defects, porphyrins accumulate in the skin
• Exposure to sunlight will lead to erythema and eruptions in the
patients
• Sunlight acting on porphyrins produces singlet oxygen, which
trigger inflammatory reaction, leading to the above symptoms
• Certain plant products, called psoralens are administered in the
treatment of psoriasis and leukoderma
Clinical significance
10. Cancer treatment
• Free radicals contribute to cancer development because of their mutagenic property
• Free radicals produce DNA damage, and accumulated damages lead to somatic mutation and malignancy
• Cancer is treated by radiotherapy
• Irradiation produces reactive oxygen species in the cells which trigger the cell death
• To increase the therapeutic effect of radiation, radio-sensitisers are administered, which increase the production of ROS
Lipid peroxidation
• Polyunsaturated fatty acids (PUFA) are normal constituents of
cellular and subcellular membranes
• The integrity of the cell membranes is affected by peroxidation
of PUFA
• Damage to cell membrane will cause increased permeability to
sodium ions, rapid influx of calcium, osmotic entrance of water
into the cell, leading to cell damage
Lipid peroxidation
• The auto-oxidation of membrane lipids proceeds as a chain
reaction and it occurs in 3 steps
– Initiation phase
– Propagation phase
– Termination phase
Initiation phase
• During this phase, the primary event is the production of R●
(PUFA radical) or ROO● (lipid peroxide radical) by the
interaction of a PUFA with free radicals generated by other
means
RH + OH● R● + H2O
ROOH ROO● + H+
• The R● and ROO●, in turn, are degraded to malondialdehyde
which is estimated as an indicator of fatty acid breakdown by
free radicals
Propagation phase• The R● rapidly reacts with molecular oxygen forming ROO●
which can attack another polyunsaturated lipid molecule
R● + O2 ROO●
ROO● + RH ROOH + R●
• The net result of two reactions is conversion of R● to ROOH
• But there is simultaneous conversion of a R● to ROO●. This would lead to continuous production of hydroperoxide with consumption of equimolecular quantities of PUFA
• The progression of this chain of events will destroy PUFA present in the membrane lipids
• Accumulation of such lipid damages lead to the destruction of fine architecture and integrity of the membranes
Termination phase
• The reaction would proceed unchecked till a peroxyl
radical reacts with another peroxy radical to form inactive
products
ROO● + ROO● RO-OR + O2
R● + R● R-R
ROO● + R● RO-OR
Role of anti-oxidants
• The damage produced by ROS may be prevented by
antioxidants
• There are two types of antioxidants
a) Preventive antioxidants – will inhibit the initial production
of free radicals. They are catalase, glutathione peroxidase,
EDTA (ethylene diamine tetra acetate)
b) Chain breaking antioxidants – once the peroxyl radicals are
generated, the chain breaking antioxidants can inhibit the
propagation phase. They include superoxide dismutase, uric
acid and vitamin E
Superoxide dismutase
• SOD is a non-heme protein
• The gene coding SOD is on chromosome 21
• Different iso-enzymes of SOD are described.
• The mitochondrial enzyme is manganese dependent;
cytoplasmic enzyme is copper-zinc dependent
• A defect in SOD gene is seen in some patients with
amylotrophic lateral sclerosis
Glutathione peroxidase
• The H2O2 generated is
removed by glutathione
peroxidase (POD)
• It is a selenium containing
enzyme
Glutathione reductase
• The oxidised glutathione is reduced by glutathione
reductase (GR) in presence of NADPH
• This NADPH is generated with the help of glucose-6-
phosphate dehydrogenase (G6PD) in HMP shunt pathway
• Therefore in G6PD deficiency the RBCs are liable to lysis,
especially when oxidising agents are administered (drug
induced hemolytic anemia)
Catalase
• When H2O2 is generated in
large quantities, the
enzyme catalase is also
used for its removal
Alpha-tocopherol
• Alpha tocopherol (T-OH) would intercept the peroxyl free
radical and inactivate it before a PUFA can be attacked
T-OH + ROO● TO● + ROOH
• The phenolic hydrogen of the alpha tocopherol reacts with the
peroxyl radical, converting it to a hydroperoxide product
• The tocopherol radical thus formed is stable and will not
propagate the cycle any further
• The tocopherol radical can react with another peroxyl radical
getting converted to inactive products
TO● + ROO● inactive products
Alpha tocopherol
• Vitamin E acts as the most effective naturally occurring chain
breaking antioxidant in tissues
• Only traces of tocopherol is required to protect considerable
amounts of polyunsaturated fat
• But in this process vitamin E is consumed
• Hence it has to be replenished to continue its activity
• This is achieved by daily dietary supply
Vitamin C
• Vitamin C, or ascorbic acid, is a water-soluble vitamin.
• This vitamin is a free radical scavenger, it is considered
to be one of the most important antioxidants in extra
cellular fluids.
• Its protective effects extend to cancer, coronary artery
disease, arthritis and aging.
THANKYOU
Recommended