Free radicals & antioxidants

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Lecture on Free Radicals

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Free radicals & antioxidants

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

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