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13. free radicals and antioxidants

  2. 2. What are free radicals? • Free radicals are ionised particles in the human body. • Free radicals can be caused by environmental toxins, stress, food additives and cooking among others. • Free radicals are also formed when oxygen is used by the body during normal metabolism. • But they are not all bad. • Free radicals actually help the body to fight viruses, bacteria, waste and toxins.
  3. 3. How Free Radicals Act • The human body is composed of many different types of cells. • Cells are composed of many different types of molecules and atoms consisting of protons, neutrons and electrons. • Electrons are involved in chemical reactions and are the substance that bonds atoms together to form molecules.
  4. 4. • Electrons surround, or "orbit" an atom in one or more shells. • The innermost shell is full when it has two electrons. When the first shell is full, electrons begin to fill the second shell. When the second shell has eight electrons, it is full, and so on. • The most important structural feature of an atom for determining its chemical behaviour is the number of electrons in its outer shell. • A substance that has a full outer shell tends not to enter in chemical reactions (an inert substance).
  5. 5. • Because atoms seek to reach a state of maximum stability, an atom will try to fill it’s outer shell by: 1. Gaining or losing electrons to either fill or empty its outer shell 2. Sharing its electrons by bonding together with other atoms in order to complete its outer shell • Atoms often complete their outer shells by sharing electrons with other atoms. By sharing electrons, the atoms are bound together and satisfy the conditions of maximum stability for the molecule.
  6. 6. How Free Radicals are Formed • Normally, bonds don’t split in a way that leaves a molecule with an odd, unpaired electron. • But when weak bonds split, free radicals are formed. • Free radicals are very unstable and react quickly with other compounds, trying to capture the needed electron to gain stability.
  7. 7. • Free radicals are highly reactive due to the presence of unpaired electron(s). • Any free radical involving oxygen can be referred to as reactive oxygen species (ROS). • Oxygen centred free radicals contain two unpaired electrons in the outer shell.
  8. 8. • In turn the newly formed radical then looks to return to its ground state by stealing electrons from cellular structures or molecules. • Thus the chain reaction continues and can be "thousand of events long."
  9. 9. • It's when the body has too many free radicals that damage can occur. • Free radicals can affect tissues, lipids, proteins and DNA. • Cells can be damaged leading to many diseases and early aging. • The effects of free radical damage can increase over time and lead to many age-related diseases.
  10. 10. Functions of Free Radicals • Some free radicals arise normally during metabolism. • Sometimes the body’s immune system’s cells purposefully create them to neutralize viruses and bacteria. • Normally, the body can handle free radicals, but if antioxidants are unavailable, or if the free-radical production becomes excessive, damage can occur. • Of particular importance is that free radical damage accumulates with age.
  11. 11. Types: Endogenous and Exogenous Free Radicals
  12. 12. TYPES OF ENDOGENOUS FREE RADICALS • The most important free radicals in the body are the radical derivatives of oxygen better known as reactive oxygen species. These include 1. Oxygen in its triplet state (3O2) or singlet state (O2), hydroxyl radical (OH), nitric oxide (NO), hypochlorous acid (HOCl), hydrogen peroxide (H2O2) and the superoxide radical (O2_). 2. Others are carbon-centered free radicals that arise from the attack of an oxidizing radical on an organic molecule. 3. Hydrogen centered radicals result from attack of the H atom (H). 4. Another form is the sulfur-centered radical produced in the oxidation of glutathione resulting in the thiyl radical.
  13. 13. Superoxide • The superoxide free radical anion is formed when oxygen is reduced by the transfer of a single electron to its outer shells. • The major source of superoxide is from the electron transfer chain of the mitochondria. • On its own it isn't particularly damaging.
  14. 14. Hydrogen peroxide • Hydrogen peroxide(H2O2) is not a free radical but falls in the category of reactive oxygen species. • It is an oxidising agent that is not particularly reactive but its main significance lies in that it is the main source of hydroxyl (OH) radicals. • It is also involved in the production of HOCl by neutrophils.
  15. 15. • In biological systems hydrogen peroxide is generated by the production of superoxide: two superoxide molecules can react together to form hydrogen peroxide and oxygen: • 2O2⁻+ 2H⁺ = H2O2 + O2 • The above reaction is called a dismutation reaction as the radical reactants produce non- radical products.
  16. 16. Hydroxyl radical • The hydroxyl radical is an extremely reactive oxidising radical that will react with most biomolecules. • The hydroxyl free radical is important in radio- biological damage. • The hydroxyl radical can damage virtually all types of macromolecules: carbohydrates, nucleic acids (mutations), lipids, and amino acids.
  17. 17. Mechanisms for scavenging hydroxyl radicals for the protection of cellular structures includes: 1. endogenous antioxidants such as melatonin and glutathione, and 2. dietary antioxidants such as phytochemicals and vitamin E.
  18. 18. Singlet oxygen • Singlet oxygen (1O2) is an electronically excited and mutagenic form of oxygen. It is similar to normal oxygen but there is one difference… it has an extra electron. • It is generated by input of energy like radiation or sunlight. • This free radical is involved in joint diseases (like arthritis) and eye diseases. • Carotenoids, such as Vitamin A and lycopene, can tame the singlet oxygen and so can Vitamin E.
  19. 19. Nitric Oxide • It is a common gaseous free radical. • It is now recognised to play a role in vascular physiology and is also known as endothelium derived relaxing factor. • Vascular endothelium produces nitric oxide from arginine using the enzyme nitric oxide synthetase (in brain, blood vessels and immune cells). • This event can be stimulated by cytokines, tumour necrosis factor, or interleukins and exercise. • Inhibition of production is known to reduce microbicidal and tumouricidal activities of macrophages.
  20. 20. Actions of Nitric Oxide free radical • Nitric Oxide is a key messenger in the cardiovascular system. • Nitric Oxide stimulates growth hormone production. • It stimulates immune cells. • It improves memory and nerve cell plasticity.
  21. 21. Peroxy-nitrite • It is produced by the reaction of nitric oxide with superoxide. • Because of its oxidizing properties, peroxy-nitrite can damage a wide array of molecules in cells, including DNA and proteins and results in cell apoptosis.
  22. 22. Hypochlorous acid • Activated polymorphonuclear cells produce HOCl as a major bactericidal agent. • This reaction occurs in the neutrophils phagocytic lysosomal vesicles. • Hypochlorous acid can cross cell membranes. • It may contribute to tissue damage during the inflammatory process.
  23. 23. Transition metals ions • Iron and copper play a major role in the generation of free radical injury and the facilitation of lipid peroxidation. • Transition metal ions generate OH from O2⁻ and H2O2. H2O2 + Fe2⁺ = OH + OH⁻ + Fe3⁺ • The reaction accelerates the non-enzymatic oxidation of molecules such as epinephrine and glutathione that generates O2- and H2O2 and subsequently OH.
  24. 24. EXOGENOUS FREE RADICALS • Drugs: • A number of drugs can increase the production of free radicals. • These drugs include antibiotics that depend on bound metals for activity (nitrofurantoin), antineoplastic agents as bleomycin, anthracyclines (adriamycin) and methotrexate. • In addition radicals derived from penicillamine, phenylbutazone and sulphasalazine might inactivate protease and deplete ascorbic acid accelerating lipid peroxidation.
  25. 25. • Radiation: • Radiotherapy may cause tissue injury that is caused by free radicals. • Electromagnetic radiation (X rays, gamma rays) and particulate radiation (electrons, photons, neutrons, alpha and beta particles) generate primary radicals by transferring their energy to cellular components such as water. • These primary radicals can undergo secondary reactions with dissolved oxygen or with cellular solutes.
  26. 26. • Tobacco smoking: • It has been estimated that each puff of smoke has an enormous amount of oxidant materials. • These include aldehydes, epoxides, peroxides, and other free radicals that may be sufficiently long lived as to survive till they cause damage to the alveoli. • In addition it also contains other relatively stable radicals in the tar phase. • It was also found that smokers have elevated amounts of neutrophils in the lower respiratory tract that could contribute to a further elevation of the concentration of free radicals.
  27. 27. • Inorganic particles: • Inhalation of inorganic particles also known as mineral dust (e.g. asbestos, quartz, silica) can lead to lung injury that seems at least in part to be mediated by free radical production. • Asbestos inhalation has been linked to an increased risk of developing pulmonary fibrosis (asbestosis), mesothelioma and bronchogenic carcinoma. • Silica particles as well as asbestos are phagocytosed by pulmonary macrophages. • These cells then rupture, releasing proteolytic enzymes and chemotactic mediators that leads to increased production of free radicals and other reactive oxygen species.
  28. 28. • Gases: • Ozone is not a free radical but a very powerful oxidising agent. Ozone (O3) contains two unpaired electrons and degrades under physiological conditions to OH, suggesting that free radicals are formed when ozone reacts with biological substrates. • Ozone can generate lipid peroxidation. • Others: • Fever, excess glucocorticoid therapy and hyperthyroidism increase metabolism. This leads to the increased generation of oxygen-derived radicals. • In addition, a wide variety of environmental agents including chemical air pollutants, pesticides, solvents, anaesthetics, exhaust fumes, also cause free radical damage to cells.
  29. 29. Free radicals: Our enemies or friends? • Although essentially cancer and degenerative diseases are to major extent caused by damage done to our DNA by them, free radicals also play an important role in cell metabolism. • The immune system is the main body system that utilizes free radicals. • Foreign invaders or damaged tissue is marked with free radicals by the immune system. • This allows for determination of which tissue need to be removed from the body. • Because of this some question the need for antioxidant supplementation, as they believe supplementation can actually decrease the effectiveness of the immune system.
  30. 30. ANTIOXIDANTS, NATURE AND CHEMISTRY • In the aerobic environment, the most dangerous by product are the species of reactive oxygen. • The role of antioxidants is to detoxify reactive oxygen intermediates (ROI) in the body. • Over the past several years, nutritional antioxidants have attracted considerable interest as potential treatment for a wide variety of disease states, including cancer, atherosclerosis, chronic inflammatory diseases and aging.
  31. 31. Definition • An antioxidant is a substance that when present in low concentrations relative to the oxidizable substrate significantly delays or reduces oxidation of the substrate (Halliwell, 1995). • Antioxidants get their name because they combat oxidation. • They are substances that protect other chemicals of the body from damaging oxidation reactions by reacting with free radicals and other reactive oxygen species within the body, hence hindering the process of oxidation.
  32. 32. • 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, whether endogenously or through supplementation in diet and exercise.
  33. 33. Antioxidant System • The body has developed several endogenous antioxidant systems to deal with the production of ROI. The enzymatic antioxidants include 1. superoxide dismutase (SOD), which catalyses the conversion of O2⁻ to H2O2 and H2O; 2. catalase, which then converts H2O2 to H2O and O2;and 3. glutathione peroxidase, which reduces H2O2 to H2O.
  34. 34. • The glutathione redox cycle is a central mechanism for reduction of intracellular hydroperoxides. • Source and Nature: • It is a tetrameric protein and has 4 atoms of selenium (Se) that confers the catalytic activity. • Glutathione peroxidase reduces H2O2 to H2O by oxidizing glutathione. • Re-reduction of the oxidized form of glutathione (GSSG) is then catalysed by glutathione reductase.
  35. 35. These enzymes also require trace metal cofactors for maximal efficiency, including selenium for glutathione peroxidase; copper, zinc, or manganese for SOD; and iron for catalase.
  36. 36. The nonenzymatic/Exogenous antioxidants This includes 1. the lipid-soluble vitamins, vitamin E and vitamin A or provitamin A (beta-carotene), 2. the water-soluble vitamin C and 3. Glutathione (GSH), a tripeptide molecule. • The enzymatic and nonenzymatic antioxidant systems are intimately linked to one another and appear to interact with one another.
  37. 37. Classification of major antioxidants Antioxidant Role Remarks Superoxide Dismutates O2⁻ to Contains Manganese dismutase (SOD) (Mn.SOD) Mitochondrial H2O2 Contains Copper & Zinc Cytoplasmic (CuZnSOD) ENZY Extracellular Contains Copper (CuSOD) MES Catalase Dismutates Tetrameric hemoprotein H2O2 to H2O present in peroxisomes Glutathione Removes Selenoproteins (contains peroxidase H2O2 and lipid Se2+) (GSH.Px) peroxides Primarily in the cytosol also mitochondria Uses GSH
  38. 38. Alpha Breaks lipid peroxidation Fat soluble tocopherol Lipid peroxide and O2⁻and OH vitamin (Vit E) scavenger VITA Beta Scavenges OH, O2⁻ and peroxy Fat soluble MINS carotene radicals vitamin Prevents oxidation of vitamin A Binds to transition metals Ascorbic Directly scavenges O2⁻, OH, and Water soluble acid H2O2 vitamin Neutralizes oxidants from stimulated neutrophils Contributes to regeneration of vitamin E
  39. 39. Antioxidant Vitamins • Vitamin E is more appropriately described as an antioxidant than a vitamin. This is because, unlike most vitamins, it does not act as a co-factor for enzymatic reactions. • Also, deficiency of vitamin E does not produce a disease with rapidly developing symptoms such as scurvy or beriberi. • Overt symptoms due to vitamin E deficiency occur only in cases involving fat malabsorption syndromes, premature infants and patients on total parenteral nutrition.
  40. 40. • The effects of inadequate vitamin E intake usually develop over a long time, typically decades, and have been linked to chronic diseases such as cancer and atherosclerosis. • Its main function is to prevent the peroxidation of membrane phospholipids, and avoid cell membrane damage through its antioxidant action.
  41. 41. • How It Acts: • Tocopherol-OH can transfer a hydrogen atom with a single electron to a free radical, thus removing the radical before it can interact with cell membrane, proteins or generate lipid peroxidation. • When tocopherol-OH combines with the free radical, it becomes tocopherol-O, itself a radical. • When ascorbic acid (Vitamin C) is available, tocopherol-O plus ascorbate yields semi-dehydro-ascorbate plus tocopherol-OH. • By this process, an aggressive ROI is eliminated and a weak ROI (dehydroascorbate) is formed, and tocopherol- OH is regenerated.
  42. 42. • Vitamin E also stimulates the immune response. Some studies have shown lower incidence of infections when vitamin E levels are high, and vitamin E may inhibit cancer initiation through enhanced immuno-competence. • Vitamin E inhibits the conversion of nitrites in smoked, pickled and cured foods to nitrosamines in the stomach. Nitrosamines are strong tumour promoters. • {Alpha-tocopherol has been shown to be capable of reducing ferric iron to ferrous iron, i.e. act as a pro- oxidant}.
  43. 43. Beta Carotene • Source and Nature: • Carotenoids are pigmented micronutrients present in fruits and vegetables. • Carotenoids are precursors of vitamin A and also have antioxidant effects. • While over 600 carotenoids have been found in the food supply, the most common forms are alpha-carotene, beta- carotene, lycopene, crocetin, canthaxanthin, and zeaxanthin. • Beta-carotene is the most widely studied. It is composed of two molecules of vitamin A (retinol) joined together. • Dietary beta-carotene is converted to retinol at the level of the intestinal mucosa.
  44. 44. • Mechanisms of Action: • The antioxidant function of beta-carotene is due to its ability to quench singlet oxygen, scavenge free radicals and protect the cell membrane lipids from the harmful effects of oxidative degradation. • The ability of beta-carotene and other carotenoids to quench excited oxygen, however, is limited, because the carotenoid itself can be oxidized during the process (autoxidation).
  45. 45. • Carotenoids also have been reported to have a number of other biologic actions, including • immuno-enhancement; • inhibition of mutagenesis and transformation; and • regression of premalignant lesions.
  46. 46. Ascorbic acid (vitamin C) • Source and Nature: • Ascorbic acid (vitamin C) is a water-soluble, antioxidant present in citrus fruits, potatoes, tomatoes and green leafy vegetables. • Mechanism of Action: • The chemopreventive action of vitamin C is attributed to two of its functions. 1. It is a water-soluble, chain breaking antioxidant. As an antioxidant, it scavenges free radicals and reactive oxygen molecules, which are produced during metabolic pathways of detoxification. 2. It also prevents formation of carcinogens from precursor compounds.
  47. 47. • It has also been shown that ascorbate is more potent than a-tocopherol in inhibiting the oxidation of LDL. • Vitamin C also contributes to the regeneration of membrane bound oxidized vitamin E. It will react with the a -tocopheroxyl radical, resulting in the generation of tocopherol in this process itself being oxidized to dehydroascorbic acid. Vitamin C supplementation leads to increased plasma and tissue levels of vitamin E.
  48. 48. Other antioxidants • Glutathione (GSH): • GSH is synthesized intra-cellularly from cysteine, glycine, and glutamate. • In addition to its role as a substrate in GSH redox cycle, GSH is also a scavenger of hydroxyl radicals and singlet oxygen. • The majority of GSH is synthesized in the liver, and approximately 40% is secreted in the bile. • The biologic role of GSH in bile is believed to be defence against dietary xenobiotics and protection of the intestinal epithelium from oxygen radical attack.
  49. 49. • CoQ10: • CoQ10 (Coenzyme Q10) is also known as ubiquinone. It is found in almost every living cell and is essential to energy production by the mitochondria. • Far beyond producing energy, CoQ10 can protect the body from destructive free radicals and enhance immune defences. • Albumin: • Albumin scavenges several free radicals and thus can be considered as one of the primary extracellular defence systems. • Plasma proteins • Namely ceruloplasmin and transferrin have also shown antioxidant activity.
  50. 50. Melatonin • Melatonin is a powerful antioxidant. • Melatonin easily crosses cell membranes and the blood-brain barrier. • Unlike other antioxidants, melatonin does not undergo redox cycling, which is the ability of a molecule to undergo repeated reduction and oxidation. • Melatonin, once oxidized, cannot be reduced to its former state. Therefore, it has been referred to as a terminal (or suicidal) antioxidant.
  51. 51. Uric acid: • Acts as an endogenous radical scavenger and antioxidant. It is present in about 0.5 mmol/L in body's fluids and is the end product of purine metabolism. • Uric acid is a powerful scavenger of singlet oxygen, peroxyl radical (ROO) and OH radical. • Concerns over elevated UA's contribution to gout must be considered as one of its many risk factors. • The effects of uric acid in conditions such as atherosclerosis, ischemic stroke, and heart attacks are still not well understood. • This might be because high levels of uric acid act as a pro- oxidant.
  52. 52. Drugs: Several pharmaceutical agents have been found to exert an antioxidant effect: 1. Xanthine oxidase inhibitors: e.g. allopurinol, folic acid. 2. NADPH inhibitors: e.g. adenosine, calcium channel blockers. 3. Albumin. 4. Inhibitors of iron redox cycling: deferoxamine, apotransferrin and ceruloplasmin 5. Statins, the anti-cholesterol drug
  53. 53. Dietary sources of antioxidants
  54. 54. POTENTIAL OF ANTIOXIDANT SUPPLEMENTS TO DAMAGE HEALTH • Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. • An antioxidant is a molecule that inhibits the oxidation of other molecules. • Antioxidants terminate these chain reactions by removing free radical intermediates, and inhibit other oxidation reactions. • Antioxidants are widely used in dietary supplements and have been investigated for the prevention of diseases such as cancer, coronary heart disease and even altitude sickness.
  55. 55. • 1. Although initial studies suggested that antioxidant supplements might promote health, later large clinical trials with a limited number of antioxidants detected no benefit and even suggested that excess supplementation with certain antioxidants may be harmful. • Antioxidants that are reducing agents can also act as pro-oxidants. • For example, vitamin C has antioxidant activity when it reduces oxidizing substances such as hydrogen peroxide, however, it will also reduce metal ions that generate free radicals. • That is, paradoxically, agents which are normally considered antioxidants can act as conditional pro-oxidants and actually increase oxidative stress.
  56. 56. • 2. Free radicals induce an endogenous response which protects against exogenous radicals (and possibly other toxic compounds). • Recent experimental evidence strongly suggests that such induction of endogenous free radical production can extend the life span. • Mito Hormesis: Most importantly, this induction of life span is prevented by antioxidants, providing direct evidence that toxic radicals may mito-hormetically exert life extending and health promoting effects.
  57. 57. HORMESIS • Hormesis is the term for generally favourable biological responses to low exposures to toxins and other stressors. • Examples in normal life are exercise, acute stress and uric acid, all of which in small amounts increase lifespan, immunity and health, but in larger amounts are harmful. • The biochemical mechanisms by which hormesis works are not well understood. • It is conjectured that low doses of toxins or other stressors might activate the repair mechanisms of the body. • Free radicals may also help by causing apoptosis of damaged cells and thus reducing aging and cancer causing cells in the body.
  58. 58. • 3. Another fact: • Antioxidant supplements have no clear effect on the risk of chronic diseases such as cancer and heart disease in the long run. • This suggests that the health benefits of fruits and vegetables come from other substances in fruits and vegetables or come from a complex mix of compounds. • For example, the antioxidant effect of flavonoid-rich foods seems to be due to fructose-induced increases in the synthesis of the antioxidant uric acid and not only to dietary antioxidants.
  59. 59. Important points • Diet plays an important role in reducing oxidative damage to the body. • The role of dietary antioxidants is not limited to its antioxidant chemicals, minerals and vitamins alone, but to an as yet unknown combination of all these. • Supplementation pills of individual antioxidants have not been shown to prolong life or improve health.
  60. 60. Homework Explain how Mithormesis works to prolong life.
  61. 61. Any Questions?