Lecture 8 & 9 Free Radicals and Anti-Oxidants PDF

‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ Free Radicals and Anti-Oxidants Definition A free radical is a molecule or molecular fragment that contains one or more unpaired electrons in its outer orbital. Oxidation reactions ensure that molecular...

‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ Free Radicals and Anti-Oxidants Definition A free radical is a molecule or molecular fragment that contains one or more unpaired electrons in its outer orbital. Oxidation reactions ensure that molecular oxygen is completely reduced to water. The products of partial reduction of oxygen are highly reactive and create havoc in the living systems. Reactive oxygen species or ROS. 1-Superoxide anion radical (O2--•) 2- Hydroperoxyl radical (HOO• ) 3- Hydrogen peroxide (H2O2) 4- Hydroxyl radical (OH•) 5-Lipid peroxide radical (ROO•) 1 ‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ 6- Singlet oxygen ( 1O2) 7- Nitric oxide (NO•) 8- Peroxy nitrite (ONOO) Important characteristics of the ROS are: a. Extreme reactivity. b. Short life span. c. Generation of new ROS by chain reaction. d. Damage to various tissues Generation of Free Radicals 1- They are constant produced during the normal oxidation of foodstuffs, due to leaks in the electron transport chain in mitochondria. About 1-4% of oxygen taken up in the body is converted to free radicals. 2- Some enzymes such as xanthine oxidase and aldehyde oxidase form superoxide anion radical or hydrogen peroxide. 3- NADPH oxidase in the inflammatory cells (neutrophils, eosinophils, monocytes and macrophages) produces superoxide anion by a process of respiratory burst during phagocytosis 4- Macrophages also produce NO from arginine by the enzyme nitric oxide synthase , This is also an important anti-bacterial mechanism. 2 ‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ 5- Peroxidation is also catalyzed by lipo- oxygenase in platelets and leukocytes. 6- lonizing radiation damages tissues by producing hydroxyl radicals, hydrogen peroxide and superoxide anion 'H2O –––– (gamma, UV radiation) ––––→ H' + OH 7- Light of appropriate wavelengths can cause photolysis of oxygen to produce singlet oxygen. 8- The capacity to produce tissue damage by H2O2 is minimal when compared to other free radicals (by definition, H2O2 is not a free radical). But in presence of free iron, H2O2 can generate OH• (hydroxyl radical) which is highly reactive. 9- Cigarette smoke contains high concentrations of various free radicals. 10- Inhalation of air pollutants will increase the production of free radicals. 11- Under hypoxic conditions, the mitochondrial respiratory chain also produce nitric oxide(NO), which can generate other reactive nitrogen species (RNS). Excess ROS and RNS can lead to oxidative stress. 3 ‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ Free Radical Scavenger Systems 1. Superoxide dismutase (SOD) , The mitochondrial SOD is manganese dependent; A defect in SOD gene is seen in patients with amyotrophic lateral sclerosis 2-Glutathione peroxidase In the next step, the H2O2 is removed by glutathione peroxidase (POD). It is a selenium dependent enzyme 3-Glutathione reductase , The oxidized glutathione, in turn, is reduced by the glutathione reductase (GR), in presence of NADPH . This NADPH is generated with the help of glucose-6-phosphate dehydrogenase (GPD) in HMP shunt pathway. Therefore in GPD deficiency, the RBCs are liable to lysis, especially when oxidizing agents are administered (drug induced hemolytic anemia) 4-Catalase When H2O2 is generated in large quantities, the enzyme catalase is also used for its removal 4 ‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ Damage Produced by Reactive Oxygen Species Free radicals are extremely reactive. Their half-life is only a few milliseconds. When a free radical reacts with a normal compound, other free radicals are generated. This chain reaction leads to thousands of events. Peroxidation of PUFA (poly unsaturated fatty acids) in plasma membrane leads to loss of membrane functions. Lipid peroxidation and consequent degradation products such as malon dialdehyde are seen in biological fluids. Almost all biological macromolecules are damaged by the free radicals , Polysaccharides undergo degradation and DNA is damaged . The DNA damage may directly cause inhibition of protein and enzyme synthesis and indirectly cause cell death or mutation and carcinogenesis 5 ‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ Thiobarbituric acid reactive substances (TBARS) CLINICAL SIGNIFICANCE 1. Chronic Inflammation: Chronic inflammatory diseases such as rheumatoid arthritis are self-disease, free radicals released by neutrophils. ROS induced tissue damage appears to be involved in pathogenesis of chronic ulcerative colitis, chronic glomerulonephritis. 2- Acute Inflammation: At the inflammatory site, activated macrophages produce free radicals. 6 ‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ 3- Respiratory Diseases: Breathing of 100% oxygen for more than 24 hrs produces destruction of endothelium and lung edema. This is due to the release of free radicals by activated neutrophils. 4- Diseases of the Eye : Retrolental fibroplasia (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. 5- Reperfusion Injury: Reperfusion injury after myocardial ischemia is caused by free radicals. During ischemia, the activity of xanthine oxidase is increased. When reperfused, this causes conversion of hypoxanthine to xanthine and superoxide anion, leading to aggravation of myocardial injury . Allopurinol, a xanthine oxidase inhibitor, reduces the severity of reperfusion injury 6- Atherosclerosis and Myocardial Infarction : Low density lipoproteins (LDL) are deposited under the endothelial cells, which undergo oxidation by free radicals. This attracts macrophages. Macrophages are then converted into foam cells. This initiates the atherosclerotic plaque formation . 7- Shock-related Injury : Release of free radicals from phagocytes damage membranes by lipid peroxidation. They release leukotrienes from platelets and proteases from macrophages. All these factors cause increased vascular permeability, resulting in tissue edema. Anti-oxidants have a protective effect 7 ‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ 8- Skin Diseases : Certain plant products, are administered in the treatment of psoriasis and leukoderma. When the drug is applied over the affected skin and then irradiated by UV light, singlet oxygen is produced with clinical benefit 9- Carcinogenesis and Treatment : Free radicals produce DNA damage, and accumulated damages lead to somatic mutations and malignancy. 10-Aging Process : Reactive oxygen metabolites (ROM) play a pivotal role in the degenerative brain disorders such as Parkinsonism, Alzheimer's dementia and multiple sclerosis. Lipid Peroxidation 1. Initiation Phase Polyunsaturated fatty acids (PUFA) present in cell membranes are easily destroyed by peroxidation. During the initiation phase, the primary event is the production of R' (carbon centered radical) (PUFA radical) or ROO' (lipid peroxide radical) by the interaction of a PUFA molecule with free radicals generated by other (Reaction no. 1) 2- Propagation Phase The carbon centered radical (R' ) rapidly reacts with molecular oxygen forming a peroxyl radical (ROO') which can attack another polyunsaturated lipid molecule. R' + O2 → ROO' (Reaction No.2) 8 ‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ ROO' + RH → ROOH + R' (Reaction No.3) The net result of reactions 2 and 3 is the conversion of R' to ROOH (a hydroperoxide). This would lead to continuous production of hydroperoxide with consumption of equimolecular quantities of PUFA. One free radical generates another free radical in the neighboring molecule. 3- Termination Phase The reaction would proceed unchecked till a peroxyl radical reacts with another peroxyl radical to form inactive products . 9 ‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ Role of Anti-Oxidants Apart from the scavenging enzymes described earlier, there are two types of anti-oxidants: 1- Preventive anti-oxidants: They will inhibit the initial production of free radicals. They are catalase, glutathione peroxidase, and ethylene diamine tetra-acetate (EDTA). 2- Chain breaking anti-oxidants: They can inhibit propagative phase. They include superoxide dismutase, uric acid and vitamin E. Anti-oxidants 1. Vitamin E is the lipid phase antioxidant. 2. Vitamin C is the aqueous phase antioxidant. 3. Ceruloplasmin can act as an antioxidant in extracellular fluid . 4. Caffeine is another effective anti-oxidant. 10 ‫د رياض حنيوه‬ ‫جامعه وارث االنبياء‬ ‫كليه الطب‬ lecture 8 & 9 ‫قسم الكيمياء الحيوية والطبية‬ 5. Cysteine, glutathione and vitamin A are minor anti- oxidants. Beta carotene can act as a chain breaking antioxidant, but is less effective than alpha tocopherol. Anti-oxidants used as therapeutic agents 1. Vitamin E 2. Vitamin C 3. Dimethyl thio urea 4. Dimethyl sulfoxide 5. Allopurinol END OF THE LECTUER Reference TEXTBOOK OF BIOCHEMISTRY Sixth Edition For Medical Students DM VASUDEVAN MBBS MD FAMS FRCPath Distinguished Professor of Biochemistry College of Medicine, Amrita Institute of Medical Sciences, Cochin, Kerala (Formerly Principal, College of Medicine, Amrita, Kerala) (Formerly, Dean, Sikkim Manipal Institute of Medical Sciences, Gangtok, Sikkim) E-mail: [email protected] SREEKUMARI S MBBS MD Professor, Department of Biochemistry Sree Gokulam Medical College and Research Foundation Thiruvananthapuram, Kerala E-mail: [email protected] KANNAN VAIDYANATHAN MBBS MD Clinical Associate Professor, Department of Biochemistry and Head, Metabolic Disorders Laboratory Amrita Institute of Medical Sciences, Kochi, Kerala Email: [email protected] 11