ROLE OF FREE RADICALS IN DIABETES A Dissertation submitted to THE JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY, ANANTAPUR In partial fulfillment for the award of degree of BACHELOR OF PHARMACY Submitted by Reg.No: 08GT5R0002 Under the supervision of K.HARI KUMAR, M.Pharm, NOVEMBER 2010 NOVEMBER 2010 DEPARTMENT OF PHARMACEUTICAL CHEMISTRY SRI VENKATESWARA COLLEGE OF PHARMACY R.V.S. NAGAR, CHITTOOR - 517127
INTRODUCTION DEFINITION OF FREE RADICALS Free radicals are atoms, molecules, or ions with unpaired electrons on an open shell configuration. Free radicals may have positive, negative or zero charge. Even though they have unpaired electrons, by convention, metals and their ions or complexes with unpaired electrons are not radicals.
Reactive Oxygen Species O 2 ⁻ Superoxide radical OH Hydroxyl radical ROO Peroxyl radical H 2 O 2 Hydrogen peroxide 1 O 2 Singlet oxygen NO Nitric oxide ONOO ⁻ Peroxynitrite HOCl Hypochlorous acid
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 in-vivo is the electron leakage that result from the electron transfer chain of the mitochondria. HYDROGEN PEROXIDE Hydrogen peroxide 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 radicals in the presence of transition metal ions. HYDROXYL RADICAL The hydroxyl radical is an extremely reactive oxidising radical that will react to most biomolecules at diffusion controlled rates, which means that reactions will occur immediately with biomolecules. SINGLET OXYGEN Singlet oxygen ( 1 O 2 ) is an electronically excited and mutagenic form of oxygen.
NITRIC OXIDE It is a common gaseous free radical. It is now recognized to play a role in vascular physiology and is also known as endothelium derived relaxing factor. PEROXYNITRITE Peroxynitrite (sometimes called peroxynitrite) is the anion with the formula ONOO−. It is an unstable structural isomer of nitrate, NO 3 −, which has the same formula but a different structure. H 2 O 2 + NO 2 − -> ONOO− + H 2 O · O 2 − + ·NO -> ONO 2 − HYPOCHLOROUS ACID Activated polymorph nuclear cells produce HOCl as a major bactericidal agent. It is generated by the action of myeloperoxidase on chloride ions in the presence of H 2 O 2 . H 2 O 2 + Cl- HOCl + OH-
PRODUCTION OF FREE RADICALS IN THE HUMAN BODY Free radicals and other reactive oxygen species are derived either from normal essential metabolic processes in the human body or from external sources such as exposure to X-rays, ozone, cigarette smoking, air pollutants and industrial chemicals. sources of free radicals are: Mitochondria Phagocytes Xanthine oxidase Reactions involving iron and other transition metals Peroxisomes Exercise & inflammation Some externally generated sources of free radicals are: Cigarette smoke Radiation Ultraviolet light Certain drugs, pesticides Anaesthetics and industrial solvents Ozone
REACTIVE OXYGEN SPECIES Reactive oxygen species (ROS) are chemically-reactive molecules containing oxygen. Examples include oxygen ions and peroxides. Damaging effects Cells are normally able to defend themselves against ROS damage with enzymes such as sup eroxide dismutases, catalases, lactoperoxidases, glutathione perioxidase and peroxiredoxins. Generally, harmful effects of reactive oxygen species on the cell are most often: Damage of DNA Oxidations of polydesaturated fatty acids in lipids (lipid peroxidation) Oxidations of amino acids in proteins Oxidatively inactivate specific enzymes by oxidation of co-factors
ROS FORMATION Reactive oxygen species are formed by several different mechanisms: The interaction of ionizing radiation with biological molecules As an unavoidable byproduct of cellular respiration. Some electrons passing &quot;down&quot; the electron transport chain leak away from the main path (especially as they pass through ubiquinone) and go directly to reduce oxygen molecules to the superoxide anion. synthesized by dedicated enzymes in phagocytic cells like neutrophils and macrophages NADPH oxidase (in both type of phagocytes) Myeloperoxidase (in neutrophils only) ROS ACTIVITY Strong oxidants like the various ROS can damage other molecules and the cell structures of which they are a part . Two common examples ◦ Another peroxyl radical on a nearby side chain cross linking them with a covalent bond. ◦ Another nearby carbon-centered radical cross linking them covalently.
The figure shows one common series of reactions. A hydroxyl radical removes a hydrogen atom from one of the carbon atoms in the fatty acid chain (only a portion of which is shown) forming A molecule of water and leaving the carbon atom with an unpaired electron (in red); thus now a radical. Several possible fates await it.
LIPID PEROXIDATION Lipid peroxidation refers to the oxidative degradation of lipids. It is the process whereby free radicals&quot;steal&quot; electrons from the lipids in cell membranes, resulting in cell damage. Initiation Propagation Termination OXIDATIVE STRESS Oxidative stress represents an imbalance between the production of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage.
OXIDATIVE STRESS Oxidative stress represents an imbalance between the production of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. CHEMICAL AND BIOLOGICAL effects Chemically, oxidative stress is associated with increased production of oxidizing species or a significant decrease in the capabilty of antioxidant defenses, such as glutathione. The effects of oxidative stress depend upon the size of these changes, with a cell being able to overcome small perturbations and regain its original state. PRODUCTION AND CONSUMPTION OF OXIDANTS One source of reactive oxygen under normal conditions in humans is the leakage of activated oxygen from mitochondria during oxidative phosphorylation. However, E. coli mutants that lack an active electron transport chain produced as much hydrogen peroxide as wild-type cells, indicating that other enzymes contribute the bulk of oxidants in these organisms.
DIABETES MELLITUS The term diabetes mellitus describes a metabolic disorder of multiple aetiology characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both. The effects of diabetes mellitus include long–term damage, dysfunction and failure of various organs. DIABETES AND OXIDATIVE STRESS In the diabetes chronic hyperglycemia was occurred. In the presence of high blood glucose levels recative oxygen spcies was produced by various processes called GLUCOSE OXIDATION GLUCOSE TOXICITY OXIDATIVE PHOSPHORYLATION The production of reactive oxygen species leads to elevation of free radicals. These free radicals will involve in process called oxidative stress and prone to beta cell dysfunction and destruction. This leads to impairment of insulin action
GLUCOSE TOXICITY Increased glucose metabolism leads to increased depositions of glucose in the cells then finally elevates of glucose toxicity. Glucose toxicity of the islet is defined as nonphysiological and potentially irreversible ß-cell damage caused by chronic exposure to supraphysiological glucose concentrations. METABOLISM OF GLUCOSAMINE Another concern has been that the extra glucosamine could contribute to diabetes by interfering with the normal regulation of the hexosamine biosynthesis pathway, but several investigations have found no evidence that this occurs. A manufacturer-supported review conducted by Anderson et al. in 2005 summarizes the effects of glucosamine on glucose metabolism in in vitro studies, the effects of oral administration of large doses of glucosamine in animals and the effects of glucosamine supplementation with normal recommended dosages in humans, concluding that glucosamine does not cause glucose intolerance and has no documented effects on glucose metabolism. Other studies conducted in lean or obese subjects concluded that oral glucosamine at standard doses does not cause or significantly worsen insulin resistance or endothelial dysfunction.
OXIDATIVE PHOSPHORYLATIONSHIP Oxidative phosphorylation is a metabolic pathway that uses energy released by the oxidation of nutrients to produce adenosine triphosphate (ATP). Although the many forms of life on earth use a range of different nutrients, almost all carry out oxidative phosphorylation to produce ATP, the molecule that supplies energy to metabolism. This pathway is probably so pervasive because it is a highly efficient way of releasing energy, compared to alternative fermentation processes such as anaerobic glycolysis. During oxidative phosphorylation, electrons are transferred from electron donors to electron acceptors such as oxygen, in redox reactions. These redox reactions release energy, which is used to form ATP. In eukaryotes, these redox reactions are carried out by a series of protein complexes within mitochondria, whereas, in prokaryotes, these proteins are located in the cells' inner membranes. These linked sets of proteins are called electron transport chains. In eukaryotes, five main protein complexes are involved, whereas in prokaryotes many different enzymes are present, using a variety of electron donors and acceptors.
MALONDIALDEHYDE IS A TOXIC STREES TO PANCREATIC BETA CELLS A considerable decrease of malondialdehyde, reduced and oxidized glutathione and reduction of the activities of Se-glutathione peroxidase and glutathione S-transferase were observed. Reactive oxygen species degrade polyunsaturated lipids, forming malondialdehyde. This compound is a reactive aldehyde and is one of the many reactive electrophile species that cause toxic stress in cells and form covalent protein adducts which are referred to as advanced lipoxidation end products (ALE), in analogy to advanced glycation end-products (AGE). The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism. Malondialdehyde reacts with deoxyadenosine and deoxyguanosine in DNA, forming DNA adducts, primarily M1G, which is mutagenic.The guanidine group of arginine residues condense with MDA to give 2-aminopyrimidines.Human ALDH1A1 aldehyde dehydrogenase is capable of oxidising malondialdehyde.
EVIDENCE THAT THE ISLET IS UNIQUELY AT RISK FOR OXIDATIVE DAMAGE The concept that the islet is unusually at risk for damage by pro-oxidant forces is not a new one . Oxidative phosphorylation during anaerobic glycolysis generates reactive oxygen species (ROS), a process that might become excessive in hyperglycemic states. The following are metabolic pathways that excess glucose might be shunted into when it accumulates beyond the levels that glycolytic enzymes can handle and that can form ROS: glycosylation (Schiff reaction)  , glucose autoxidation, and the glucosamine pathway. ROS that might be formed include superoxide, hydrogen peroxide, nitric oxide, and hydroxyl radicals. Among these, the hydroxyl radical is the most toxic because it easily passes through membrane barriers to the cell's nucleus and strongly reacts mutagenically with DNA.
OXIDATIVE STRESS AND DIABETIC COMPLICATIONS Hyperglycemiaderived oxygen free radicals as mediators of diabetes-associated complications. Current studies have specified that a hyperglycemia-induced overproduction of superoxide appears to be the major event in the development of complications of diabetes. Superoxide overproduction is associated with increased generation of nitric oxide and, as a result, formation of the strong oxidant peroxynitrite and by poly (adenosine diphosphate-ribose) polymerase activation, which in turn further initiates the pathways implicated in the development of diabetes-related complications. In addition, this procedure consequence in severe endothelial dysfunction and initiation of inflammation in blood vessels of individuals with diabetes, and these aspects contribute to the development of complications of diabetes. Furthermore, in vivo evidence supports the major contribution of hyperglycemia in producing oxidative stress and, eventually, severe endothelial dysfunction in blood vessels of individuals with diabetes.
OXIDATIVE-STRESS-ACTIVATED SIGNALING PATHWAYS MEDIATORS OF INSULIN RESISTANCE AND BETA-CELL DYSFUNCTION Insulin resistance is a condition in which impaired glucose utilization of peripheral tissues. In creased levels of FFAs are positively corrlated with both insulin resistence and the deterioration of beta cell function. The FFAs are involved in the ROS formation because their ability to directly oxidize and damage DNA of pancreas, proteins and lipids. Then ROS functioning as signaling molecules to activate a number of cellular stress pathways that cause cellular damage of pancreas. The activation of stress sensitive pathways such as NF-KA P38 MAPK JNK/SAPK HEXOSAMINE by elevated glucose The other proposed mediators are AGE, SORBITOL, CYTOKINES AND PROTONOIDS, PKC ACTIVATION, DAG (diaclyglycerol)
CONCLUSION Free radicals are atoms, molecules, or ions with unpaired electrons on an open shell configuration. Free radicals may have positive, negative or zero charge. Even though they have unpaired electrons, by convention, metals and their ions or complexes with unpaired electrons are not radicals. In the hyperglycemic condition i.e. increased glucose levels involve process called autooxidation leads to formation of reactive oxygen species nothing but free radicals. These free radicals will again involve in pancreatic damage. This pancreatic damage finally leads to diabetic complication like neuropathy, nephropathy, and cardiopathy. The FFA levels contribute to the pathophysiology of diabetes via the generation of ROS and consequent activation of numerous stress-sensitive pathways.
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