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Premium member Presentation Transcript A Presentation by Dr. Akif Ahsan: A Presentation by Dr. Akif AhsanFree Radicals: Free Radicals Free radical damage has been implicated in a wide variety of age related chronic diseases such as atherosclerosis, diabetes, ophthalmic and neurodegenerative disease as well as being involved in the ageing process. Obesity has been described as a state of chronic oxidative stress.Diseases / Conditions associated with increased free radicals: Diseases / Conditions associated with increased free radicalsIntroduction: Introduction A free radical is any species capable of independent existence that contains one or more unpaired electrons. They are unstable, very reactive and short-lived as they tend to catch an electron from other molecules.Introduction: Introduction This definition includes the hydrogen atom and most of the transition metal ions. It also includes the oxygen molecule, which is biradical since its outer two electrons are in different orbitals and have parallel spins (they are not paired). Free radicals may be electrically neutral or either positively or negatively charged.Introduction: Introduction They attack sites of increased electron density such as: the nitrogen atom present in proteins and DNA predominantly and carbon-carbon double bonds present in polyunsaturated fatty acids and phospholipids to produce additional free radical, often reactive, intermediates.Introduction: Introduction The presence of low concentrations of free radicals is important for normal cellular redox status, immune function and intracellular signaling. However, excessive production can damage lipids, proteins and DNA and compromise cell function leading to cell death by necrosis or apoptosis .History: History The first organic free radical identified was triphenylmethyl radical, by Moses Gomberg in 1900. Historically, the term radical has also been used for bound parts of the molecule, especially when they remain unchanged in reactions. These are now called functional groups . For example, methyl alcohol was described as consisting of a methyl "radical" and a hydroxyl "radical". Neither are radicals in the modern chemical sense.Formation of Free Radicals: Formation of Free Radicals Homolysis of covalent bonds Addition of a single electron to a neutral atom Loss of a single electron from a neutral atomFormation of Free Radicals: Formation of Free Radicals As a rule, a radical needs to pair its unpaired electron with another, and will react with another molecule in order to obtain this missing electron. If a radical achieves this by "stealing" an electron from another molecule, that other molecule itself becomes a radical (Reaction 1), and a self propagating chain reaction is begun (Reaction 2). If a radical pairs its unpaired electron by reacting with a second radical, then the chain reaction is terminated, and both radicals "neutralize" each other (Reaction 3).Common Free Radicals: Common Free Radicals Most common free radicals are reactive oxygen (ROS) and reactive nitrogen (RNS) species such as:Other examples: Other examples Certain non-radical molecules have been included in this definition. These are oxidizing agents or molecules that can be easily converted into radicals.Role of transition metals in converting less reactive to more reactive species: Role of transition metals in converting less reactive to more reactive speciesHalf-life of Common Free Radicals: Half-life of Common Free RadicalsCellular enzymatic and non enzymatic antioxidants:: Cellular enzymatic and non enzymatic antioxidants: Superoxide dismutase (SOD) Cu-Zn SOD (in cytosol, SOD1) Mn -SOD (in mitochrondria , SOD2) EC-SOD (Cu-Zn SOD, SOD3) Glutathione peroxidase (GPX) Glutathione reductase (GR) Catalase (CAT) Glutathione (GSH) Glucose-6-phosphate dehydrogenaseCellular enzymatic and non enzymatic antioxidants (2):: Cellular enzymatic and non enzymatic antioxidants (2): GSSG-S- transferase Proteindisulphide isomerase Thioredoxin Thioredoxin reductase Glutaredoxin Peroxiredoxin Heme oxygenase Methionine sulphoxide reductase Paroxonase (PON-1) Thiol -disulphide oxidoreductaseMajor dietary antioxidants: Major dietary antioxidants Vitamin C ( ascorbate ) Vitamin A ( α, β and γ carotenes) Vitamin E (8 different isomers) α- lipoic acid Phytochemicals ( flavanoids , lignans , phenols) Selenium (for GPX activity) Copper (for Cu-Zn SOD activity) Zinc (for Cu-Zn SOD activity and protects S-H groups) Manganese (for Mn -ZN SOD activity)Metal - Binding Proteins: Metal - Binding Proteins Transition metals are tightly bound to various proteins that prevent them from reacting with peroxides to form free radicals. These include: Ceruloplasmin Lactoferrin Metallothionein Transferrin Haemoglobin Myoglobin Cytochrome oxidases FerritinAnalytes with Anti-oxidant properties: Analytes with Anti-oxidant properties Some commonly measured analytes have antioxidant activities: Gamma GT Uric acid Bilirubin HDL CK MyoglobinMolecular Adaptations: Molecular Adaptations Organisms respond to oxidative stress by upregulating a large number of genes that encode for proteins involved in antioxidant reactions. In the yeast ( Saccaromyces cerevisiae ) exposure to H2O2 leads to the altered transcription of ~70 genes.Molecular Adaptations: Molecular Adaptations GAPDH has been identified as a target of oxidative modification in many cellular systems and it may have a regulatory role as a sensor of oxidative stress conditions.Generation of Free Radicals: Generation of Free RadicalsPotential sources of reactive oxygen species (ROS) in skeletal muscle and locations of the major intracellular and extracellular antioxidants.: Potential sources of reactive oxygen species (ROS) in skeletal muscle and locations of the major intracellular and extracellular antioxidants.Cellular sources of ROS: Cellular sources of ROSOther cellular sources of ROS: Other cellular sources of ROS Other oxidases and oxygenases Cytochrome P450 Auto-oxidation reactions eg : FMH2, FAD, catecholamines , tetrahydropteridines (such as tetrahydrobiopterin ) and thiol compounds such as cysteine . Auto-oxidation greatly accelerated by transition metal ions especially manganese, iron and copper ions. Haem proteins: estimated that ~3% of Hb in erythrocytes undergoes oxidation every day exposing these cells to a constant flux of O2.-Mitochondrial production of ROS: Mitochondrial production of ROS In the electron transport chain, electrons are passed through a series of proteins via oxidation-reduction reactions, with each acceptor protein along the chain having a greater reduction potential than the previous. The last destination for an electron along this chain is an oxygen molecule.Mitochondrial production of ROS: Mitochondrial production of ROS 95-99% of oxygen consumed is reduced into water catalyzed by coenzyme Q ( CoQ ): However 1-5% will form the superoxide radical where CoQ is turned into a superoxide generator:Positive Effects of Free Radicals: Positive Effects of Free Radicals Immune system: neutrophils and macrophages use ROS to destroy engulfed microorganisms.Positive Effects of Free Radicals: Positive Effects of Free Radicals Can serve as second messengers or modify oxidation-reduction ( redox ) states. Involved in some enzyme activation. Involved in drug detoxification. Play an essential role in muscle contraction.Negative Effects of Free Radicals: Negative Effects of Free Radicals Because of their reactive nature free radicals can provoke inflammation or altered cellular functions through: lipid peroxidation protein modification DNA modificationInflammation and Oxidative Stress: Inflammation and Oxidative StressFree Radical Products: Free Radical Products Lipid peroxidation Protein modification DNA modificationLipid Peroxidation: Lipid PeroxidationLipid Peroxidation: Lipid Peroxidation Lipid peroxidation is a complex process and a wide range of products are formed in variable amounts. The major products are: α,β- unsaturated reactive aldehydes 4-hydroxy-2-nonenal (HNE) malondialehyde (MDA) 2-propenal ( acrolein ) isoprostanesLipid Aldehydes: Lipid Aldehydes The lipid aldehydes are relatively stable, but reactive, and can diffuse within or escape from the cell and attack targets far from their site of formation. They can be regarded as “second cytotoxic messengers”. They can react with various biomolecules including protein, DNA and phospholipids generating stable end-products. They react with amino acids, mainly Cys , His and Lys, to modify protein structure and function. They can cross-link lipids in cell membranes interrupting structure and fluidity. They can react with DNA to form a number of DNA adducts having mutagenic and carcinogenic effects.Isoprostanes: Isoprostanes Isoprostanes resemble the prostaglandins but are formed in vivo by the non-enzymatic free radical-catalyzed peroxidation of polyunsaturated fatty acids with three or more double bonds: linolenic acid (C18:3 ω3 ) arachidonic acid (C20:4 ω6 ) eicosapentanoic acid (C20:5 ω3).Isoprostanes cause their biological effects by:: Isoprostanes cause their biological effects by: Vasoconstriction, seemingly by activation of receptors, analogous or identical to those for thromboxane . Adduct formation with thiol groups forming glutathione. Adduct formation with lysine groups on proteins and induce cross-links.Protein Modification: Protein Modification Proteins are major targets of free radical attack because of their high abundance and because they are primarily responsible for most functional processes within cells. Protein modification may alter every level of protein structure from primary to quaternary causing major structural changes.Protein Modification: Protein Modification Oxidative damage is induced either directly or indirectly: by reaction of secondary products leading to peptide backbone cleavage or fragmentation, cross-linking, altered susceptibility to proteolytic enzymes, and/or modification of the side chains of virtually every amino acid. by the formation of new reactive groups (from tyr , cys , his, pro, lys , arg , trp , phe , val ) or the formation of protein carbonyls.Protein Modification: Protein Modification Most damage is irreparable and may have a wide range of downstream consequences affecting the function of receptors, enzymes, transport proteins etc. and may generate new antigens provoking an immune response. In turn it can result in secondary damage to other biomolecules such as inactivation of DNA repair enzymes and loss of fidelity of damaged DNA polymerase in replicating DNA.DNA Modification: DNA Modification Free radicals induce several types of DNA damage including strand breaks, DNA-protein cross-links and a large range of base and sugar modifications. Of the free radicals the highly reactive hydroxyl radical (.OH) is the most prominent in the development of base and sugar modifications. DNA damage also occurs through reactive nitrogen species undergoing mainly nitration and deamination of purinesDNA Modification: DNA Modification The net result is an increased risk of mutagenesis and carcinogenesis. Oxidative damage to DNA is repaired by cellular repair systems and DNA base damage is thought to be repaired mainly by base-excision repair.Measurement of Free Radicals: Measurement of Free Radicals Direct measurement Biomarkers of free radical damageDirect Measurement: Direct Measurement Electron spin resonance (ESR) detects the presence of unpaired electrons. By itself it only detects fairly unreactive radicals as reactive radicals do not accumulate to any significant degree. The method can be modified by adding “trapping”, agents that intercept reactive radicals and react with them to form stable radicals, which can then be measured by ESR. However, the technique is not suitable for clinical laboratories because of the need for expensive equipment, expertise and time.Indirect Measurement (Biomarkers): Indirect Measurement (Biomarkers) Measurement of free radical mediated damage on: Total Antioxidant Activity Lipids Proteins DNATotal antioxidant Activity (TAC): Total antioxidant Activity (TAC) is a measure of the total capacity of a plasma sample to quench an oxidative burst of a “free radical” ( peroxynitrate ). The peroxynitrate is neutralised by antioxidants in the sample and this “activity” is measured by its reaction with Phalosin , a photoprotein that emits an intense luminescence upon oxidation. The luminescence is read off a standard curve made using an antioxidant ( Trolox , a vitamin E analogue).Limitations:: Limitations: The TAC assay does not measure total antioxidant activity. Generally measure the low molecular weight antioxidants and exclude the contribution of antioxidant enzymes and metal binding proteins. The major contributor to the TAC assay is urate , often accounting for >50% of the activity. But urate is of limited importance as an antioxidant in vivo . A number of other compounds exist that can react but are not antioxidants. Different TAC assays may not correlate with each other because the various antioxidants react differently in different assays.DNA Modification: DNA Modification Numerous markers are available for measurement of DNA modification. The most used marker is 8-hydroxy-2’-deoxyguanine (8-OHdG), produced by free radical induced guanine oxidation and used as a marker of “whole body” oxidative DNA damage.DNA Modification: DNA Modification The oxidized DNA is continually repaired, and oxidized nucleotides such as 8-OHdG are excreted via blood and urine. Assayed by HPLC and MS techniques. However, 8-OHdG can also arise from degradation and oxidation of guanine in the DNA precursor pool. There are many more products of oxidative DNA damage thus 8-OHdG is a partial measure of damage to guanine residues in DNA and may not truly reflect rates of oxidative damage to DNA. Also likely is artefactual production of 8-OHdG by auto-oxidation during and after sample extraction.Protein Modification: Protein Modification Glutathione and S- glutathionylated proteins Tyrosine oxidation, nitration and halogenation Carbonylated proteinsProtein Modification: Protein Modification Protein carbonyls (C=O) may be generated by the oxidation of several amino acids ( lys , arg , pro and thr ). Carbonyls can arise as a result of protein glycation by sugars, by binding of aldehydes such as lipid peroxidation products and by direct oxidation of amino acid side chains by free radicalsProtein Modification: Protein Modification Protein carbonyl content (PCC) is the most widely used marker of oxidative modification of proteins. There are several methodologies for the quantitation of PCC; in all of them 2,4 dinitrophenyl hydrazine is allowed to react with the protein carbonyls to form the corresponding hydrazone . Measured readily by spectrophotometric and immunoassay techniques.Limitations:: Limitations: Oxidized amino acids can be absorbed from the diet. The general lack of knowledge in oxidized amino acid kinetics, as they may decompose in complex ways ( eg . fragmentation, crosslinking and unfolding) which may accelerate or hinder proteolytic and proteosome mediated turnover. Only a small number of proteins may be carbonylated .Lipid Peroxidation: Lipid Peroxidation 4-hydroxy-2-nonenal (HNE) Malondialdehyde (MDA) 2-propenal ( acrolein ) Isoprostanes Expired pentane, ethane or hexaneMalondialdehyde (MDA): Malondialdehyde (MDA) Common measurement based on the reaction of malondialdehyde (MDA) with thio-barbituric acid (TBA); forming a MDA-TBA2 adduct that absorbs strongly at 532 nm (TBARS assay). Limitations of TBARS assay : Most of the TBA reactive material in body fluids are not related to lipid peroxidation . The TBARS assay can be used as a general marker of lipid peroxidation but noting that it may over-estimate lipid peroxidation .Limitations of assaying MDA: Limitations of assaying MDA MDA is only one of the many aldehydes formed during lipid peroxidation and MDA can also arise from free radical attack on sialic acid and deoxyribose . The concentration of free MDA is probably low in vivo as they readily conjugate to proteins. Lipid aldehydes undergo further metabolism by cells. Lipid peroxides can also be absorbed from the diet.Isoprostanes: Isoprostanes Isoprostanes are specific end-products of free radical peroxidation of unsaturated acids. Of the isomers the most studied are the F2-isoprostanes from arachidonic acid, specifically 8-iso-PGF2a . Isoprostanes are measured by immunoassay or LCMS. At present the measurement of 8-iso-PGF2a is regarded as one of the most reliable approaches to the assessment of free radical-mediated lipid peroxidation in vivo . A tissue that does not contain isoprostanes has yet to be reported.Limitations:: Limitations: They are minor end-products of lipid peroxidation . They are chemically stable in vivo and ex vivo but once they are released into the circulation they are rapidly metabolized undergoing hydrolysis by various phospho -lipases and then by β- oxidation . They are also present in plasma in two forms: esterified to lipids and as free acids, with the esterified form being the most abundant. In urine they exist as only as the free.Free-radical theory of aging: Free-radical theory of aging The free-radical theory of aging (FRTA) states that organisms age because cells accumulate free radical damage over time. For most biological structures, free radical damage is closely associated with oxidative damage. Earlier, this theory was only concerned with free radicals such as superoxide (O 2 - ), but it has since been expanded to encompass oxidative damage from ROS such as H 2 O 2 , or OH - .Free-radical theory of aging: Free-radical theory of aging Denham Harman first proposed the free radical theory of aging in the 1950s, and in the 1970s extended the idea to implicate mitochondrial production of reactive oxygen species. In later years, the free radical theory was expanded to include not only aging per se , but also age related diseases. Free radical damage within cells has been linked to a range of disorders including cancer, arthritis, atherosclerosis, Alzheimer’s disease and diabetes.Evidence: Evidence Mutant strains of the roundworm that are more susceptible to free radicals have shortened lifespan, and those with less susceptibility have longer lifespan. In some model organisms, such as yeast and Drosophila, there is evidence that reducing oxidative damage can extend lifespan. In mice, interventions that enhance oxidative damage generally shorten lifespan. However, in roundworms, blocking the production of the naturally occurring antioxidant (superoxide dismutase) has recently been shown to increase lifespan.Controversies: Controversies Consumption of high levels of antioxidants, which should increase lifespan under the theory, may extend average but not maximum lifespan in mice. The effect, if present, is weak and only inconsistently observed. In one laboratory, Phenybutylnitrone (PBN) was shown to produce about a 10% extension of maximum lifespan in experimental animals. However, this finding has not been reproduced by other laboratories. Antioxidant supplementation has not been conclusively shown to produce an extension of lifespan in a mammal. Whether reducing oxidative damage below normal levels is sufficient to extend lifespan remains an open and controversial question. Remember It all started with O2: Remember It all started with O2The same thing that makes you live can kill you in the end…: The same thing that makes you live can kill you in the end… Thank Yoy … You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.