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Free radicles
 

Free radicles

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    Free radicles Free radicles Presentation Transcript

    •  Definition: Free radical is a molecule or molecular fragment that contains one or more unpaired electrons in its outer orbits.
    •  Free radicals conventionally represented by super script dot R•  Characteristics of free radicals : 1 )Tendency of free radicals to acquire electrons from other substance makes it more reactive. 2 )Short life span 3 )Generation of new free radicals by chain reaction. 4 )Damage to various tissues.
    •  Partial reduction of oxygen leads to formation of free radicals called as reactive oxygen species .The following are members of this group. Super oxide anion radical (O2 · ) Hydroperoxy radicals ( HOO· ) Hydroxyl radical ( OH· ) Lipid peroxyl radical ( ROO· ) Nitric oxide ( NO· ) , Peroxy nitrite (ONOO · ) H2O2 , singlet oxygen (are not free radicals)
    •  Free radicles are generated in oxidative metabolism due to leak of electrons .  Flavoprotein linked oxidases like xanthine oxidase , L α amino acid oxidase .  Super oxide is formed by autooxidation of hemoglobin to methemoglobin (approximately 3 % of the Hb has been calculated to autooxidise per day )
    •  Cyclooxygenase & lipoxygenase reactions in metabolism of eicosanoids.  NADPH oxidase system of inflammatory cells by process of respiratory burst during phase of phagocytosis.  Free iron causes increased production of free radicals .
    •  Free radicals are formed cytochrome P450 reductase enzyme complex durinrg metabolism of xenobiotics .  β oxidation of very long chain fatty acids in peroxisomes produces H2O2 .
    •  Transfer of 4 electrons from reduced cytochrome C to molecular oxygen assisted by cytochrome oxidase  Transfer of 4 electrons lead to safe product H2O .  Site of electron escape appears to be ubiquinone & cytochrome C .
    •  Cytochrome C oxidase does not release partially reduced intermediates , this crucial criterion meets by holding O2 tightly between Fe & Cu atoms .
    •  Although Cyt C oxidase & other protiens that reduce O2 are remarkably successful in not releasing intermediates , small amounts of super oxide & peroxyl radicals are unavoidably formed.  About 1-4 % of oxygen taken up in the body is converted to free radical .
    •  Flavoprotien linked oxidases 1 ) Xanthine oxidase , 2) L α amino acid oxidase , 3 ) Aldehyde dehydrogenase .  Reduction of isoalloxazine ring of flavin nucleotides takes place in 2 steps via a semiquinone ( free radical ) intermediate.
    • xanthine oxidase Hypoxanthine xanthine O2 O2 · acetaldehyde dehydrogenase Acetaldehyde acetate O2 O2 ·
    •  NADPH oxidase inflammatory cell produce supere oxide anion by a process of respiratory burst during phagocytosis.  This is the deliberate production of free radicals by the body .
    • activation of inflammatory cell drastic increase in consumption of oxygen (respiratory burst ) 10% of oxygen uptake by macrophage is used for free radical generation .
    •  In chronic granulomatous disease the NADPH oxidase is absent in macrophages & neutrophils .  Streptococci & pneumococci themselves produce H2O2 therefore they are destroyed by myeloperoxidase system .
    •  Staphylococci being catalase + ve can detoxify H2O2 in the macrophages & they are not destroyed .  Hence recurrent pyogenic infections by staphylococci are common in CGD .
    •  Prodstaglandin H synthase & lipooxygenase enzyme catalysed reactions produce free radicals , by producing peroxide .  Macrophages produce NO from arginine by enzyme nitric oxide synthase , this is also an important anti bacterial mechanism .
    •  Super oxide ion can release iron from ferritin .
    •  The capacity to produce tissue damage by H2O2 is minimal because this is not a free radical . But in the presence of free iron H2O2 can generate hydroxyl free radical (OH ·)which is highly reactive.
    •  Ionising radiation damages tissues by producing hydroxylradical , H2O2 ,super oxide anion .  Light of appropriate wave length can cause photolysis of oxygen to produce singlet oxygen .  Cigarette smoking contains high concentrations of free radicals.  Other toxic compounds CCl4 drugs & inhalation of air pollutants will increase free radical production .
    •  Polyunsaturated fatty acids present in cell membranes are destroyed by peroxidation.  This occurs by three phases. 1 )intiation phase 2 )prolangation phase 3 )termination phase
    •  Production of carbon centered free radical R· ( or ) ROO· (lipid peroxide radical ) 1 )RH +OH· R· + H2O metal ion 2 )ROOH ROO· + H+ R· , ROO· degraded to malon dialdehyde . It is estimated as an indicator of fatty acid break down by free radical .
    •  Carbon centered radical rapidly reacts with molecular oxygen forms peroxyl radical (ROO· ) which can attack another PUFA . R· + O2 ROO· ROO· + RH ROOH + R·  One free radical generates another free radical in the neighbouring molecule a chain reaction (or) propagation is intiated .
    •  The above reactions would proceed unchecked till a peroxyl radical reacts with another peroxyl radical to form inactive products . ROO· + ROO· RO- -OR+O2 R· + R· R - - R ROO· + R· RO- -OR
    •  Super oxide dismutase  Catalase  Glutathione peroxidase  Cytochrome oxidase
    •  Chief amongst the enzymes that defense against ROS is super oxide dismutase .  Super oxide dismutase is present in all major aerobic tissues .  Eukaryote contains 2 forms of this enzyme, 1 ) Copper Zinc dependent cytosolic enzyme 2 ) manganese containing mitochondrial enzyme .
    •  The active site of cytosolic enzyme in eukaryotes contains a copper ion & Zinc ion coordinated to the side chain of a histidine residue .  The negatively charged superoxide is guided electrostatically to a very positively charged catlytic site at the bottom of the channel .
    •  H2O2 formed by SOD & by other processes is scavenged by catalase ( a ubiquitous heme protein that catalyze the dismutation of H2O2 into H2O & O2.)  Catalase is found in blood bone marrow mucous membranes , liver & kidney .  SOD & catalase are remarkably efficient , performing their reactions at or near the diffusion limited rate.
    •  The Kcat / Km ratio of enzyme super oxide dismutase is 7x 10 9 enzymes that have high K cat / K m ratio at the uper limits have attained kinetic perfection.  Their catalytic velocity restricted only by the rate at which they encounter the substrate in the solution .
    •  For catalytically perfect enzymes , every encounter between enzyme & substrate is productive .  Any rate in catalytic rate can come only by decreasing the diffusion .  Circe effect : In this case the electrostatic attractive forces on the enzyme entice the substrate to the active site .
    •  Catalase decreases the free energy of activation ∆G 1 of H2O2.  In the absence of catalase ∆G1 free energy of activation is 18Kcal / mol where as in the presence of catalase 7 Kcal / mol .  K cat / Km value of catalase is 4 X 107.
    •  Catalase is a heme protein containing 4 heme groups .  In addition to possessing peroxidase activity , it is able to use one molecule of H2O2 as a substrate electron donor & another molecule of H2O2 as oxidant or electron acceptor . catalase 2H2O2 2H2o+O2
    •  This enzyme is remarkable in containing a modified aminoacid selenocystein at its active site in which selenium has replaced the sulphur .  The enzyme catalyzes the destruction of H2O2 & lipid hydroperoxides by reduced glutathione , protecting the membrane lipids & hemoglobin against oxidation by peroxides .
    •  Vitamin E : lipid soluble , chain breaking antioxidant.  βcarotene & its anologues (lycopene & retinyl stearate ): lipid soluble radical scavenger & singlet oxygen quencher .  Coenzyme Q : may acts as antioxidant in addition to its major role in energy metabolism .
    •  Transferrin : binds ferric ions ( 2 per mole of protein )  Lactoferrin : binds ferric ions at low pH ( 2 per mole of protein )  Haptoglobins : binds hemoglobin  Albumin : binds copper , heme , scavenges OH.  Ceruloplasmin : ferrooxidase activity – stoichiometricO2 scavenging ,binds copper ions utilizes H2O2 for reoxidation of copper .
    •  Ascorbic acid OH radical scavenger  Bilirubin : scavenges peroxyl radicals, open chain tetra pyrroles are effective singlet oxygen quenchers .  Urate : radical scavenger & metal binder  Mucus : scavenges OH radicals  Glucose : OH radical scavenger .
    •  Water soluble : urate , ascorbates , thiols ,bilirubin, flavanoids.  Lipid soluble : tocopherol , ubiquinol 10 , β carotene .  Urate & vitamin E acts in lipid phase to trap ROO· radicals .
    •  Preventive antioxidants reduces the rate of chain intiation .  Preventive antioxidants include Catalase , peroxidases , Ceruloplasmin , transferrin , albumin.  EDTA , DTPA acts anti xidants by chelating metal ions .
    •  Vitamin E is most important natyral antioxidant .  Vitamin E apear to be the 1st line defense against peroxidation of PUFA contained in cellular & subcellular membrane phospholipids .  The phospholipids of mitochondria, endoplasmic reticulum & plasmamembranes possess affinities for αtocoferol & vitamin appears to concentrate at these sites.
    •  The tocopherools acts as antioxidants by breaking free radical chain reactions as a result of their ability to tranfer a phenolic hydrogen to peroxyl free radical of a peroxidized PUFA .  The phenoxy free radical may react with vitaminC to regenerate tocopherol or it react with further peroxyl free radicl so that the chromane ring & the side chain are oxidized to the non free radical product.
    • ROO· +TocOH ROOH +TocO· ROO· +TocO· ROOH + non free radical product
    • ROO· +TocOH ROOH +TocO· ROO· +TocO· ROOH + non free radical product
    •  The antioxidant action of vitamin E is effective at high oxygen concentrations , & it is concentrated in lipid structures exposed to highO2 partial pressures such as the erythrocyte membrane , membranes of respiratory tree & the retina.
    •  Glutathione peroxidase contains Selenium, provides a second line of defense against hydroperoxides.  Tocopherol & selenium reinforce each other in their action against lipid peroxides.  Selenium is required for normal pancreatic function thus promoting absorption of lipids & vit E  Vitamin E reduces selenium requirement by preventing loss of selenium from the body or maintaining it in an active form .
    •  Carotenoids are capble of quenching singlet molecular oxygen .  Carotenoids like lycopene ,β carotene , are important biological molecules that can inactivate electronically excited molecules by process called quenching.  βcarotene & related compounds can acts as chain breaking antioxidant.  Can acta as preventive antioxidant by decreasing the formation of methyl linoleate hydroperoxide .
    •  Singlet oxygen is capable of inducing damage to the DNA .  Lycopene shows greater quenching ability than βcarotene (double ability )  Comparing the structures opening of the β ionine ring increases the quenching ability.
    •  Quenching ability of cartenoids not only depends on triplet energy state that is the length of the conjugated double bond system but also on the functional groups .