FREE RADICALES

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Free radicals
M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
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Free radicals

• Definition
• Free radical is a molecule or molecular
  fragment that contains one or more unpaired
  electrons in its outer orbits.

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Electron configuration in free radial
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Introduction

• 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.

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Reactive oxygen species

• 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)

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Endogenous generation of free radicles

• Free radicles are generated in oxidative
  metabolism due to leak of electrons .
• Flavoprotein linked oxidases like xanthine
  oxidase , L a 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 )

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Endogenous generation of free radical

• 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 .

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contd

• Free radicals are formed cytochrome P450
  reductase enzyme complex durinrg metabolism of
  xenobiotics .
• ß oxidation of very long chain fatty acids in
  peroxisomes produces H2O2 .

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Electron leak generates free radical

• 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 .

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contd

• Cytochrome C oxidase does not release partially
  reduced intermediates , this crucial criterion
  meets by holding O2 tightly between Fe Cu atoms
  .

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contd

• 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 .

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Electrons can be gained by univalent reduction
which may account for 1 -5 of total oxygen
consumption .
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Free radicals from flavoprotien linked oxidases.

• Flavoprotien linked oxidases
• 1 ) Xanthine oxidase ,
• 2) L a amino acid oxidase ,
• 3 ) Aldehyde dehydrogenase .
• Reduction of isoalloxazine ring of flavin
  nucleotides takes place in 2 steps via a
  semiquinone ( free radical ) intermediate.

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contd

• xanthine oxidase
• Hypoxanthine xanthine
• O2 O2
• acetaldehyde dehydrogenase
• Acetaldehyde
  acetate
• O2 O2

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Respiratory burst

• 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 .

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contd

• activation of inflammatory cell
• drastic increase in consumption of oxygen
  (respiratory burst )
• 10 of oxygen uptake by macrophage is used for
  free radical generation .

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contd

• In chronic granulomatous disease the NADPH
  oxidase is absent in macrophages neutrophils .
• Streptococci pneumococci themselves produce
  H2O2 therefore they are destroyed by
  myeloperoxidase system .

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contd

• Staphylococci being catalase ve can detoxify
  H2O2 in the macrophages they are not destroyed
  .
• Hence recurrent pyogenic infections by
  staphylococci are common in CGD .

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Free radicals from metabolism of eicosanoids

• 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 .

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Free iron producing free radicals

• Super oxide ion can release iron from ferritin .

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contd

• 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.

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Cytochrome P 450 reductase
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Other factors

• 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 .

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Lipid peroxidation

• 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

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Initiation 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 .

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Propagation phase

• 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 .

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Termination phase

• The above reactions would proceed unchecked till
  a peroxyl radical reacts with another peroxyl
  radical to form inactive products .
• ROO ROO RO- -ORO2
• R R R - - R
• ROO R RO- -OR

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Intracellular antioxidants

• Super oxide dismutase
• Catalase
• Glutathione peroxidase
• Cytochrome oxidase

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Super oxide dismutase

• 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 .

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Super oxide dismutase
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contd

• 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 .

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Catalase

• 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.

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contd

• 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 .

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Contd

• 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 .

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contd

• 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.

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Contd

• 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 2H2oO2

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Glutathione peroxidase

• 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 .

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Membrane antioxidants

• Vitamin E lipid soluble , chain breaking
  antioxidant.
• ßcarotene 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 .

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Extra cellular antioxidants

• 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
  stoichiometric O2 scavenging ,binds copper ions
  utilizes H2O2 for reoxidation of copper .

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contd

• 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 .

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Chain breaking antioxidants

• Water soluble urate , ascorbates ,
• thiols ,bilirubin,
• flavanoids.
• Lipid soluble tocopherol ,
• ubiquinol 10 ,
• ß carotene .
• Urate vitamin E acts in lipid phase to trap
  ROO radicals .

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Preventive antioxidants

• 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 .

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Vitamin E

• 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 atocoferol vitamin appears to concentrate
  at these sites.

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contd

• 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 vitamin C
  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.

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contd

• ROO TocOH ROOH TocO
• ROO TocO ROOH non free
• 
  radical product

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contd

• ROO TocOH ROOH TocO
• ROO TocO ROOH non free
• 
  radical product

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ss
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contd

• The antioxidant action of vitamin E is effective
  at high oxygen concentrations , it is
  concentrated in lipid structures exposed to high
  O2 partial pressures such as the erythrocyte
  membrane , membranes of respiratory tree the
  retina.

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contd

• 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 .

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Vitamin A as antioxidant

• Carotenoids are capble of quenching singlet
  molecular oxygen .
• Carotenoids like lycopene ,ß carotene , are
  important biological molecules that can
  inactivate electronically excited molecules by
  process called quenching.
• ßcarotene related compounds can acts as chain
  breaking antioxidant.
• Can acta as preventive antioxidant by decreasing
  the formation of methyl linoleate hydroperoxide .

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contd

• Singlet oxygen is capable of inducing damage to
  the DNA .
• Lycopene shows greater quenching ability than
  ßcarotene (double ability )
• Comparing the structures opening of the ß
  ionine ring increases the quenching ability.

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contd

• 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 .

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