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Free radicals and antioxidants

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Free radicals and antioxidants
• What is „free radical“?
• Reactive oxygen and nitrogen species
(RONS)
• Are the RONS always dangerous?
• Well known term „oxidative stress“ - what
is it?
• Antioxidants - types and appearance
• Markers of oxidative stress
• Disorders Associated with Oxidative stress
Free radical - what is it?
Atom: proton, neutron, electronic shell (orbital)
Free radical
• particles with an unpaired electron spinning
around the nucleus. (can be atom, ions,
molecule).
• tend to reach equilibrium, plucks an electron
from the nearest intact molecule.
• most of biomoleculs are not radicals
Free radical and „science“
• chemist
during the thirties - there is superoxide
• biochemist
during the sixties - make a discovery of
superoxid dismutase (SOD)
• doctor
free radicals are associated with many
disorders
Mechanism of radical reactions
Radicals are highly reactive species
Three distinc steps
• initiation (homolytic covalent bonds
cleavage)
• propagation (chain propagation)
• termination
ROS (reactive oxygen species)
Free radicals
superoxide, O2 В· hydroxyl radical, OH В·
peroxyl, ROO В·
alkoxyl, RO В·
hydroperoxyl, HO2 В·
Particals, which are not
free radicals
hydrogen peroxide, H2O2
(FentonВґs reaction)
hypochlorous acid, HClO
ozone, O3
singlet oxygen, 1O2
RNS (reactive nitrogen species)
Particals, which are not free
Free radicals
radicals
nitrogen(II) oxide, NO .
+
nitrosyl,
NO
.
nitrogen(IV) oxide, NO2
nitrous acid, HONO
nitogen(III) oxide, N2O3
peroxynitrite, ONOO alkylperoxinitrite, ROONO
The main sources of free radicals
membranes enzymes and/or coenzymes with
flavine structures, hem coenzymes,
enzymes containing Cu atom in an active
site
1. respiratory chain mitochondria : mainly
superoxide and then H2O2
• approx 1- 4% O2 entres into resp. chain
(mainly complexes I a III)
The main sources of free radicals II
2. Endoplasmic reticulum
superoxide creation (by cytochrome P- 450)
3. special cells (leukocytes)
superoxide creation by NADP-oxidas
4. hemoglobin to methemoglobin oxidation
(erytrocyte is „full“ of antioxidants)
Free radicals physiological function
Used by oxides a oxygenes
• cytochromoxidase (toxic intermediates, H2O2
and superoxide, are bound to an enzymu)
• monoxygenases - activate O2 in liver ER or in
adrenal gland mitochondria ; hydroxylation
Free radicals physiological function II
ROS a RNS against bacteria
• enzyme complex NADPH-oxidase of leukocytes
• myeloperoxidase - catalysis of the following
reaction
H2O2 + Cl- + H+ = HClO + H2O
Free radicals physiological function III
• Signal molecules
first messenger пѓћ second messenger пѓћ
information net
This info net function is affected by the redox state
of cells
• redox state : antioxidant capacity, reduction
equivalent availlability, RONS rate
пѓћ ROS: second messenger
Immunity vs. regulation
a massive production of ROS as immunity
instrument
x
an induction of the changes low concentration
ROS, which are probably regulation
mechanism
Antioxidant defence system
3 levels
inhibition of production the abundance of RONS
capture of radicals (scavengers, trappers, quenchers)
correction mechanism of destroyed biomoleculs
Antioxidants and scavengers review
1. Endogennous antioxidants
• enzymes (cytochrome c,SOD, GSHPx, catalase)
• nonenzymatic
- fixed in membranes (пЃЎ -tocopherol, пЃўcaroten, coenzym Q 10)
- out of membranes (ascorbate, transferrin,
bilirubin)
Antioxidants and scavengers review II
2. Exogennous antioxidants
• FR scavengers
• trace elements
• drugs and compounds influence to FR
metabolism
Enzymes defence mechanism
Superoxid dismutase
(EC 1.15.1.1, SOD)
2O2. - + 2H+ п‚® H2O2 + O2
SOD - is present in all oxygen-metabolizing
cells, different cofactors (metals)
an inducible in case of superoxide overproduction
Superoxid dismutase
Mn 2+ SOD (SOD1)
tetramer
matrix mitochondria
lower stability then Cu, Zn - SOD
Superoxid dismutase
Cu 2+/Zn 2+ SOD (SOD 2)
dimer, Cu = redox centr
cytosol, intermitochondrial space
hepatocyt, brain, erytrocyte
high stability, catalysation at pH 4,5-9,5
Glutathion peroxidases
elimination of intracellular hydroperoxides
and H2O2
2 GSH + ROOH п‚® GSSH + H2O + ROH
• cytosolic GSH - glutathionperoxidasa (EC
1.11.1.9, cGPx)
• extracelullar GSH - glutathionperoxidasa
(eGSHPx)
• phospholipidhydroperoxide GSH peroxidase (EC 1.11.1.12, PHGPx)
Catalasa
(EC 1.11.1.6, KAT)
2 H2O2 п‚® 2 H2O + O2
high affinity to H2O2 : peroxisomes
hepatocytes mitochondria, cytoplasm of
erytrocytes
tetramer with Fe, needs NADPH
High-molecula endogennous
antioxidants
• transferrin
• ferritin
• haptoglobin
• hemopexin
• albumin
Low-molecule endogennous antioxidats I
Ascorbate (vitamin C)
Alfa-tocopherol a vitamin E
collagen synthesis
localise in membranes
dopamine to epinephrine produces hydroperoxides,
conversion
which are changes by
GSHPx
reduction agent
Fe absorption
antioxidant = reduction O2 В· OH В·, ROOВ·, HO2 В·
tocopheryl radical
regeneration
prooxidant
Ascorbic acid and its metabolites
Low-molecule endogennous antioxidats II
• ubiquinone (coenzyme Q)
electron carrier in respisratory chain
co-operates with tocopheryl
• carotenoides, -caroten, vitamin A
removing the radicals from lipids
Low-molecule endogennous antioxidats III
• glutathione (GSH, GSSG)
in all mammalian cells (1-10 mmol/l)
important redox buffer
2 GSH пѓ› GSSG + 2e- + 2H+
ROS elimination, stabilisation in reduction form (
SH- groups, tocopheryl and ascorbate
regeneration)
substrate of glutathione peroxidases
Low-molecule endogennous antioxidats IV
• Lipoic acid (lipoate)
PDH cofactor
tocopheryl and ascorbate regeneration
• melatonin
lipophilic ; hydroxyl radicals scavenger
Low-molecule endogennous antioxidats V
• uric acid (urates)
• bilirubin
• flavonoids
Trace elements influence to FR
metabolism
Selenium
influence to vitamin E resorption, part of
selenoproteins
пѓџ of Se = insufficient immun. respons,
erytrocytes hemolysis, methemoglobin synthesis
Zinc
cell membrane stabilisation
Fe antagonist
Oxidative stress
Equilibrium failure between creation and
a elimination of RONS leads to
oxidative stress
Be carefull - this equilibrium can be
disbalance in both sides
Oxidative damage to lipid
Damage
• unsaturated bonds loss
• arising of reactive
metabolites (aldehydes)
Sequel
• changes in fluidity and
permeability of
membranes
• membranes integral
enzymes are
influenced
The peroxidation of linoleic acid
Oxidative damage to proteins
Damage
Sequel
• agregation, fragmentation
and cleveage
• reaction with hem iron
ion
• functional group
modification
• changes in: enzymes
activity, ions transport
• proteolysis
Oxidative damage to DNA
Damage
• saccharide ring
cleveage
• bases modification
• chain breakeage
Sequal
• mutation
• translation mistakes
• protoesynthesis
inhibition
Oxidative stress markers
Free radicals detection
• very difficult, because of chem-phys.
properties
Oxidative stress products detection
• more simple, a wide range of techniques
Oxidative stress markers II
Lipoperoxidation markers:
malondialdehyde (MDA), conjugated diens,
isoprostanes
Oxidative damage to protein markers :
protein hydroperoxides
Oxidative damage to DNA :
modified nucleosides
Antioxidants determination
ascorbate
tocopheryl
SOD
GSHPx
glutathion
Disorders Associated with
Oxidative stress
Neurological
Alzheimers Disease
Parkinson�s Disease
Endocrine
Diabetes
Gastrointestinal
Acute Pancreatitis
Disorders Associated with
Oxidative stress
Others conditions
Obesity
Air Pollution
Toxicity
Inflammation
Literature
Е tГ­pek Stanislav a kol.: Antioxidanty a volnГ©
radikГЎly ve zdravГ­ a nemoci, Grada, 2000
Free radicals and antioxidant protocols
edited by Armstrong D., Methods in
Molecular biology, volume 108, HUMANA
PRESS, Toronto, New Yersey, 1998
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