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Free Radicals -Rizky

Disruption Abdulah-
FREE RADICALS

•Free radicals are reactive oxygen species (ROS) that


produced in most of oxidation reactions in our body.
Most of reactions happens in mitochondria.

•In small amount ROS play important role as intracellular


second messenger, encounter disease, etc. But in
higher amounts of ROS can cause many
patophysiologycal conditions.

•Free radicals has been associated with diverse
pathophysiologycal events. More recently, it also play
role in the development of vasculopathies, including
atherosclerosis, hypertension, and restenosis after
angioplasty.

Arterial Sclerosis Cancer

Cardiac Arrest Immune Reactions

Stroke Inflamatory
Illnessess
Cardiovascular Free Diabetes
Diseases Radicals
Radiation Sickness Aging

Poisoning Alzheimer Diseases

Smoking Illnessess Parkinson Diseases


Potential Sources of ROS
Fenton Reaction

Highlighted in gray are some of the most important ROS in vascular cells.
Oxidases convert oxygen to O2 ・ -, which then dismutated to H2O2 by
superoxide dismutase (SOD). H2O2 can be converted to H2O by catalase or
glutathione peroxidase (GSH-Px) or to hydroxyl radical ( ・ OH) after reaction
with Fe2+. In addition, O2 ・ - reacts rapidly with nitric oxide (NO ・ ) to form
peroxynitrite (OONO-).
Free Radicals :

1.Nitric oxide (NO ・ )


§Normally produced by endothelial nitric oxide synthase
(eNOS) in the vasculature
§Patophysiologycally (inflammatory states) produced by
inducible NOS, can be expressed in macrophages and
smooth muscle cells.
§NO ・ is a crucial mediator of endothelium-dependent
vasodilatation by activating the vascular smooth muscle cell
(VSMC) guanylate cyclase.
§NO ・ also play a role in platelet aggregation and in
maintaining the balance between smooth muscle cell
growth and differentiation.
2. Peroxynitrite (ONOO ・ -)
§ONOO ・ - is an important mediator of lipid peroxidation
and protein nitration , including oxidation of LDL. These
events are some of the earliest atherogenic process.
-
§When O2 ・ in concert with NO ・ , they rapidly react to
form the highly reactive molecule ONOO ・ -.
3. Superoxide (O2 ・ -)
-
§All types of vascular cells produce O 2
・ as a mitochondrial
source of ROS.
-
§O2 ・ can be made from one electron reduction of oxygen
by a variety of enzymes (oxidases), such as cytochrome
P450 and membrane-associated NAD(P)H oxidase(s).
§Under some circumstances, eNOS becomes uncoupled and
producing O2 ・ - rather than NO ・ .
-
§O2 ・ is a mediator of VSMC growth, differentiation, and
apoptosis.
4. Hydrogen peroxide (H2O2)
-
•H2O2 is a more stable ROS, a dismutation product of O2 ・
by superoxide dismutase (SOD), which is then converted
to H2O by either catalase or glutathione peroxidase (GSH-
Px).
-
•Similar with O 2
・ , H2O2 can be produced in reactions
catalyzed by cytochrome P450 and membrane-associated
NAD(P)H oxidase(s).
•H2O2 is important mediator for VSMC growth,
differentiation, and apoptosis.
5.Hydroxyl radical ( ・ OH)
•・ OH can be produced by various mechanisms, especially
by the Fenton reaction of hydrogen peroxide (which
diffuses into the nucleus) and metals and other
endogenous and exogenous ROS.
•The ・ OH attacks DNA strands when it is produced
adjacent to cellular and mitochondrial DNA causing the
addition of DNA bases new radicals, which lead to the
generation of a variety of oxidation products.
Intracelullarly low amount of ROS can act as intracellular second
messengers, modulating the function of biochemical pathways.

Higher amounts of ROS can cause


DNA damage, significant toxicity,
or even apoptosis.
ENERGY

•Energy-rich molecules, such as glucose, are


metabolized by a series of oxidation reactions
yielding CO2 and water.

•The metabolic intermediates of these reactions
donate to a specific coenzymes NAD+ and FAD to
form the energy-rich reduced coenzymes, NADH
and FADH2

•These reduced coenzymes donate a pair of
electrons to enter electron transport chain and a
process calles oxidative phosphorylation.


Electron Transport Chain and Oxidative Phosphorylation

Electron transport chain is present in mitochondria, happens in ATP synthase


complexes, consist of five separated protein complexes. Complexes I to IV
need great portion of oxygen to donate or receive electrons to form water.
In addition, these complexes also release partially reduced oxygen (ex: O2-),
Is dealt with enyzme such as SOD. Complex V catalyzes ATP synthesis.
Oxidative Stress

Oxidative stress is characterized by an imbalance


between increased exposure to free radicals,
principally derived from oxygen, and antioxidant
defenses, comprised of both small molecular weight
antioxidants, such as glutathione, and antioxidant
enzymes, such as GPx and SOD.
Oxidative Stress as the Connection Between Nutrition
Overload, Diabetes, and Related Cardiovascular Complication
Biomarker of Oxidative Stress

1.Lipid peroxidation assays : Lipid hydroperoxydes, TBARS,


Malondialdehyde (MDA), F2-isoprostanes, various HETEs.

Lipid hydroperoxydes (LOOH)


LOOH are the result of oxidation of fatty acids by any species
sufficiently reactive to abstract hydrogen from a methylene
group, forming a carbon-centered radical that reacts with
molecular oxygen to form the lipid peroxide.

Thiobarbituric Acid Reactive Substances (TBARS)


TBARS is a harmful substances formed by lipid peroxidation, as
degradation products of fats in foodstuff. Plasma concentrations
of TBARS are an index of lipid peroxidation and oxidative stress.
Malondialdehyde (MDA)
MDA is a naturally occurring product (end product) of lipid peroxidation
and prostaglandin biosynthesis that is mutagenic and carcinogenic. It
reacts with DNA to form adducts to deoxyguanosine and
deoxyadenosine. Therefore, MDA is a marker for oxidative stress.

Isoprostanes
Isoprostanes are prostaglandin (PG)–like substances that are
produced in vivo independently of cyclooxygenase (COX) enzymes,
primarily by free radical-induced peroxidation of arachidonic acid.
F2-isoprostanes is the most reliable approach to assess oxidative
stress status in vivo, providing an important tool to explore the
role of oxidative stress in the pathogenesis of human disease.
Biomarker of Oxidative Stress

2.Protein oxidation assays : Protein carbonyls, various


tyrosine products, methionine sulfoxidation.

Protein carbonyls
Protein carbonyls are formed by a variety of oxidative
mechanisms and
are sensitive indices of oxidative injury. It can be used to
measure oxidized protein in plasma, serum, cell lysates, and
tissue homogenates.

Various tyrosine products


Tyrosine products are found in several proteins as a
product of UV irradiation, -irradiation, aging, exposure to
oxygen free radicals, nitrogen dioxide, peroxynitrite, and
lipid hydroperoxides. Interest of dityrosine in proteins is
based on its potential as a specific marker for oxidatively
damaged proteins and their selective proteolysis, hence it
could be used as a marker for oxidative stress
Biomarker of Oxidative Stress

3.DNA oxidation assays : 8-OHdG, oxidation changes by


the Comet assay, M1G.

8-hydroxy-2' -deoxyguanosine (8-OHdG)


The interaction of ・ OH with the nucleobases of the DNA strand,
such as guanine, leads to the formation of C8-hydroxyguanine (8-
OHGua) or its nucleoside form deoxyguanosine (8-OHdG). Although
the other nucleobases of DNA react with ・ OH in a similar manner,
the 8-OHdG lesion is the most abundant DNA lesion because it is
relatively easily formed and is promutagenic, and therefore a
potential biomarker of oxidative stress.

M1G (pyrimido[1,2-a]purin-10(3H)-one)
The major adduct to DNA is a pyrimidopurinone called M1G. M1G is
a tool for detecting DNA damage that may lead to cancer. Free M1G
is also biomarker for oxidative stress.
Biomarker of Oxidative Stress

4.Antioxidant : Ascorbic acid, tocopherols, GSH, GSSG,


uric acid, TAS (total antioxidant status)
Biomarker of Oxidative Stress

Antioxidant measurement

• TAS and endogen antioxidant (SOD, GPx, catalase) measurement


are biomarkers that represent oxidative stress.
• These biomarkers can be used to measure the efficacy of antioxidant
therapy.
• Measurement of all known antioxidants separately and the interactions
among different antioxidants species find difficulties, the total
antioxidant status in vivo may be an important protective factor.
The Antioxidant Defen s e
System
1 . Primary Antioxidants
•Decrease the Formation of New Free Radicals
(SOD,GPx)
•Metallothionein (Ceruloplasmin )

GPx Dependent to Selenium

SOD Dependent to Zinc, Copper, Iron and


Manganese.
The Antioxidant Defen s e
System
2 . Secondary Antioxidants
•Trap Radicals Preventing the Amplification of
Radical Species (Vit E, Vit C,  Carotene, Uric
Acid, Bilirubin, Albumin)
The Antioxidant Defen s e
System
3 . Tertiary Antioxidants
•Biomolecule Repair Mechanism
DNA Repair Enzymes
Methionine Sulphoxide Reductase
-Abdulah R, 2010-