Oxidative Stress Analytical Aspects

Analytical Aspects
of Oxidative Stress
ByBy
Prof. Dr. Moustafa RizkProf. Dr. Moustafa Rizk
Ass. Prof. of Clinical PathologyAss. Prof. of Clinical Pathology
Faculty of Medicine, University of Alexandria.Faculty of Medicine, University of Alexandria.

What is oxidative stress (OS)?What is oxidative stress (OS)?
Analytical Aspects of Oxidative Stress
Set of intracellular or extracellularSet of intracellular or extracellular
conditions that leads to chemical orconditions that leads to chemical or
metabolic generation of reactive oxygen.metabolic generation of reactive oxygen.

The susceptibility of an organ to OS is aThe susceptibility of an organ to OS is a
function of the overall balance between thefunction of the overall balance between the
factors that exert OS and those that exhibitfactors that exert OS and those that exhibit
antioxidant capability.antioxidant capability.
When an organ will be susceptible to OS?When an organ will be susceptible to OS?

OS is a disturbance in the equilibrium statusOS is a disturbance in the equilibrium status
of pro-oxidant/antioxidant systems in intactof pro-oxidant/antioxidant systems in intact
cells with the increase at the end of thecells with the increase at the end of the
reactive oxygen species (ROS).reactive oxygen species (ROS).

ROS (Free radicals)ROS (Free radicals)
ROS are chemical species possessingROS are chemical species possessing
one or more unpaired electrons in theirone or more unpaired electrons in their
outer most shell.outer most shell.
OO22 && HH && OHOH
The free radical nature is usuallyThe free radical nature is usually
denoted by the subscript dot.denoted by the subscript dot.

1.1. Homolytic cleavageHomolytic cleavage
X:YX:Y →→ X + YX + Y
2.2. OO22 + e+ e →→ OO22
3.3. NO - eNO - e →→ NONO
4.4. Electron transfer is far common processElectron transfer is far common process
than homolytic cleavage.than homolytic cleavage.
-
+
How are free radicals formed?How are free radicals formed?
-
-

1.1. X + YX + Y →→ XYXY Termination ReactionTermination Reaction
2.2. Donate its unpaired electron to anotherDonate its unpaired electron to another
molecule or it might take an electron.molecule or it might take an electron.
What is the sequele of free radicals?What is the sequele of free radicals?

As part of normal intracellular biologicAs part of normal intracellular biologic
processes.processes.
When the normal defenses that serve toWhen the normal defenses that serve to
protect tissues are inadequate.protect tissues are inadequate.
Can be released by inflammatory cells, intoCan be released by inflammatory cells, into
their local environment.their local environment.
Can be initiated by exposure to pollution,Can be initiated by exposure to pollution,
radiation, UV, cigarette smoke.radiation, UV, cigarette smoke.
What are the sources of free radicals?What are the sources of free radicals?

OFR and ROSOFR and ROS
OFR: oxygen is considered to be one of theOFR: oxygen is considered to be one of the
most important free radicals in biochemistrymost important free radicals in biochemistry
(O(O22
--
& OH)& OH)
ROS: collective one that includes not only OROS: collective one that includes not only O22
radicals but also non radical derivatives ofradicals but also non radical derivatives of
oxygen (Hoxygen (H22OO22 & singlet oxygen).& singlet oxygen).

Examples of Reactive Oxygen Species (ROS)Examples of Reactive Oxygen Species (ROS)
1. Hydroxyl radical (HO1. Hydroxyl radical (HO..
))
[The most reactive and damaging of the oxygen radicals][The most reactive and damaging of the oxygen radicals]
produced in living cells as follows:produced in living cells as follows:
-
H-O-HH-O-H H + HOH + HO Hydroxy radicalHydroxy radicalb.
a. FeFe2+2+
+ H+ H22OO22 FeFe3+3+
+ HO + HO+ HO + HO
(Fe-catalysed Haber Weiss reaction)(Fe-catalysed Haber Weiss reaction)
-
c. OO22 + H+ H 22OO22 HO + HO + OHO + HO + O22
(Non catalysed Haber Weiss reaction)(Non catalysed Haber Weiss reaction)
-
High
energy
radiation

2.2. Superoxide radical OSuperoxide radical O22
-
OO22 + e+ e OO22
Transfer of electron to oxygenTransfer of electron to oxygen
(reduction reaction)(reduction reaction)
-
a.a. Leakage of electrons during the normal process ofLeakage of electrons during the normal process of
mitochondrial electron transportmitochondrial electron transport
OO22 + 4H+ 4H++
+ 4e+ 4e 2H2H22OO
b.b. Respiratory burst of neutrophils.Respiratory burst of neutrophils.
c.c. Auto-oxidation of reduced transition metalsAuto-oxidation of reduced transition metals
FeFe2+2+
+ O+ O22 FeFe3+3+
+ O+ O22
-
d.d. By endothelial cells and macrophagesBy endothelial cells and macrophages
-
-

3.3. Hydrogen PeroxideHydrogen Peroxide
OO22 + 2e+ 2e OO22
(2 electron reduction of oxygen)(2 electron reduction of oxygen)
-
HH22OO22 is not a free radical but it can breakdown inis not a free radical but it can breakdown in
presence of transition metal ionspresence of transition metal ions
-
2 O2 O22 + 2H+ 2H HH22OO22 + O+ O22
- +
HH22OO22 + O+ O22 OH + OH + OOH + OH + O22
CuCu+2+2
FeFe +2+2
-

Thus the keyplayers in the biochemistry of oxygenThus the keyplayers in the biochemistry of oxygen
free radical, or reactive oxygen species are oxygenfree radical, or reactive oxygen species are oxygen
itself, superoxide, hydrogen peroxide, transitionitself, superoxide, hydrogen peroxide, transition
metal ions and the hydroxyl radical. The first 4 ofmetal ions and the hydroxyl radical. The first 4 of
which conspire by a variety of reactions to generatewhich conspire by a variety of reactions to generate
the last (Hydroxyl radical).the last (Hydroxyl radical).

Effect of ROS on cellular injuryEffect of ROS on cellular injury
Effect of the hydroxyl radicalEffect of the hydroxyl radical
a.a. OH generation on DNA continue by theOH generation on DNA continue by the
reaction of Hreaction of H22OO22 with transition metal ionswith transition metal ions
bound to DNA.bound to DNA.
OH attack purines, pyramidines bases ofOH attack purines, pyramidines bases of
DNADNA →→ breakage, mutations, alterations,breakage, mutations, alterations,
protein degradation.protein degradation.

b.b. OH interacts with unsaturated fatty acidsOH interacts with unsaturated fatty acids
XX
(reactive free radical)(reactive free radical)
AbstractAbstract
Methylene hydrogenMethylene hydrogen
XHXH
LL
LH (PUSFAs)LH (PUSFAs)
OO22
LOOH (LOOH (Lipid peroxide)Lipid peroxide)
LOOLOO LHLH
(Lipid peroxy radical)(Lipid peroxy radical)
(Lipid radical)(Lipid radical)

Transition metal ions are involved in lipidTransition metal ions are involved in lipid
peroxidation in 2 ways:peroxidation in 2 ways:
1.1. Can participate in the first initiationCan participate in the first initiation
2.2. Decompose lipid peroxides into peroxyl andDecompose lipid peroxides into peroxyl and
alkoxylalkoxyl
LOOHLOOH LOLO LOO, LOLOO, LO
LHLH
LL
LOOHLOOH
peroxidation chain canperoxidation chain can
become self perpetuatingbecome self perpetuating
FeFe2+2+

Lipid peroxides and tissue injury :Lipid peroxides and tissue injury :
Double bonds of PUSFAs are susceptible to beDouble bonds of PUSFAs are susceptible to be
attacked by oxidation.attacked by oxidation.
- CH- CH - C -- C - ++ OO22 C + CHC + CH
OO22
CarbonCarbon
centeredcentered
radicalradical
OHOH
HydroxylHydroxyl
radicalradical
- C -- C -- C -- C -
OO22HH
++
Lipid hydroxyperoxideLipid hydroxyperoxide

Disrupt membrane function.Disrupt membrane function.
- C -- C -
OO22HH
LipidLipid
hydroxyperoxidehydroxyperoxide
Decompose to highly cytotoxicDecompose to highly cytotoxic
products as aldehydesproducts as aldehydes
Inactivation of membraneInactivation of membrane
receptorsreceptors
Membrane rigidityMembrane rigidity

Oxidative Modification of LDL :Oxidative Modification of LDL :
Native LDL undergoes oxidative modification byNative LDL undergoes oxidative modification by
intracellular oxygen free radicalintracellular oxygen free radical →→ peroxidation ofperoxidation of
(PVFAs) in LDL lipids.(PVFAs) in LDL lipids.
If the process continueIf the process continue →→ highly reactive productshighly reactive products
accumulate in the LDL particles as malon-accumulate in the LDL particles as malon-
dialdehyde, lipid peroxides, lysophosphotides.dialdehyde, lipid peroxides, lysophosphotides.

Oxidative Modification of LDL :Oxidative Modification of LDL :
These products induce functional changes in theThese products induce functional changes in the
endothelial wallendothelial wall →→ favor penetration offavor penetration of
monocytes and movement of oxidized LDL intomonocytes and movement of oxidized LDL into
the subendothelial spacethe subendothelial space →→ ↑↑ fatty streak.fatty streak.
The previous cascade of events have been termedThe previous cascade of events have been termed
the oxidative hypothesis of atherosclerosis.the oxidative hypothesis of atherosclerosis.

Lipid Peroxides and ProstaglandinsLipid Peroxides and Prostaglandins
ROOHROOH Arachidonic acidArachidonic acid
PGEsPGEs
ProstacyclinProstacyclin TXATXA22
PlateletPlatelet AggregationAggregation
AtherosclerosisAtherosclerosis ThrombosisThrombosis
IschemiaIschemia
+
-
- +

Estimation of free radicals & lipid peroxidesEstimation of free radicals & lipid peroxides
1.1. Direct assaysDirect assays
a.a. HH22OO22 in exhaled airin exhaled air
b.b. HH22O in human urineO in human urine
c.c. NMR spectroscopy for metal ionsNMR spectroscopy for metal ions
d.d. Electron spin resonance (ESR)Electron spin resonance (ESR)

2.2. Indirect assaysIndirect assays
a.a. Trapping assay:Trapping assay:
Salicylate (2 hydroxybenzoate) used as a trapSalicylate (2 hydroxybenzoate) used as a trap
for OHfor OH →→ 2, 3 dihydroxybenzoate2, 3 dihydroxybenzoate
b.b. Uric acid oxidationUric acid oxidation
Uric acid acts as antioxidantUric acid acts as antioxidant in vivoin vivo..
The major end product of uric acid oxidation isThe major end product of uric acid oxidation is

c.c. Finger print assays:Finger print assays:
ROS can produce chemical changes when theyROS can produce chemical changes when they
react with biological molecules such as DNAreact with biological molecules such as DNA
and lipids. Measurement of end products canand lipids. Measurement of end products can
be regarded as a finger print assay.be regarded as a finger print assay.

1.1. Measurement of DNA damage can beMeasurement of DNA damage can be
estimated by examining urine samples forestimated by examining urine samples for
the presence of thymidine glycol (Tg) usingthe presence of thymidine glycol (Tg) using
HPLC.HPLC.

2.2. Measurement of end products of lipidMeasurement of end products of lipid
peroxidation:peroxidation:
• They are often the first parameters to whichThey are often the first parameters to which
researchers turn when they wish to prove theresearchers turn when they wish to prove the
involvement of free radicals in call damage.involvement of free radicals in call damage.

• TBA assay is the most popular and easiestTBA assay is the most popular and easiest
method used as an indicator of lipidmethod used as an indicator of lipid
peroxidation.peroxidation.

• The method measured the amount of MDA and MDA-The method measured the amount of MDA and MDA-
like substances formed by the lipid peroxidationlike substances formed by the lipid peroxidation
process. It involved heating the sample under acidicprocess. It involved heating the sample under acidic
condition with TBA which react with MDA to form a redcondition with TBA which react with MDA to form a red
MDA-TBA complexMDA-TBA complex

A measure of total serum lipid peroxidationA measure of total serum lipid peroxidation
has proven to be a simple, inexpensive andhas proven to be a simple, inexpensive and
accurate means of reflecting whole bodyaccurate means of reflecting whole body
free radical activity. This test is gainingfree radical activity. This test is gaining
general acceptance in the researchgeneral acceptance in the research
laboratory as a simple, standard means oflaboratory as a simple, standard means of
assessing the body's antioxidant capabilityassessing the body's antioxidant capability
or overall oxidative stress.or overall oxidative stress.

Data show design & preparation by : Dr. El-Sayed Amr - (012) 3106023Data show design & preparation by : Dr. El-Sayed Amr - (012) 3106023

Oxidative Stress Analytical Aspects

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