Analytical Aspects of Oxidative Stress

Analytical Aspects of Oxidative Stress

ByByProf. Dr. Moustafa RizkProf. Dr. Moustafa Rizk

Ass. Prof. of Clinical PathologyAss. Prof. of Clinical PathologyFaculty of Medicine, University of Alexandria.Faculty of Medicine, University of Alexandria.

What is oxidative stress (OS)?What is oxidative stress (OS)?


Set of intracellular or extracellular Set of intracellular or extracellular conditions that leads to chemical or conditions that leads to chemical or

metabolic generation of reactive oxygen.metabolic generation of reactive oxygen.

The susceptibility of an organ to OS is a The susceptibility of an organ to OS is a function of the overall balance between the function of the overall balance between the factors that exert OS and those that exhibit factors 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 status OS is a disturbance in the equilibrium status of pro-oxidant/antioxidant systems in intact of pro-oxidant/antioxidant systems in intact cells with the increase at the end of the cells 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 possessing ROS are chemical species possessing one or more unpaired electrons in their one or more unpaired electrons in their outer most shell.outer most shell.


OO22 & & H H & & OHOH

The free radical nature is usually The free radical nature is usually denoted by the subscript dot.denoted by the subscript dot.


1.1. Homolytic cleavageHomolytic cleavage

X:Y X:Y →→ X + Y X + Y

2.2. OO22 + e + e →→ O O22

3.3. NO - e NO - e →→ NO NO

4.4. Electron transfer is far common process Electron transfer is far common process than homolytic cleavage.than homolytic cleavage.

-

+

How are free radicals formed?How are free radicals formed?

-

-

1.1. X + Y X + Y →→ XY XY Termination ReactionTermination Reaction

2.2. Donate its unpaired electron to another Donate 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 biologic As part of normal intracellular biologic processes.processes.

When the normal defenses that serve to When the normal defenses that serve to protect tissues are inadequate.protect tissues are inadequate.

Can be released by inflammatory cells, into Can 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 the OFR: oxygen is considered to be one of the most important free radicals in biochemistry most important free radicals in biochemistry

(O(O22-- & OH) & OH)

ROS: collective one that includes not only OROS: collective one that includes not only O2 2

radicals but also non radical derivatives of radicals 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-H H-O-H H + HO H + HO Hydroxy radicalHydroxy radicalb.

a. FeFe2+2+ + H + H22OO22 Fe Fe3+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

Analytical Aspects of Oxidative Stress-

OO22 + e + e O O22

Transfer of electron to oxygenTransfer of electron to oxygen(reduction reaction)(reduction reaction)

-

a.a. Leakage of electrons during the normal process of Leakage of electrons during the normal process of mitochondrial electron transportmitochondrial electron transport

OO22 + 4H + 4H++ + 4e + 4e 2H 2H22OOb.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 Fe Fe3+3+ + O + O22-

d.d. By endothelial cells and macrophagesBy endothelial cells and macrophages

-

-

3.3. Hydrogen PeroxideHydrogen Peroxide


OO22 + 2e + 2e O O22

(2 electron reduction of oxygen)(2 electron reduction of oxygen)

-

HH22OO22 is not a free radical but it can breakdown in is 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 + O OH + OH + O22CuCu+2+2

Fe Fe +2+2

-

Thus the keyplayers in the biochemistry of oxygen Thus the keyplayers in the biochemistry of oxygen free radical, or reactive oxygen species are oxygen free radical, or reactive oxygen species are oxygen itself, superoxide, hydrogen peroxide, transition itself, superoxide, hydrogen peroxide, transition metal ions and the hydroxyl radical. The first 4 of metal ions and the hydroxyl radical. The first 4 of which conspire by a variety of reactions to generate which 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 the OH generation on DNA continue by the reaction of Hreaction of H22OO22 with transition metal ions with transition metal ions

bound to DNA.bound to DNA.

OH attack purines, pyramidines bases of OH attack purines, pyramidines bases of DNA DNA →→ 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 hydrogenXHXH

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 lipid Transition 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 initiation2.2. Decompose lipid peroxides into peroxyl and Decompose lipid peroxides into peroxyl and

alkoxylalkoxyl

LOOH LOOH LO LO LOO, LOLOO, LOLHLH

LL

LOOHLOOHperoxidation chain can peroxidation 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 be Double bonds of PUSFAs are susceptible to be attacked by oxidation.attacked by oxidation.

- CH - CH - C - - C - ++ O O22 C + CH C + CH

OO22

Carbon Carbon centered centered radicalradical

OHOH

Hydroxyl Hydroxyl radicalradical

- C -- C -- C -- C -

OO22HH

++Lipid hydroxyperoxideLipid hydroxyperoxide


Disrupt membrane function.Disrupt membrane function.

- C -- C -

OO22HH

Lipid Lipid hydroxyperoxidehydroxyperoxide

Decompose to highly cytotoxic Decompose to highly cytotoxic products as aldehydesproducts as aldehydes

Inactivation of membrane Inactivation of membrane receptorsreceptors

Membrane rigidityMembrane rigidity

Oxidative Modification of LDL :Oxidative Modification of LDL :


Native LDL undergoes oxidative modification by Native LDL undergoes oxidative modification by intracellular oxygen free radical intracellular oxygen free radical →→ peroxidation of peroxidation of (PVFAs) in LDL lipids.(PVFAs) in LDL lipids.

If the process continue If the process continue →→ highly reactive products highly 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 the These products induce functional changes in the endothelial wall endothelial wall →→ favor penetration of favor penetration of monocytes and movement of oxidized LDL into monocytes and movement of oxidized LDL into the subendothelial space the subendothelial space → → ↑↑ fatty streak. fatty streak.

The previous cascade of events have been termed The 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 assays Direct assays

a.a. HH22OO22 in exhaled air in 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 assays Indirect assays

a.a. Trapping assay:Trapping assay:

Salicylate (2 hydroxybenzoate) used as a trap Salicylate (2 hydroxybenzoate) used as a trap

for OH for OH →→ 2, 3 dihydroxybenzoate 2, 3 dihydroxybenzoate

b.b. Uric acid oxidationUric acid oxidation

Uric acid acts as antioxidant Uric acid acts as antioxidant in vivoin vivo..

The major end product of uric acid oxidation is The major end product of uric acid oxidation is

allantoin.allantoin.




c.c. Finger print assays:Finger print assays:

ROS can produce chemical changes when they ROS can produce chemical changes when they

react with biological molecules such as DNA react with biological molecules such as DNA

and lipids. Measurement of end products can and 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 be Measurement of DNA damage can be

estimated by examining urine samples for estimated by examining urine samples for

the presence of thymidine glycol (Tg) using the presence of thymidine glycol (Tg) using

HPLC.HPLC.





2.2. Measurement of end products of lipid Measurement of end products of lipid

peroxidation:peroxidation:

• They are often the first parameters to which They are often the first parameters to which

researchers turn when they wish to prove the researchers 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 easiest TBA assay is the most popular and easiest

method used as an indicator of lipid method 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 peroxidation like substances formed by the lipid peroxidation

process. It involved heating the sample under acidic process. It involved heating the sample under acidic

condition with TBA which react with MDA to form a red condition with TBA which react with MDA to form a red

MDA-TBA complexMDA-TBA complex



A measure of total serum lipid peroxidation A measure of total serum lipid peroxidation has proven to be a simple, inexpensive and has proven to be a simple, inexpensive and accurate means of reflecting whole body accurate means of reflecting whole body free radical activity. This test is gaining free radical activity. This test is gaining general acceptance in the research general acceptance in the research laboratory as a simple, standard means of laboratory as a simple, standard means of assessing the body's antioxidant capability assessing 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

Analytical Aspects of Oxidative Stress

Health & Medicine

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