Oxidative stress and aging


Oxidative stress and aging (library project)

The general “free radical theory of aging” has been in circulation for over 30 years. The detailed mechanisms in specific diseases are now being identified and understood. The objectives of this project will be to identify named diseases associated with aging and oxidative stress and to critically review the mechanisms proposed.


1. BL Wilkinson, GE Landreth. The microglial NADPH oxidase complex as a source of oxidative stress in Alzheimer's disease. JOURNAL OF NEUROINFLAMMATION 3: Art. No. 30 NOV 9 2006

2. Halliwell, Barry. Free radicals in biology and medicine / Barry Halliwell and John M.C. Gutteridge. Edition 4th ed.

Publisher Oxford : Oxford University Press, 2007. (see library catalogue).

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In his work on health information Smith (1975) states…


Title - Oxidative stress and the biochemistry of ageing. “Life is pleasant. Death is peaceful. It's the transition that's troublesome.” Isaac Asimov “(And that transition is ageing)”


Halliwell and Gutteridge (2007) pg1. claimed that ‘over 2.2 billion years ago oxygen appeared in significant amounts in the earth's atmosphere almost entirely due to the evolution of photosynthesis by cyanobacteria.' Previously called blue-green algae, this phylum of bacteria are prokaryotes that combine plant-type photosynthesis and cytochrome oxidase-based respiration in the same cell. This species of bacteria evolved to split water by use of the sun's energy to thereby drive their metabolism.

O2 being the by-product, was then discarded in tonnes amount into the atmosphere.

Architects of earth's atmosphere



“Oxygen has been a trouble-maker since the very beginning.” Doris Aberlo

The chemical symbol for a single atom of oxygen is ‘O', however the oxygen element surrounds us as a diatomic molecule thus being O2. More than 99% of the atmosphere consists of the isotope oxygen-16 O2 however traces of oxygen-17 and oxygen-18 do also exist.

Apart from anaerobic non oxygen requiring organisms, O2 is required by most organisms for the efficient production of energy by the use of electron transport chains that ultimately donate electrons to O2. This process occurs in such processes like in the mitochondria of eukaryotic cells or many bacterial cell membranes.

Even though oxygen can be a good component in it being essential for most living species to survive, Halliwell and Gutteridge (2007) deduced that due to the need for O2 this obscures the fact that it is a toxic, mutagenic gas and a serious fire risk. Survival by aerobes is only present because they have evolved antioxidant defences. The favourable aspects of aerobic life are in contrast linked to potentially dangerous oxygen-linked oxidation processes.

“The same thing that makes you live can kill you in the end” Neil Young

These methods are thought to form the basis of a number of physiological and pathophysiological phenomena and are participating in processes as diverse as inflammation, ageing, carcinogenesis, drug action and drug toxicity, defence against protozoa and many others. (Sies, H 1985)

Historical Introduction:

Oxidative Stress

The term oxidative stress was introduced by Sies in 1985 (HnG) and he defined it 6 years later in 1991 as a disturbance in the prooxidant - antioxidant balance in favour of the former, leading to potential damage. Such damage is often called oxidative damage.

In principle oxidative stress can result from mainly

1. Diminished antioxidants and/or

2. Increased production of RS (Sies,H)

Oxidative stress could be equated to the amount of free radical damage that is being produced in a organ/organelle/cell/organism. The majority of free radicals that cause damage to the biological systems are oxygen radicals. There is a balance of many different factors that determine the amount of free radicals being produced in various conditions and some of which are shown in the following figure.

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(Scan figure table on pg 135 in critical review book)

Reactive Species.

Reactive species play physiological roles and are involved in many diseases and in the action of several toxins but these evanescent species are difficult to measure. There is an old Chinese saying,

“ It is difficult to find a black cat in a dark room, especially if there is no cat.”

Reactive Oxygen species (ROS) are produced in the cell by various environmental stimuli such as infection of microbes (viruses, bacteria, etc.), ionizing and UV irradiation, and pollutants (ie.oxidants), which are collectively called “oxidative stress”. These ROS are highly reactive with biological macromolecules to result in most commonly producing lipid peroxides (which are often radicals), inactivating proteins and mutating DNA (by producing 8-OH-dG or breaking nucleic acid chains). (not in own words) (Tetsuka and Okamotochapter 7 pg 112)

Sies,H (1991) Oxidative stress II. Oxidants and Antioxidants. Academic press, London. 82(2):291-5.

Free Radicals

Dr. Denham Harmon became very famous for having "discovered" free radicals and more accurately, for the proposal of the pioneering idea of the free radical theory of ageing.

Harman is widely known as the "father of the free radical theory of ageing". Dr Denham Harman

Radical [radicalis from Late Latin, having roots] originally basically means oriented towards the origin or root or to have a foundation or principle. In chemistry however it is defined as a group of atoms acting as a single unit, passing without change from one compound to another, but not able to exist in a free state.

In 1900, Gomberg first reported of a free radical (triphenylmethyl) (first free radical image). The discovery was made of the proposal that free radicals cause ageing in 1956.

Free radical

An atom or group of atoms containing at least one unpaired electron. These molecules contain an open bond or half bond and are highly reactive. The odd electron is denoted in chemical formulae by a superscript dot, for example, the simplest free radical atomic hydrogen H. . Free radicals are capable of independent existence, occupy only by themselves atomic or molecular orbitals and can exist for a brief period of time before reacting to produce stable molecules.

As well as breakage of a covalent bond by homolytic fission, radicals can exist naturally in their unpaired forms. Unpaired electrons can have a spin of either +1/2 or -1/2.

Presence of one or more unpaired electrons commonly causes free radicals to be attracted slightly to a magnetic field (that is to be paramagnetic). Even though the chemical reactivity of radicals varies widely this sometimes makes them highly reactive.

If two radicals react, both are eliminated. Atomic hydrogen forms diatomic hydrogen when the two unpaired electrons of each atom form a covalent bond ;

Supplementarily a more biologically relevant example is the fast reaction of nitric oxide (NO. ) and to form a non-radical product, peroxynitrite.

If a radical reacts with a nonradical, another free radical must be produced. This type of reaction may become a chain reaction.

In ischemic injury to tissues, for example myocardial infarction, free radical production may play an important role at certain stages in progression of the injury. (Taber's cyclopedic medical dictionary)

In the human body covalent bonds can get broken down to form free radicals. Merely cutting your fingernails can cleave disulphide bonds to generate sulphur radicals. When fracturing or grinding of the bone occurs, this can generate free radicals by mechanisms of damage to the covalent bonds which is mostly present in the collagen. We are made up of proteins which are essential for repair and growth. However, the process of freeze-drying (lyphophilization) can generate radicals capable of damaging many biomolecules. The protein can be directly damaged by radicals or during the lyophilisation procedure or by products generated from radical attack upon molecules added .


“Age is an issue of mind over matter. If you don't mind, it doesn't matter.” Mark Twain

"Life would be infinitely happier if we could only be born at the age of eighty and gradually approach eighteen." Mark Twain

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Age is not a diagnosis. Being of a certain age, especially of an age wherein deterioration of the mind and body is assumed to have taken place, denotes ageing. Statistically and in general, the condition of persons in their 70's and 80's will be different in many respects from their conditions in the 30's to 60's. Nevertheless, just because one has attained a certain chronological age is not of itself, reason to believe an individual is mentally or physically infirm, incompetent, handicapped or disabled.

Growing old, maturing is ageing and it implies progressive changes related to the passage of time.

“When I was a boy of 14, my father was so ignorant I could hardly stand to have the old man around. But when I got to be 21, I was astonished at how much the old man had learned in seven years.” Mark Twain

There is no precise method for determining the rate or degree of ageing. In a study of 1500 persons' age 100 years or more, it was concluded that longevity is not inheritable. (dictionary)

Ageing is associated with an increased incidence, susceptibility and prevalence of numerous diseases, many of them chronic. Ageing is deleterious, progressive, intrinsic and universal. Ageing is a multifactorial and heterogeneous phenomenon. As such, characteristics of ageing have the potential to differ vastly between species, and are non uniform even between individuals of the same species. Although death is a clear end point, ageing is much more difficult to describe.

Mildred :- “George, I'm worried about your ageing!”

George :- “Which one dear, physical or mental?”

Mildred :- “Physical, of course! Morons don't age!”

It is now known that free radicals cause extensive damage to cellular components that can lead to serious dysfunctions and death. Recently, free radicals have been associated with the ageing process, several clinical disorders, and a range of age related diseases including atherosclerosis, cancer, and neurodegenerative diseases such as Parkinson's and Alzheimer's. (Preface critical review pg ix) The fundamental idea of the oxidative theory in ageing has been tested in a wide range of ageing modelling systems and in comparative studies. Orange bits are by (Miwa, satomi. Muller,F L. Beckman, K.B 2008)

As a result, a new market is emerging; Oxidative Stress diagnostics and Therapeutics. This new discipline in the field of Clinical Age Management and Longevity Medicine is revolutionising the economics, science and practice of health care in new ways.

Virtually every medical discipline will be reshaped by longevity research and its social implications.

Spin Trapping

Type spin trapping into google and number 3 and picture of girl

The use of radical-addition reactions to detect short-lived radicals was first proposed by E. G. Janzen in 1965.

The versatile spin trapping technique has become a valuable tool in the study of transient free radicals.

Due to the short lifetimes and broad line widths of radicals, with particular interest in oxygen radicals, make most of them difficult to detect, if not impossible, by direct electron spin resonance (ESR) ( about ESR on page 268 but 270 in detail H n G) in room temperature aqueous solutions. With of course pitfalls and limitations, spin trapping however provides a means, in principle to overcome these problems under various experimental conditions. (Buettner and Mason 2003).

In this technique, a long-lived radical (more persistent paramagnetic species called the spin adduct) is produced when a short lived radical reacts with a trap(the spin trap). The identification of two types of spin traps have been developed, nitrone and nitroso compounds. (Halliwell and Gutteridge 2007) The reaction of nitroso (R-NO) compounds with short-lived radicals can produce longer-lived nitroxide (sometimes calles aminoxyl) radicals.

The electron delocalisation between the nitrogen and oxygen atoms associated with their stability.

Nitroxide radicals are also produced by nitrone traps.

The resulting spin adducts can be detected and identified by Electron Paramagnetic Resonance (EPR) spectroscopy.

The standard by which novel spin traps are evaluated is PBN, (N-tert-butyl-alpha-phenyl nitrone) which has been shown to extend the lives of experimental animals

Spin trapping can be used to determine the efficiency of various antioxidants toward specific types of free radicals, by measuring the amounts of various free radicals before and after treatment.

Later it was discovered that these "spin traps" had powerful free radical quenching abilities in living systems and could treat a variety of conditions, including inflammatory and degenerative age-related diseases because they inhibit fundamental pathogenic mechanisms.2

Do you need to talk about why nitrone spin traps are by far the most popular? P273 H n G and pg 28 o n D critical review.

Nitrone spin traps are considered, by far, the most popular (Buettner and Mason 2003) and more commonly used.(Halliwell and Gutterage 2007) 1 in book In nitroso traps the radical detected adds directly to the nitrogen, whereas with nitrones it adds to the adjacent carbon. (Buettner and Mason 2003) ESR spectrum is more easily influenced by the trapped radical of the nitrose trap and this usually generates hyperfine splittings. The spectra tend to be broadly similar whatever the radical trapped with nitrone traps. Overall nitroso compounds have a higher proportion of disadvantages as they are more toxic to animals or cells and especially when oxygen radicals are trapped, nitroso compounds often give less stable adducts than nitrones.

Spin Traps

Spin trapping can be used to determine the efficiency of various antioxidants toward specific types of free radicals, or the presence of these radicals under various experimental conditions

There are numerous applications in chemistry and biochemistry. Spin trapping is a versatile technique for detecting transient radical species. The method is based on the addition of a compound (the spin trap) to the transient radical species. This reaction yields a more persistent paramagnetic species called the spin adduct, which can be detected and identified by Electron Paramagnetic Resonance (EPR) spectroscopy. The standard by which novel spin traps are evaluated is PBN, (N-tert-butyl-alpha-phenyl nitrone) which has been shown to extend the lives of experimental animals

How and why Spin Traps are used

Spin Traps were originally utilized in measuring free radical activity because they react with free radicals both in vitro and in vivo, producing stable complexes that can be measured by a variety of techniques.1 They are frequently used to measure the efficacy of other antioxidants by measuring the amounts of various free radicals before and after treatment. Later it was discovered that these "spin traps" had powerful free radical quenching abilities in living systems and could treat a variety of conditions, including inflammatory and degenerative age-related diseases because they inhibit fundamental pathogenic mechanisms.2

New evidence surfaces almost daily that free radical pathology underlies many disease processes and aging itself. Antioxidants have become one of the cornerstones of any health maintenance and longevity program. "Spin traps" could provide unique protection against free radical damage that complements and enhances the activities of the classical antioxidants such as vitamin C, vitamin E, glutathione, R-Lipoic Acid and a wide variety of phytochemically derived free radical fighters.3

Spin traps are currently being explored as potential therapeutic agents, as they have been shown to block or reverse the damage associated with a variety of disease states in animal models. It has recently been shown that they exert this benefit by altering signal transduction pathways, and reducing systemic inflammation, one of the primary culprits associated with the chronic degenerative diseases of aging. They are effective at doses much lower than those necessary to trap free radicals, and may be more effective with concurrent antioxidant treatment.

Recently, researchers found that the underlying mechanism of "spin trap" activity differs from antioxidants. Spin traps suppress gene transcriptional factors associated with a variety of pathophysiological states.4 In particular, spin traps modulate NF kappa-B regulated cytokines and inducible nitric oxide synthase that are implicated in AIDS, arthritis, arteriosclerosis, Alzheimer's disease and other pro-inflammatory disease conditions.5 Arguably, this mechanism involves actions at a level proximal to oxidatively sensitive signal amplification systems rather than simple neutralization of free radicals.6

PBN Overview

Alpha Phenyl-N-Tert Butyl Nitrone

PBN is the most extensively researched and widely used Spin Trap

The most commonly used spin trap and the standard which measures new ones is PBN - alpha phenyl-N-tert butyl nitrone. Hundreds of studies have been conducted over the last ten years that have tested PBN and other "spin traps" in numerous conditions.4 Spin traps have been shown to affect cellular oxidation states and oxidatively sensitive enzyme systems.5

* PBN has been shown to be an excellent neuroprotective and anti-inflammatory agent.6

* PBN has extended the life span of mice,7 improved cognitive performance in rats,8 reversed protein oxidation in aged gerbils and returned them to youthful states.9

* PBN has attenuated hydroxyl radical formation in ischemia-reperfusion injury,10 blocked nitric oxide synthase, which minimized the exitotoxicity of peroxynitrite radical.11 Paradoxically, PBN acted as a delivery system for transporting beneficial nitric oxide to targeted areas undergoing oxidative stress.12

Low toxicity

PBN has been shown to be of extremely low toxicity even at the highest dosages and with long-term use.13a,13b The effective dosage of PBN in humans for treating age and ischemic related disorders is expected to be between approximately 1 and 10 mg/70 kg body weight. This equates to 70-700mg for a 70 kg man. Toxicity tests have demonstrated that PBN is completely innocuous, with such low toxicity that it was not possible to determine an LD.sub.50.13c Spin Traps work differently than antioxidants

Recently, researchers found that the underlying mechanism of "spin trap" activity differs from antioxidants. Spin traps suppress gene transcriptional factors associated with a variety of pathophysiological states.14 In particular, spin traps modulate NF kappa-B regulated cytokines and inducible nitric oxide synthase that are implicated in AIDS, arthritis, arteriosclerosis, Alzheimer's disease and other pro-inflammatory disease conditions.15 Arguably, this mechanism involves actions at a level proximal to oxidatively sensitive signal amplification systems rather than simple neutralization of free radicals.16
PBN has a wide range of practical applications

Because PBN alters several fundamental cellular processes and metabolic pathways, it may be efficacious for a wide range of conditions. Between the years of 1991 and 2000, researchers, John Carney at University of Kentucky Research Foundation and Robert Floyd at Oklahoma Medical Research Foundation, filed a series of U.S. patents for methods of using PBN. They recommended PBN for many diverse conditions that involve oxidative stress and free-radical pathology.16-23


Aging has been demonstrated to be associated with the production of abnormally high levels of oxidized proteins. The consequence of this increased level of protein oxidation is an abnormally low level of critical enzymes in the affected cells. Most, if not all, cells in the body will undergo abnormally high levels of protein oxidation and there will be decreases in antioxidant systems and abnormally low levels of mitochondrial function. The protein oxidation is thought to arise from oxygen free radicals, largely generated via a metal catalyzed reaction within the cell. Studies have now been conducted that daily administration of a free radical spin trapping compound, PBN, for fourteen days completely reverses this process. Not only is the level of protein oxidation decreased, but the abnormally low level of enzyme activity is restored to normal.

PBN has shown to be efficacious in the process and problems of aging including: Alzheimer's disease, dementia, Parkinson's disease, loss of neurotransmitters and receptor sensitivity and macular degeneration. use 1 or 2 egs of diseases such as diabetes from PBN overview spin traps for cosmetic applications and healthy skinPBN Cosmetic Applications

The Standard Nitrone Spin Trap for Cosmetic Applications


PBN (N-tert-butyl-alpha-phenylnitrone) is the established nitrone "spin trap". The term "spin trap" is derived from the technique used to detect and identify free radicals, electron spin resonance. Reactive free radicals are attracted and bound to the beta carbon atom in the spin trap, forming a spin adduct and effectively "trapping" the free radical, allowing the structure of the trapped radical to be deduced.

Spin traps are exceptional molecules that can trap and detoxify damaging free radicals which age the skin. PBN blocks and detoxifies harmful free radical molecules from damaging cells and reduces overall systemic inflammation - the culprit recently discovered to underlie all the degenerative diseases of aging.

PBN has been shown to be an excellent neuroprotective and anti-inflammatory agent and transforms skin damaging free radicals into productive regenerative activity.
Cosmetic Applications and Benefits of PBN Powder

* PBN can provide unique protection against free radical induced skin damage.

* PBN can be administered topically to control inflammatory processes; an underlying cause of skin aging.

* PBN is useful for enhancing the activities of topically applied antioxidants such vitamin C, vitamin E and Lipoic Acid and is commonly incorporated into preparations which include other antioxidants.

* Spin Traps show promise as blocking agents for inflammation caused by UV exposure. Unlike traditional sunscreens, which prevent UV mediated inflammation when used before exposure, spin trap compounds scavenge free radicals and minimize the inflammatory response during and for up to 12 hours after exposure to UV light. By interfering with the inflammatory cascade, these compounds may prevent collagen and elastin degradation, and thus premature aging of the skin.


PBN powder for cosmetic application is doubly recrystalised from high purity solvents and is greater than 98% pure (HPLC). PBN is a white crystalline solid; melting point 73°-74° C. PBN powder is poorly soluble in water, but soluble in alcohols. PBN powder is hydrolyzed at low pH into benzaldehyde and N-tert-butyl-hydroxylamine.

Dr. Denham Harman, the father of the "Free Radical Theory of Aging", and keynote speaker at the 2nd Annual Anti-aging Conference in Monaco, referred to PBN and spin traps as a "breakthrough in anti-aging therapy with the potential to significantly slow down the aging process."

In his bestselling book, The Wrinkle Cure, Dr. Nicholas Perricone notes that spin traps are capable of "Stopping Free-Radical Damage Before it Begins..." and that such agents 'create a barrier - a trap - that holds free radicals in place' so that they can be "stopped before they scar the cells that make up your skin." He also claims that researchers "found that the traps actually prevented the free radicals from moving from place to place and damaging cells... and that 'the odds are these free-radical fighters will become a highly effective method of maintaining a youthful, radiant complexion throughout life... Thanks to a spin trap, we will make quantum leaps in the effort to control the effects of aging..."


PBN is available in gram to multi-kilogram packaging for the cosmetic industry.

Spin Trap Research

The severely used radical traps for the study of oxygen centred free radicals is 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which has been extensively utilized to study superoxide and hydroxyl radicals in biological and chemical systems (Buettner and Mason 2003). Other traps include a-Phenyl-tert-butylnitrone PBN and 5-Diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO).


Hydroxyl pg 28, 29 -34 in Critical book and pg 273 - h n g also stuff about SOD-which needs mentioning

Do you need to talk about spin trapping for Hydroxyl radical or superoxide.


The superoxide theory of O2 toxicity is the most popular way to describe oxygen toxicity itself. This theory proposes that excess O2 causes over production of O2.-. This in turn leads to damage to metabolically important enzymes and other biomolecules.

A highly reactive form of oxygen (Ō2.), the superoxide anion, produced when oxygen is reduced by a single electron. Superoxide is produced during normal catalytic function of certain enzymes, by the oxidation of haemoglobin to methemoglobin, and when ionizing radiation passes through water. It is also produced when granulocytes phagocytose bacteria. Superoxide is destroyed by the enzyme superoxide dismutase (SOD), which catalyses the conversion of two molecules of superoxide anion to one molecule of oxygen and one of hydrogen peroxide:

Ō2. + Ō 2. + 2H+ ↔ H2O2. + O2.


An antioxidant was defined as any substance that, when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate. The term oxidizable substrate includes almost every molecule found in vivo.

This definition emphasise the importance of the damage target studied and the source of RS used when antioxidant action is examined, it is also imperfect like, us all. This definition does not take into account chaperones, repair systems or inhibitors of RS generation. Thus we now simplify the definition to any substance that delays, prevents or removes oxidative damage to a target molecule.

For Friedovich and McCord reference

Superoxide dismutase(SOD); discovered by Fridovich and McCord (1969), is an enzyme that destroys superoxide.

The types of SOD are as follows:- 1 Copper-zinc SOD

2. Manganese SOD (sometimes called SOD2)

3. Iron and cambialistic SODs

4. Nickel-containing SODs

Further to this catalase enzyme can disintegrate H2O2 into H2O and O2

Type in Proff Bisby equation

The SOD-catalysed dismutation of superoxide may be written with the following half-reactions:

* M(n+1)+−SOD + O2− → Mn+ − SOD + O2

* Mn+ − SOD + O2− + 2H+ → M(n+1)+ − SOD + H2O2.

where M = Cu (n=1); Mn (n=2); Fe (n=2); Ni (n=2).

In this reaction the oxidation state of the metal cation oscillates between n and n+1.

One form of the enzyme contains manganese and another contains copper and zinc.

Lipid Peroxidation

In his work on health information Smith (1975) states…

A continuous scientific researcher, Dr A.L. Tapell, defined Lipid peroxidation as the oxidative deterioration of polyunsaturated lipids. (HnG)

It is an incredibly important process contributing to oxidative stress whereby free radicals "steal" electrons from the lipids in cell membranes. With addition to this, lipid peroxidation proceeds by a free radical chain reaction mechanism, resulting in cell damage.(wiki)

It most often affects polyunsaturated fatty acids (PUFAs), since they contain two or more double bonds positioned in between methylene -CH2- groups that possess especially reactive hydrogens. (wiki) (HnG)

The main targets of attack in lipid peroxidation are membrane lipids, as a large amount of PUFAs side-chains exist in the membranes surrounding cells and organelles. On the contrary to this the chief constituents of biological membranes are lipids and therefore we are at constant risk of going rancid. (H n G)

The main targets of attack in lipid peroxidation are membrane lipids, as a large amount of PUFAs side-chains exist in the membranes surrounding cells and organelles. On the contrary to this the chief constituents of biological membranes are lipids and therefore we are at constant risk of going rancid. (H n G)

In animal cell membranes the dominant lipids are phopholipids, esters based on the alcohol glycerol, the comonest in animal cell membranes being lecithin(phosphatidylcholine. Some membranes particularly plasma membranes, contain significant proportions of sphingolipids and cholesterol. By contrast, the membranes of subcellular organelles such as mitochondria or nuclei rarely contain much sphingolipids or cholesterol. Mitochondrial inner membranes contain cardiolipin. Peroxidation of PUFAs in this lipid may contribute to age-related decline in mitochondrial function and can facilitate cytochrome c release during apoptosis.

The fatty-acid side-chains of membrane lipids in animal cells have unbranched carbon chains and contain even numbers of carbon atoms, usually 14 to 24 and the double bonds are cis configuration, causing ‘kinks' in the structure.

Since membrane lipids are amphipathic, on exposure to water they aggregate with their hydrophobic regions clustered together and their hydrophobic regions in contact with H2O.how this is achieved depends on relative amounts of lipid and water. When phospholipids are shaken or sonicated in aqueous solution they form micelles but as more phospholipids are added liposomes result, bags of aqueous solution bounded by a lipid bilayerliposomes can be surrounded by a single bilayer(unilamellar) or several bilayers (multilamellar) the membrane fluidity is largely due to the prescence of PUFA side-chainswhich lower the melting point of the membrane interior so that it has the chemical nature and viscosity of a light oil. Damage to PUFAs tend to decrease membrane fluidity, which is essential for the proper fynctioning of biological membranes.

When phospholipid are shaken or sonicated in aqueus solution they form micelles but as more phospholipids are addedb liposomes result, bags of aqueous solution bounded by a lipid bilayer. (hnG)

Target of attack:dietry lipids,and lipoproteins

We can see that stable lipids in the membrane are vulnerable towards free radical oxidation however it can be visualised that externally derived fats from food have to be digested, absorbed and transported around the body. These fats can be oxidized during storage or cooking and perhaps in the gastrointestinal tract itself.

Lipoproteins involved in fat transport may contain small amounts of oxidized lipids from the diet and can undergo further oxidation in vivo, contributing to atherosclerosis.. fatty acids circulating in the blood are also potential targets for oxidation. (H n G)

Oxidative stress that occurs in the cells, as a consequence of an inequity between the prooxidant/antioxidant systems, causes injure to biomolecules such as nucleic acids, proteins, structural carbohydrates, and lipids [Sies & Cadenas 1985].

Among these targets, the peroxidation of lipids is basically damaging because the formation of lipid peroxidation products leads to spread of free radical reactions (scitopic)

Lipid peroxidation refers to the oxidative degradation of lipids. It is the process whereby free radicals "steal" electrons from the lipids in cell membranes, resulting in cell damage. This process proceeds by a free radical chain reaction mechanism. It most often affects polyunsaturated fatty acids, because they contain multiple double bonds in between which lie methylene -CH2- groups that possess especially reactive hydrogens. As with any radical reaction the reaction consists of three major steps ( or could write The general process of lipid peroxidation consists of three stages (scitopic)) initiation, propagation and termination. (Wikipedia)

In his work on health information Smith (1975) states…

Lipid Peroxidation

Q. In the good old days, what was a common cause of fires in textile mills?

In 1820, attempting to study oil oxidation (hng), the Swiss chemist (cyberlipid), de Saussure used a simple mercury monometer, which in outcome demonstrated that a layer of walnut oil on water exposed to air was able to absorb about 145 times its own volume of oxygen (O2) during a 1 year period. Parallel with these changes, oil became viscous and had a bad smell (became rancid).

oxygen-dependent deterioration of lipids , leading to rancidity has been recognized since antiquity as a problem in the storage of fats and oils and was often dealt with by using antioxidant spices(HnG). Parmentier AA, a pharmacist who introduced potato culture in France, hypothesized that oxygen in combining with fats was the agent of rancidity (cited by Braconnot H, 1815) Rancidity is even more relevant today with the popularity of polyunsaturated margarine and cooking oils and the importance of paints,plastics,lacquers,waxes and rubberall of which can undergo oxidative damage. (Surprisingly enough) even oils within breast implants can oxidize.

Commenting on the above experiment of de Saussure, the famous chemist Berzelius ( who also discovered selenium) ‘suggested' that oil auto-oxidation might be involved in the spontaneous ignition of wool lubrified with linseed oil, a common cause of fires in textile mills at the time.

The sequence of reactions which is now recognized as the ‘core' of lipid peroxidation was worked out in detail by scientists at the British Rubber Producers' Association research laboratories in the 1940s. the relevance of these reactions to biological systems was not appreciated until later, however. Food scientists have also made a substantial contributions to understanding lipid peroxidation. (H n G) (Cyberlipid)


A continuous scientific researcher, Dr A.L. Tapell, defined Lipid peroxidation as the oxidative deterioration of polyunsaturated lipids. (HnG)

It is an incredibly important process contributing to oxidative stress whereby free radicals "steal" electrons from the lipids in cell membranes. With addition to this, lipid peroxidation proceeds by a free radical chain reaction mechanism, resulting in cell damage.(wiki)

It most often affects polyunsaturated fatty acids (PUFAs), since they contain two or more double bonds positioned in between methylene -CH2- groups that possess especially reactive hydrogens. (wiki) (HnG) The hydrogen atoms contain only one electron, therefore leaves at the back an unpaired electron on the carbon, - ●CH-. The presence of a double bond in the fatty acid weakens the C-H bonds on the carbon atom nearby to the double bond and thus facilitates H ● subtraction.(Scitopic)

The predominantly susceptible targets in peroxidation are membrane lipids, mainly phospholipids, as a large amount of PUFAs side-chains exist in the membranes surrounding cells and organelles. The first demonstration of free radical oxidation of membrane phospholipids was given in 1980(porter NA et al Lipids 1980, 15, 163) leading to a new fruitful era with a continuos flow of innumerable works devoted to chemistry, biochemistry and medicine.

On the contrary to this, the chief principle constituents of biological membranes are lipids and therefore we are at constant risk of going rancid. (H n G)

The membrane lipids, mainly phospholipids, containing polyunsaturated fatty acids are predominantly susceptible to peroxidation because abstraction from a methylene (-CH2-) group of a hydrogen atom, which contains only one electron, leaves at the back an unpaired electron on the carbon, - ●CH-. The presence of a double bond in the fatty acid weakens the C-H bonds on the carbon atom nearby to the double bond and thus facilitates H ● subtraction. (Scitopic)

The initial reaction of ●OH with polyunsaturated fatty acids produces a lipid radical (L ●), (scitopic)

which in turn reacts with molecular oxygen to form a lipid peroxyl radical (LOO ●). (Scitopic)

Initiation is the first stage whereby a fatty acid radical is produced. The initiation phase of lipid peroxidation includes most commonly hydrogen atom abstraction from a methylene (CH2) group .(scitopic)or by ROS addition to the polyunsaturated fatty acid.. In both cases, a carbon radical results

The most notably reactive oxygen species (ROS), is mainly hydroxyl (OH●) , Several other ROS species include the radicals: alkoxyl (RO ●), peroxyl (ROO ●), and possibly HO 2 ● but not H 2O 2 or O 2 − ● (scitopic)

(HnG) Formaula

Or by H● abstraction

Nitrogen dioxide can perform similar reactions whereas HOCL is more likely to chlorinate lipids by addition across double bonds, although chloramines can form on -NH2 groups on some lipids. Ozone directly oxidizes lipids, forming ozonides. A double bond weakens the bond energy of the C - H bonds present on the next carbon atom(the allylic hydrogens. The reduction potential of a bis-allylic PUFA●/ PUFA couple at PH7 (Eo') has been estimated as about 0.6V. hence OH●, HO●2, RO● and RO●2 radicals are (thermodynamically at least) capable of oxidizing PUFAs at allylic hydrogens (HnG)

Hydroxyl radical readily initiates peroxidation of fatty acids, lipo proteins and membranes, although OH● generated outside a membrane will also attack extrinsic carbohydrates and proteins(e.g cell surface glycoproteins) and head groups of phospholipids. Hence irradiation of biological material stimulates lipid peroxidation;this has been shown not only for membranes and lipoproteins but also for food lipids(a problem in attempts to sterilize or preserve food by irradiating it.) radiation-induced peroxidation is inhibited to some extent by scavengers of OH●, such as mannitol and formate, which will compete with the lipids for any OH● generated in free solution. However H2O crosses membranes and any water undergoing homolysis within the membrane will generate OH● not acca=essible to scavengers. The rate constant for reaction of OH● with artificial lecithin bilayers has been measured as about 5 x 108 M-1s-1.

By contrast, neither NO● nor O2●- is sufficiently reactive to abstract H from lipids; in any case the charge of O2●- tends to preclude it from entering the lipid phase of membranes. Indeed O2●- does not readily cross biological membranes, except where specific channels exist, nor does it appear to react with any membrane constituents on its passage through such channels. However HO●2 is more reactive and can abstract H● from some PUFAs,such as linoleic and arachidonic acids( rate constants 1.2, 1.7 and 3.0 x 103 M-1s-1 respectively).


Protonated O2●-, being uncharged, should enter membranes more easily than O2●- and several papers have described HO2● - dependent peroxidation of liposomes and lipoproteins. In addition HO2● can stimulate peroxidation by reaction with preformed lipid hydroperoxides to generate peroxyl radicals.


(H n G)


The fatty acid radical is not a very stable molecule, so it reacts readily with molecular oxygen, thereby creating a peroxyl-fatty acid radical. This too is an unstable species that reacts with another free fatty acid producing a different fatty acid radical and a lipid peroxide or a cyclic peroxide if it had reacted with itself. This cycle continues as the new fatty acid radical reacts in the same way. (Wikipedia)

The peroxyl radical LOO ● can abstract hydrogen from a neighboring fatty acid to produce a lipid hydroperoxide (LOOH) and a second lipid radical [Catala, 2006]. The LOOH formed can suffer reductive cleavage by reduced metals, such as Fe ++, producing lipid alkoxyl radical (LO ●). Both alkoxyl and peroxyl radicals stimulate the chain reaction of lipid peroxidation by abstracting additional hydrogen atoms [Buettner, 1993]. Peroxidation of lipids can disturb the assembly of the membrane, causing changes in fluidity and permeability, alterations of ion transport and inhibition of metabolic processes [Nigam & Schewe, 2000]. Injure to mitochondria induced by lipid peroxidation can direct to further ROS generation [Green & Reed, 1998]. In addition, LOOH can break down, frequently in the presence of reduced metals or ascorbate, to reactive aldehyde products, including malondialdehyde (MDA), 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (4-HHE) and acrolein [Esterbauer et al 1991; Parola et al, 1999; Uchida, K, 1999; Kehrer, J. P.; Biswal, 2000; Lee et al 2001], Figure 1. (Scitopic)

Carbon radicals often stabilize by molecular rearrangement to form conjugated dienes. If two carbon radicals collide within a membrane they might cross-link the fatty acid side chains


However the most likely fate of carbon radicals under aerobic conditions is to combine with O2, especially as O2 concentrates inside membranes. Reaction with O2 gives a peroxyl radical, ROO● (or RO●2) sometimes shortened to peroxy radical:


Of course very low O2 concentrations might favour self-reaction of carbon-centred radicals, or their reactions with other membranes components, such as -SH groups on proteins. Hence the O2 concentration affects the pathway of peroxidation, for this and other reasons.

Peroxyl radicals can abstract H● from an adjacent fatty-acid side-chain. Formula

This is the propogation stage of lipid peroxidation. It forms new carbon radicals that can react with O2 to form new peroxyl radicals, and so the chain reaction of lipid peroxidation continues.

The RO●2 combines with the H● that it abstracts to give a lipid hydroperoxide (ROOH). This name is sometimes shortened to lipid peroxide, although the latter term also includes cyclic peroxides, which result when a peroxyl radical attacks another double bond in the same fatty acid residue.

A single initiation event thus has the potential to generate multiple peroxide molecules by a chain reaction. The initial H● abstraction from a PUFA can occur at different points on the carbon chain. Thus peroxidation of linoleic acid gives two hydroperoxides, that of linolenic acid four. Peroxidation of arachidonic acid gives six lipid hydroperoxides. Similarly, EPA can give eight hydroperoxides and DHA ten. All these products are formed as a racemic mixtures of optical isomers, i.e. lipid peroxidation is not stereospecific. Cyclic and bicyclic peroxides can also form
[edit] Termination

When a radical reacts with a non-radical it always produces another radical, which is why the process is called a "chain reaction mechanism." The radical reaction stops when two radicals react and produce a non-radical species. This happens only when the concentration of radical species is high enough for there to be a high probability of two radicals actually colliding. Living organisms have evolved different molecules that speed up termination by catching free radicals and therefore protect the cell membrane. One important such antioxidant is vitamin E. Other anti-oxidants made within the body include the enzymes superoxide dismutase, catalase, and peroxidase. (Wikipedia)
[edit] Hazards

If not terminated fast enough, there will be damage to the cell membrane, which consists mainly of lipids. Phototherapy may cause hemolysis by rupturing red blood cell cell membranes in this way[1]

In addition, end products of lipid peroxidation may be mutagenic and carcinogenic [2] . For instance, the end product malondialdehyde reacts with deoxyadenosine and deoxyguanosine in DNA, forming DNA adducts to them, primarily M1G. (Wikipedia)

A great variety of compounds are formed during lipid peroxidation of membrane phospholipids. Lipid peroxidation is one of the major outcomes of free radical-mediated injury to tissue. Peroxidation of fatty acyl groups occurs mostly in membrane phospholipids. Peroxidation of lipids can greatly alter the physicochemical properties of membrane lipid bilayers, resulting in severe cellular dysfunction. In addition, a variety of lipid byproducts are produced as a consequence of lipid peroxidation , some of which can exert adverse and/or beneficial biological effects. (scitopic)


Certain diagnostic tests are available for the quantification of the end products of lipid peroxidation, specifically malondialdehyde (MDA) [2] The most commonly used test is called a TBARS Assay (thiobarbituric acid reactive substances assay). Thiobarbituric acid reacts with malondialdehyde to yield a fluorescent product. However, there are other sources of malondialdehyde, so this test is not completely specific for lipid peroxidation. (Wikipedia)

Lipid Peroxidation and its role in atherosclerosis

Esterbauer,H., Wäg,G. and Puhl,H. Lipid peroxidation and its role in atherosclerosis. British Medical Bulletin (1993) VOL 49, No 3 pp. 566-576 (Esterbauer et al.1993) (Scitopic)



for free radical theory of ageing Denham Hardman

Marnett, L.J. (1999) Lipid peroxidation—DNA damage by malondialdehyde. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis424(1-2) pp83-95 Found at http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T2C-3VXYRD2-8&_user=899537&_coverDate=03%2F08%2F1999&_rdoc=1&_fmt=high&_orig=search&_sort=d&_docanchor=&view=c&_acct=C000047642&_version=1&_urlVersion=0&_userid=899537&md5=54248b1cb91e38728933e4093f399ab1

Dr. Denham Harmon, M.D., Ph.D., first proposed a theory of aging as the indiscriminate chemical re-activity of free radicals possibly leading to random biological damage. His idea has had much experimental success, and it is now considered a major theory of aging. Dr. Harmon's theory implies that antioxidants such as vitamin C and vitamin E, which prevent free radicals from oxidizing (removing electrons from) sensitive biological molecules, will slow the aging process. Dr. Harmon launched his theory by showing, for the first time, that feeding a variety of antioxidants to mammals extended their life spans. http://www.healingdaily.com/conditions/free-radicals.htm

Fentons Reaction

Fenton's reagent is a solution of hydrogen peroxide and an iron catalyst that is used to oxidize contaminants or waste waters. Fenton's reagent can be used to destroy organic compounds such as trichloroethylene (TCE) and tetrachloroethylene (PCE).

It was developed in the 1890s by Henry John Horstman Fenton.

Ferrous Iron(II) is oxidized by hydrogen peroxide to ferric iron(III), a hydroxyl radical and a hydroxyl anion. Iron(III) is then reduced back to iron(II), a peroxide radical and a proton by the same hydrogen peroxide (disproportionation).

(1) Fe2+ + H2O2 → Fe3+ + OH· + OH−

(2) Fe3+ + H2O2 → Fe2+ + OOH· + H+

Reaction (1) was first suggested by Haber and Weiss in the 1930s,[1] but is commonly referred to as 'the Fenton reaction'. In the net reaction the presence of iron is truly catalytic and two molecules of hydrogen peroxide are converted into two hydroxyl radicals and water. The generated radicals then engage in secondary reactions. Iron(II) sulfate is a typical iron compound in Fenton's reagent. The exact mechanisms are debated (also non-OH· oxidizing mechanisms of organic compounds have been suggested) and, therefore, it may be appropriate to broadly discuss 'Fenton chemistry' rather than a 'Fenton reaction'.

In the Electro-Fenton process, hydrogen peroxide is produced in the required amount from the electrochemical reduction of oxygen.[2]

Fenton's reagent is also used in organic synthesis for the hydroxylation of arenes in a radical substitution reaction such as the classical conversion of benzene into phenol.

(3) C6H6 + FeSO4 + H2O2 → C6H5OH

A recent hydroxylation example involves the oxidation of barbituric acid to alloxane.[3] Another application of the reagent in organic synthesis is in coupling reactions of alkanes. As an example tert-butanol is dimerized with Fenton's reagent and sulfuric acid to 2,5-dimethyl-2,5-hexanediol.[4]

The mismanagement of iron in cellular systems can lead to the toxic accumulation of iron in organ systems such as the liver and brain. It is believed that this build up of iron eventually leads to the production of free radicals leading to oxidative stress, cellular damage and eventual cellular death via apoptotic signaling.

Fenton's Reagent Revisited Cheves Walling Vol 8 1975


Oxidative stress can induce apoptotic death, and mitochondria have a central role in this and other types of apoptosis, since cytochrome c release in the cytoplasm and opening of the permeability transition pore are important events in the apoptotic cascade.

Experimental evidence of respiratory chain defects and of accumulation of multiple mtDNA deletions with ageing is in accordance with the mitochondrial theory, although some other experimental findings are not directly ascribable to its postulates.

Two major developments opened breakthroughs in mitochondrial pathology: first, the discovery that mitochondrial DNA (mtDNA) mutations are at the basis of diseases [1], and second, the unexpected role of mitochondria in the mechanisms of cell death [2 N. Zamzami, P. Marchetti, M. Castedo, C. Zanin, J.L. Vayssière, P.X. Petit and G. Kroemer, J. Exp. Med. 181 (1995), pp. 1661-1672. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (774)2]. A common denominator of mtDNA mutations is the role of reactive oxygen species (ROS).

Mitochondrial damage

The mitochondrial DNA theory of ageing is in the midst of the longest-standing mechanistic proposals for why we age at the rate we do. It postulates that mitochondrial DNA (mtDNA) mutations might be a key force in determining the rate of human ageing which was proposed by Harman in 1972.

He wrote that:

“Free radicals ‘escaping' from the respiratory chain … would be expected to produce deleterious effects mainly in the mitochondria … Are these effects mediated in part through alteration of mitochondrial DNA functions?” (Aubrey)

In ageing Harman quoted that mitochondrial production of Ros determines ageing. This theory is called the mitochondrial theory and this means that mitochondria are the major producers of Ros and also they are the major targets due to their proximity to the ros production site. Damage to mitochondria and it's DNA would result in accumulation of the mitochondria that produce even more Ros, causing a viscious cycle.

(Miwa, satomi. Muller,F L. Beckman, K.B 2008)pg 4)

Cellular respiration

Chemically, a substance is oxidized when electrons are removed and reduced when electrons are added. All chemical reactions involve the transfer of electrons. The body generates energy during cellular respiration by gradually oxidizing its food in a controlled manner and storing it in the form of chemical potential energy, called adenine triphosphate (ATP).

Mitochondria are small membrane enclosed regions of a cell that perform essential multiple cellular functions and predominantly produce more than 90% of the ATP that cells require. Other functions include maintanence of intracellular homeostasis of inorganic ions, mineral support for SOD and GSX as well as providing structural support of enzymes of the electron transport chain. (Singh et al. 2003) Mitochondria accomplish the task of ATP manufacture through a mechanism called the "electron transport chain." In this mechanism, electrons are passed between different molecules, with each pass producing useful chemical energy. Oxygen occupies the final position in the electron transport chain. Occasionally, the passed electron incorrectly interacts with oxygen, producing oxygen in radical form.

Glycolysis = Co2 + h20

Do I need to write about electron transport chain/cellular respiration?

Mitochondria are deeply involved in the production of Reactive oxygen species (ROS) through one-electron carriers in the respiratory chain; mitochondrial structures are also very susceptible to oxidative stress as evidenced by massive information mitochondrial DNA (mtDNA) mutations.

A common denominator of mtDNA mutations is the role of ROS (Lenaz).

this means that mitochondria are the major producers of Ros and also they are the major targets due to their proximity to the ros production site.

The respiratory chain is a powerful source of ROS, primarily the superoxide radical and consequently hydrogen peroxide, either as a product of superoxide dismutase [7] or by spontaneous disproportionation.(Lenaz)

There are two major respiratory chain regions where ROS are produced, one being complex I (NADH coenzyme Q reductase) and the other complex III (ubiquinol cytochrome c reductase)

Complex I (NADH dehydrogenase, also called NADH:ubiquinone oxidoreductase; EC removes two electrons from NADH and transfers them to a lipid-soluble carrier, ubiquinone (Q). The reduced product, ubiquinol (QH2) is free to diffuse within the membrane. At the same time, Complex I moves four protons (H+) across the membrane, producing a proton gradient. Complex I is one of the main sites at which premature electron leakage to oxygen occurs, thus being one of main sites of production of a harmful free radical called superoxide.

The pathway of electrons occurs as follows:

NADH is oxidized to NAD+, reducing Flavin mononucleotide to FMNH2 in one two-electron step. The next electron carrier is a Fe-S cluster, which can only accept one electron at a time to reduce the ferric ion into a ferrous ion. In a convenient manner, FMNH2 can be oxidized in only two one-electron steps, through a semiquinone intermediate. The electron thus travels from the FMNH2 to the Fe-S cluster, then from the Fe-S cluster to the oxidized Q to give the free-radical (semiquinone) form of Q. This happens again to reduce the semiquinone form to the ubiquinol form, QH2. During this process, four protons are translocated across the inner mitochondrial membrane, from the matrix to the intermembrane space. This creates a proton gradient that will be later used to generate ATP through oxidative phosphorylation.


complex I and II pictures

or type in complex 1 electron transport into google

Complex I

Complex III (cytochrome bc1 complex; EC removes in a stepwise fashion two electrons from QH2 at the QO site and sequentially transfers them to two molecules of cytochrome c, a water-soluble electron carrier located within the intermembrane space. The two other electrons are sequentially passed across the protein to the Qi site where quinone part of ubiquinone is reduced to quinol. A proton gradient is formed because it takes 2 quinol (4H+4e-) oxidations at the Qo site to form one quinol (2H+2e-) at the Qi site. (in total 6 protons: 2 protons reduce quinone to quinol and 4 protons are released from 2 ubiquinol). The bc1 complex does NOT 'pump' protons, it helps build the proton gradient by an asymmetric absorption/release of protons.

When electron transfer is reduced (by a high membrane potential, point mutations or respiratory inhibitors such as antimycin A), Complex III may leak electrons to molecular oxygen, resulting in the formation of superoxide, a highly-toxic reactive oxygen species, which is thought to contribute to the pathology of a number of diseases and to processes involved in aging.[citation needed]

In complex III, antimycin is known not to completely inhibit electron flow from ubiquinol to cytochrome c: the antimycin-insensitive reduction of cytochrome c is mediated by superoxide radicals; the source of superoxide in the enzyme may be either cytochrome b566, [31] or ubisemiquinone [32] or Rieske's iron-sulfur center [33]. Ubisemiquinone is relatively stable only when protein bound [34], therefore the coenzyme Q (CoQ) pool in the lipid bilayer is no source of ROS.

The role of ubiquinone within ROS production deserves some comments (cf. [18]), since it has been described both as a prooxidant [22 A. Boveris and B. Chance, Biochem. J. 134 (1973), pp. 707-716. View Record in Scopus | Cited By in Scopus (943)[22], [28] and [32]] and as a powerful antioxidant [[35], [36] and [37]]; the former action has been ascribed to either oxidized or reduced quinone, whereas the latter exclusively to ubiquinol.

In some instances, a prooxidant effect may be ascribed only apparently to CoQ: for example the enhanced ROS production when CoQ-depleted mitochondria oxidizing succinate are reconstituted with CoQ [22] is a consequence of the increased rate of electron feeding to complex III via the quinone, and presumably complex III itself is the source of ROS generation.

Complex III


Being a major source of endogenous oxidative damage, throughout normal oxidative phosphorylation, the mitochondrian produces about 107 reactive oxygen species (ROS)/mitochondrion/day. (Singh et a.l 2003)

The continuous generation of ROS by mitochondria throughout cell life produces an age-related “chronic” oxidative stress, which plays a key role in cellular aging. It is now well established that oxidative damage to mitochondrial DNA occurs upon aging (Vinu et al. 2006)

More than 90% of the oxygen used by aerobic cells is consumed in mitochondria and about 1-2% of oxygen used by mammalian mitochondria in state four does not form water but superoxide anion which is converted to hydrogen peroxide within mitochondria either spontaneously or by Mn-superoxide dismutase. Hydrogen peroxide is released to the extramitochondrial space. Studying different mammalian species, Sohal et al. found that mitochondria from shorter-lived species produce higher amounts of hydroperoxide than those from the longer-lived species . Thus, oxygen free radicals and hydroperoxides are generated continuously in the mitochondrial respiratory chain and they, particularly extremely aggressive hydroxyl radical, cause oxidative damage to proteins, lipids and DNA.

According to the free radical theory of aging, cells continuously produce free radicals, and constant radical damage eventually kills the cell. When radicals kill or damage enough cells in an organism, the organism ages.1 (Gradual loss of mitochondrial function due to oxidative stress is a common feature of ageing.)

This theory is supported by experimental evidences such as the extension of lifespan obtained by increasing the antioxidant defense as well as the inverse relationship between the rate of reactive oxygen species (ROS) production and the maximum lifespan of species.(Vina et al. 2006) The fact that mitochondria are damaged inside intact cells was almost simultaneously reported by Vina et a.l 2006 and by Hagen et al. Administration of antioxidants can increase the average lifespan of flies [10]. Orr and Sohal reported that simultaneous overexpression of copper-zinc superoxide dismutase and catalase genes transgenic Drosophila extend their mean and maximum lifespan.

The primary site of free radical oxygen damage is mitochondrial DNA (mtDNA). This DNA is found in the nucleus of the cell, which serves as the "command centre" of the cell, as well as in the mitochondria. Mitochondrial DNA (mtDNA) is constantly exposed to ROS produced within the mitochondria and alongside that contains no introns as well as having no protective histones.(Singh)

A major development that opened breakthroughs in mitochondrial pathology was the discovery that mtDNA mutations are at the basis of diseases (lenaz).

The cell fixes much of the damage done to nuclear DNA. However, the mitochondrial genome is extremely susceptible to genetic changes occurring spontaneously and at increases frequency by exogenous oxidants and cannot be readily fixed. Therefore, extensive mtDNA damage accumulates over time and shuts down mitochondria, causing cells to die and the organism to age.

With the exception of erythrocytes, all human cells contain mitochondria. Hence, this free radical generation process can disrupt all levels of cell function. Each mitochondrian contains several copies of mtDNA. Of the 3000 genes that participate in biogenesis of a single mitochondrian, no more than 37 of these genes encode thirteen proteins, all of which are subunits of the electron transport chain. In addition a minimal set of 22 transfer RNAs and 2 ribosomal RNA's necessary for translation in mitochondrian are encoded by mtDNA.

Furthermore MtDNA is maternally inherited and is the basis of hereditary mitochondrial cytopathies. Individuals with mitochondrial disease can possibly either inherit their mutation from the mother or experience a mutation during oogenesis or early embryogenesis. A homoplasmic condition exists when all wild-type or all mutant mtDNA exist in a cell. On the other hand a heteroplasmic condition exists when a heterogeneous mtDNA population, mutant and wild-type mtDNA coexist in the same cell. The heteroplasmic condition is frequently associated with mitochondrial disease.

Ironically this oxidative phosphorylation mechanism which is the predominant source of energy for various tissues, is also unfortunately the most significant source of endogenous ROS in the cell because they carry the electron transport chain during oxidative phosphorylation which reduces oxygen to water by addition of electrons. Under certain pathological conditions and by environmental oxidants ROS production can be considerably enhanced. The sparks in this analogy are free electrons escaping the transport system. These unpaired electrons readily form free radical molecules which are chemically reactive and highly unstable.

The endogenous rate of oxidation is estimated to be approximately 150 000 events/cell/day in human cells. Many different mutations of mitochondrial DNA have been found to be pathogenic in humans. Several general classes of mutations have been reported. These include point mutations, deletions, duplication and insertions and rearrangements. The deletions removing tRNAs and protein coding genes gi