Medical Biology 62:71-77,
Oxygen Radicals: A
Commonsense Look at Their Nature and Medical Importance
From the Department of Biochemistry, University of London King's
College, London, U.K.
"Oxygen radicals" are now
popular subjects for research papers; several hundred are published each
year. Many of these pass rapidly into oblivion, joining the great mass
of unread scientific literature that clogs library shelves and dilutes
important research findings to an increasingly great extent. The basic
chemistry of oxygen-derived species was established years ago by
radiation chemists (1,6), but "superoxide" is still endowed with
miraculous properties by the uninitiated.
Demonstration that the action of a disease or toxin in vivo produces
increased lipid peroxidation (a currently-popular scientific activity)
means nothing more than the fact that its action produces increased
lipid peroxidation: it does not automatically follow that the lipid
peroxidation causes the damaging effects of the drug or disease.
The purpose of this paper is to explain:
i) what oxygen radicals are
ii) the evidence that oxygen radicals are important in vivo
iii) what needs to be done to establish a role for oxygen radicals and
lipid peroxidation in human disease.
What are the oxygen radicals and how are they produced?
Electrons within atoms and molecules occupy regions of space known as "orbitals".
Each orbital can hold a maximum of two electrons. A single electron
alone in an orbital is said to be "unpaired" and a radical is defined as
any species that contains one or more unpaired electrons. Such a
definition embraces the atom of hydrogen (one unpaired electron) and the
ions of such transition metals as iron, copper and manganese (cf.
Holmberg, this volume).
The diatomic oxygen molecule, O2, has two unpaired electrons
and thus qualifies as a radical. Most of the oxygen taken up by human
cells is reduced to water by the action of the cytochrome oxidase
complex in mitochondria. This requires the addition of four electrons to
each oxygen molecule,
O2 + 4 H+ + 4e- ---> 2 H2O (1)
For chemical reasons (reviewed in ref. 21 and 28), O2 likes
to receive its electrons one at a time, producing a series of partially
reduced intermediates O2 add le- O2-
add le- H2O2 add le---> ---> ---> 2H (two unpaired
superoxide hydrogen peroxide electrons) (one unpaired (no unpaired
electron) OH + OH- hydroxyl radical hydroxyl ion (2) (one
unpaired electron) (no unpaired electron)
| | | add le- H+
| add H+ H2O2 H2O2
Cytochrome oxidase keeps the partially reduced intermediates on the
pathway to water tightly bound to its active site (21); they do not
escape into free solution.
Superoxide ion is the one-electron reduction product of oxygen.
Dissolved in organic solvents, it is an extremely reactive species, e.g.
it can displace chlorine from such unreactive chlorinated hydrocarbons
as carbon tetrachloride (CCl4) (40). In aqueous solution O2-
is poorly reactive, acting as a reducing agent (e.g. it will reduce
cytochrome c or nitro-blue tetrazolium) and slowly undergoing the
dismutation reaction, in which one molecule of superoxide reduces
another one to form
hydrogen peroxide (H2O2 ). The dismutation
reaction occurs in stages; O2- must first combine
with a proton to yield the hydroperoxyl radical, HO2,
O2- + H+ ---> HO2 (3)
HO2 + O2 + H+ ---> H2O2
+ O2 (4)
overall O2- + O2- + 2H+
---> H2O2 + O2 (5)
At physiological pH the low concentration of H+ ions slows
the rate of dismutation.
Despite the low reactivity of O2- in aqueous
solution, systems producing it do a great deal of damage in vitro (e.g.
they fragment DNA and polysaccharides, kill bacteria and animal cells in
culture) and in vivo (e.g. when O2- generating
systems are injected into the footpads of rats inflammation is produced,
their instillation into the lungs of rats and rabbits produces oedema
and cell death, and infusion of them into vascular beds produces
endothelial cell damage and extensive leakage from the blood vessels)
(21,26,28). Depending on the circumstances, damage caused by O2-
generating systems might be attributed to
itself, e.g. exposure of tissue fluids to O2-
causes formation of a factor chemotactic for neutrophils that brings
more of them into the area and hence can potentiate inflammation
(ii) HO2 radical,
which is more reactive than O2- (6).
Formation of HO2 is favoured at pH values lower than
"physiological", but the phagocytic vacuole operates at an acid pH and
the pericellular pH of macrophages has been reported to be 6 or less
(iv) hydroxyl radical (see below)
(v) singlet oxygen.
Singlet O2 is an
especially reactive form of oxygen capable of rapidly oxidising many
molecules, including membrane lipids. Its formation in O2--
generating systems has often been proposed but clear-cut evidence
for a damaging role of singlet O2 in such systems has not
been obtained. One of the problems is that the "scavengers" of singlet O2
frequently used react with other radical species as well (for reviews
see ref. 26 and 28).
What is the evidence that O2- is formed in vivo
in human cells?
Any electron transport chain operating in the presence of O2
"leaks" some of the electrons, passing them directly onto O2
Since O2 prefers to take electrons one at a time, O2-
is produced. Such O2- production can be
demonstrated in vitro using mitochondria and microsomes from a range of
animal tissues. The rate at which O2- is produced
rises as the concentration of O2 in the system is raised
(e.g. see ref. 20). A number of compounds slowly become oxidised on
exposure to O2 and O2- is generated;
these include adrenalin, tetrahydrofolate, reduced FMN and
Since human cells contain mitochondria, endoplasmic reticulum,
oxidisable compounds and oxygen, it is likely that O2-
is formed within them in vivo. Backing up this evidence, for those who
do not like extrapolating from in vitro experiments, is the fact that
human cells contain high levels of superoxide dismutase (SOD) activity
(45). This enzyme, for which O2- is the specific
substrate (35), is known to be a very important anti-oxidant in bacteria
and small mammals (26) and its presence in human cells is good evidence
that O2- is formed in vivo. During the maturation
of erythrocytes most enzymes are lost, but SOD remains. It is not a
great stretch of the imagination to associate this with the ability of
oxyhaemoglobin to release O2- radical and
Another source of O2- in vivo is the respiratory
burst of phagocytic cells such as neutrophils, monocytes, eosinophils
and macrophages (3, 16, 25). The amount of O2-
produced might sometimes be controlled by the O2 tension of
body fluids (14). Host defence against invading bacteria is dependent on
the circulating neutrophils, which respond to contact with particles
they recognise as foreign by producing a "burst" of O2
radical. The particle is engulfed (the piece of membrane surrounding it
being the segment that produces O2- on contact;
cf. Segal, this volume), and other vesicles then fuse with the
phagocytic vesicle. This exposes the engulfed particle to other
anti-bacterial mechanisms, including cationic proteins, lysosomal
enzymes and myeloperoxidase (3, 16, 25).
Which of these processes is the most important in bacterial killing?
Human and other animal neutrophils can kill some strains of bacteria
under anaerobic conditions, when O2- cannot form.
Obviously, the other mechanisms are important here. Many other bacterial
strains are not killed in the absence of O2,
however, even though engulfment and vesicle fusion proceed normally. In
chronic granulomatous disease (CGD), an inborn error of metabolism, the
respiratory burst does not occur but other aspects of phagocytic action
proceed normally. CGD was first described in humans because it is
accompanied by severe and recurrent infections affecting lymph nodes,
skin, lungs and liver (43). The symptoms of CGD provide direct evidence
for the production of O2- by human phagocytic
cells in vivo and for its role in bacterial killing.
It follows therefore that if neutrophils become activated in the wrong
place, or to excessive extents (as in the autoimmune diseases, 25) then
the oxygen radicals they release could do a lot of damage. It must be
remembered, however, that phagocytic cells also produce hydrolytic
enzymes (elastase, neutral proteases etc.), chemotactic factors,
prostaglandins, leukotrienes and other chemicals, so that damage by
activated phagocytes could be due to any one of these factors or to any
combination of them. It cannot be attributed a priori to oxygen
O2- generating systems produce H2O2
by the dismutation reaction (eqn. 5) and a number of oxidase
enzymes produce H2O2 directly, examples being
glycollate oxidase and amino acid oxidases. SOD enzymes remove O2-
by greatly accelerating the dismutation reaction, so if we accept
that O2- is formed in vivo in humans
then we must accept that H2O2 vapour is present in
expired human breath (48), a likely source being H2O2
released from alveolar macrophages (3, 25) although a contribution from
peroxide-producing oral bacteria (10) cannot be ruled out.
That H2O2 is formed in vivo in humans is further
supported by the presence of enzymes specific for its removal, such as
catalase and glutathione peroxidase. The latter enzyme requires selenium
for its activity (13; cf. Diplock, this volume). H2O2
is probably more damaging than is O2- in in vitro
experiments in aqueous solution, but many cells seem to tolerate its
presence and bacteria often produce H2O2 (e.g.
ref. 10). On the other hand, the toxicity of O2-
generating systems to several animal cells in culture has been
attributed to formation of H2O2 (e.g. ref. 44).
Why this should be so is discussed in the next section.
Hydroxyl radical is produced when water is exposed to high-energy
ionising radiation and hence its properties have been well documented by
radiation chemists (6, 49). Unlike the hydroxyl ion, the hydroxyl
radical is fearsomely reactive, combining with most molecules found in
vivo at near diffusion-controlled rates. Hence any OH produced in vivo
will react at or close to its site of formation. The extent of the
damage done would therefore depend on what the site of formation was
(e.g. production of OH close to DNA could lead to strand breakage
whereas production close to an enzyme molecule already present in excess
in the cell, such as lactate dehydrogenase, might have no biological
Hydroxyl radical is produced whenever H2O2 comes
into contact with copper (I) ions (Cu+) or iron (II) ions (Fe2+).
Dr. Gutteridge has reviewed in this volume the substantial evidence that
metal complexes capable of causing hydroxyl radical formation are
present in vivo in human cells (also see ref. 28). Particularly
important in vivo are complexes of iron salts with phosphate esters such
as ATP and GTP (17, 19) or with DNA (18). Organisms take great care to
ensure that as much iron or copper as possible is bound to transport
proteins or functional proteins such as transferrin, caeruloplasmin or
haemoglobin. Metals bound to these proteins are inactive or only weakly
active in catalysing OH production (28, 50).
Since both H2O2 and metal complexes are present in
vivo in humans, it is logical to assume that OH radicals can form.
Direct evidence for this is difficult to obtain. Many methods exist for
demonstrating the existence of OH in vitro (see ref. 24 and 28 for
reviews) but in vivo any OH formed is likely to react so close to its
site of formation that the use of these methods is impractical, although
some new techniques (such as the ability of OH to convert
dimethylsulphoxide into methane (36) or its ability to hydroxylate
aromatic rings in characteristic ways (37) show promise for in vivo use.
One can also attempt to infer the formation of OH radical in vivo by
observing the damage done (as in rheumatoid arthritis, see below). In
vitro, phagocytic cells have been shown to produce OH radical (11-13)
and the killing of
bacteria can sometimes be prevented by reagents that react with this
species (3, 16, 25).
It was mentioned in the previous section that the killing of animal
cells in culture by O2- generating systems can
sometimes be attributed to H2O2. It could, of
course, be achieved by H2O2 itself; some enzymes
are known to be inactivated by H2O2 although the
best examples come from plant rather than animal systems
(11). There is another possibility, however, H2O2
generated externally crosses cell membranes easily and could penetrate
inside the cell and cause OH to be formed. Externally added scavengers
of OH would not prevent this since they could not reach the correct
place. By contrast, O2- crosses cell membranes
only slowly (42) unless there is a specific channel for it (the only
known example of this being the erythrocyte membrane, which has an
"anion channel" through which O2- can move(3).
Hydroxyl radical will never cross a membrane: it will react with
whatever membrane component if meets first.
What is lipid peroxidation and is it of medical importance?
Lipid peroxidation has been broadly defined by A. L. Tappel in the USA
as "oxidative deterioration of polyunsaturated fatty acids", i.e. fatty
acids that contain more than two carbon-carbon double bonds.
Oxygen-dependent deterioration, leading to rancidity, has been long
recognised as a problem in the storage of fats and oils and is even more
relevant today with the popularity of "polyunsaturated" food products.
Some of the best studies on peroxidation chemistry have been carried out
by food chemists.
Initiation of peroxidation in a membrane or polyunsaturated fatty acid
is due to the attack of any species that can "pull off" a hydrogen atom
from one of the - CH2 - groups in the carbon chain. Hydroxyl
radical and possibly HO2 can do this, but H2O2
and O2- cannot. Hence O2-
does not initiate lipid peroxidation. Since a hydrogen atom has only one
electron, removing it leaves behind an unpaired electron on the carbon.
The resulting carbon radical - CH -, undergoes molecular rearrangement
to form a conjugated diene, which then combines rapidly with O2
to give a O2 | peroxy radical, - CH -. Peroxy radicals are
capable of abstracting a hydrogen atom from other fatty acids and so
setting off a chain reaction that can continue until the membrane fatty
acids are completely oxidised to hydroperoxides (eqn. 6) O2 |
- CH- + - CH2 - ---> peroxy adjacent fatty acid
radical carbon chain O2H | - CH - + - CH - (6) carbon
radical, lipid forms another hydroperoxide peroxy radical.
Lipid hydroperoxides are stable under physiological conditions until
they come into contact with transition metals such as iron or copper
salts. Cu2+, Fe2+ or Fe3+ salts as well
as haem and haem proteins (e.g. cytochromes, haemoglobin) can interact
with lipid peroxides. These metals or their complexes cause lipid
hydroperoxides to decompose in very complicated ways, producing radicals
that can continue the chain reaction of lipid peroxidation (as in eqn.
6), as well as cytotoxic aldehydes and hydrocarbon gases. Most attention
is paid in the literature to malonaldehyde, but this is a very minor
endproduct of lipid peroxidation (for reviews see ref. 4, 26, 32).
Does lipid peroxidation occur normally in vivo in humans? This question
is surprisingly difficult to answer: little evidence for lipid peroxides
or their decomposition products can be found in healthy human tissues
(28). Expired human breath contains gaseous hydrocarbons that might have
originated from decomposition of lipid hydroperoxides, but they might
also have been produced by bacteria in the gut or even on the skin.
Animal cell membranes contain tocopherol (vitamin E), which is a
powerful inhibitor of lipid peroxidation, and proteins such as
caeruloplasmin and glutathione peroxidase probably help to protect
against this process in vivo (27).
Diseased tissues, or tissues isolated after exposure of animals to such
toxins as ethanol, phenylhydrazine and paraquat often show evidence of
increased peroxidation. Simple in vitro experiments demonstrate quite
clearly that dead or damaged tissues peroxidise more rapidly than living
ones, presumably because of membrane disruption by enzymes released from
lysosomes, release of metal ions from their storage sites and failure of
antioxidant mechanisms. Thus evidence that a toxin increases lipid
peroxidation in vivo does not prove the sequence of events toxin --->
lipid peroxidation ---> damage (7) but is equally explained by the
sequence toxin ---> cell damage or death ---> lipid peroxidation (8)
Of course, toxins released by dead or dying cells undergoing
peroxidation might cause further damage to healthy cells, although there
is little evidence for this in vivo. Among the many claims I have seen
in the literature for lipid peroxidation as an agent of the damage
induced by a toxin, I have seen clear evidence for sequence 7 only in
the case of the hepatotoxic effects of carbon tetrachloride (32).
Sequence 8 is a much better explanation of the in vivo effects on
membrane lipids of, for example, paraquat.
An often quoted illustration of the importance of lipid peroxidation in
vivo is the accumulation of "age pigment" in various human tissues.
Chemical analysis of age pigment shows convincingly that it is an
endproduct of oxidative damage to lipids (41). However, the lipids in
question seem to be taken into lysosomes before they are degraded; they
are not "normal cell lipids". The exposure of lipids to hydrolytic
enzymes and metal ions within lysosomes no doubt facilitates their
peroxidation, and so more peroxidised material accumulates within cells
as lysosomes get older and have engulfed more lipid material.
The TBA test
The TBA (thiobarbituric acid) test is one of the most widely used (and
abused!) tests for measuring lipid peroxidation. The simplicity of
performing the test (the material under study is merely heated with acid
and TBA and the formation of a pink colour measured at 523 nm) conceals
its essential complexity.
Consider a typical experiment. A lipid system, perhaps with added metal
ions, chelating agents or other reagents, in incubated in the presence
of air. Then TBA plus acid are added and the mixture heated at 100
degrees Celcius. The air, metals and other reagents are still present,
so as much or even more oxidative damage to the lipid can be done during
the TBA test itself as happened during the initial incubation.
The pink colour is due to the formation of an adduct between TBA and
malonaldehyde (MDA) under acidic conditions. Indeed, the TBA assay is
often calibrated with MDA and the results of peroxidation assays are
often expressed as "amounts of MDA formed". Some papers in the
literature give the mistaken impression that TBA reacts only with free
MDA and so measures the production, but it was shown as long ago at 1958
in studies with peroxidising fish oil that 98 % of the MDA that reacts
in the TBA test was not present in the original sample assayed but forms
from lipid peroxides that decomposed during the acid-heating
stage of the TBA assay. More recent studies confirm this and show that
the apparent "TBA reactivity" of say, serum, varies with the exact
concentration of acid, type of acid and period of heating used in the
TBA assay (23). The amount of MDA formed during the initial incubation
of the system as opposed to during
the assay depends on such factors as the iron salt concentration (4, 23,
32). An apparent "inhibitor" of lipid peroxidation as detected by the
TBA test might actually inhibit the peroxidation process, but could
equally well interfere with decomposition of the peroxides during the
acid-heating stage of the assay. Similarly, absolute values for the "TBA
reactivity" of body fluids or tissue extracts are meaningless, although
changes in these values may be significant provided that the same assay
in employed in the same way each time.
Of course, many scientists are aware of these problems with the TBA
assay and there are ways around them (2, 41), including the use of other
assay systems in conjunction with the TBA test (4, 27). I have included
these cautions to encourage a more critical attitude to some of the
Oxygen Radicals and Disease
Free radicals have been suggested to be involved in the pathology of a
number of diseases. In several cases the evidence consists only of
observations of increased lipid peroxidation in diseased tissues, which
is ambiguous (see above). I have chose to look in detail at two cases
where the evidence at first sight is more convincing, cancer and
inflammatory joint disease.
Any substance that reacts with DNA is potentially carcinogenic. Exposure
of DNA to O2- generating systems causes extensive
strand breakage and degradation of deoxyribose (9, 39), an effect shown
in vitro to be due to formation of OH. Both bacteria and animal cells in
culture suffer DNA damage on exposure to O2-
generating systems, which can be shown to be mutagenic (46, 47). It is
therefore tempting to attribute the increased risk of development of
cancer in chronically inflamed tissues to generation of oxygen radicals
by phagocytic cells, although there is no direct evidence for this.
Great excitement was generated by reports that cancer cells in culture
and from some transplantable tumours in animals are deficient in SOD
activity, especially in their mitochondria (for a review see ref. 34).
The relevance of these studies to human cancer is not at all clear,
however, since human tumours biopsied during surgery show no defects in
any SOD activity (31, 45).
I have already speculated on the role of oxygen radicals in the
autoimmune diseases. Rheumatoid arthritis has some of the features of an
autoimmune disease but its exact cause is unknown. The synovial fluid of
the inflamed joint swarms with neutrophils. Since the fluid contains
increased concentrations of products
that activated neutrophils release (including lactoferrin, 5) and
end-products of arachidonic acid metabolism), then at least some of
these neutrophils must be activated and thus producing superoxide, and
hence H2O2 in vivo. Human synovial fluid is poor
in SOD, catalase and glutathione peroxidase activities (8) but
does contain iron complexes capable of catalyzing a reaction between O2-
and H2O2 to form OH (38). There is as yet no
direct proof that OH is formed in vivo, but evidence consistent with its
formation includes the observation that the hyaluronic acid in synovial
fluid is degraded in rheumatoid joints, and the type of degradation
observed can be reproduced by exposing pure hyaluronic acid in vitro to
OH radical (22). TBA-reactive material is also present in serum and
synovial fluid of rheumatoid patients. There are significant
correlations (38) between the content of TBA-reactive material in
synovial fluid, its content of catalytic iron complexes and both
clinical ("knee score") and laboratory ("white cell count" and "fluid
content of C-reactive protein") assessments of disease activity.
Thus there is certainly evidence for oxygen radicals being produced in
the rheumatoid joint and having some deleterious effects. The question
to be answered in how important are oxygen radicals in relation to other
agents of damage. The pathology of rheumatoid arthritis is very complex
and the number of potentially damaging agents, including hydrolytic
enzymes, prostaglandins and leukotrienes, is enormous (29). Some
scientist have tried to assess the importance of oxygen radicals by
examining the effects of injecting SOD directly into inflamed joints
(33; see Marklund, this volume), whereas our group, reasoning that iron
complexes are required for O2- dependent formation of highly
reactive OH radical, is examining the effect of iron-chelating drugs
that can prevent OH formation (such as
desferrioxamine, 12) on animal models of acute and chronic inflammation
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