Medical Biology 62:71-77, 1984

Oxygen Radicals: A Commonsense Look at Their Nature and Medical 
Importance

B. Halliwell

From the Department of Biochemistry, University of London King's 
College, London, U.K.

Introduction

"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   +   4H+   +   4e-   --->   2H2O                   (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)
                      electron)

                     OH             OH-

      hydroxyl radical            hydroxyl ion                (2)
  (one unpaired electron)     (no unpaired electron)

            |                          |
            |                          |
            |  add le- H+              |     add H+
           H2O                        H2O 

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

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

 (i)   O2- 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 (15)
 (iii) H2O2 (see below)
 (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 
oxyhaemoglobin (21,24).

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 methaemoglobin.

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 
radicals.


Hydrogen Peroxide

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

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 
consequences).

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 published literature.


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.

Cancer

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).

Rheumatoid arthritis

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 (7).


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37. Richmond R, Halliwell B, Chauhan J, Darbre A: Superoxide-
    dependent formation of hydroxyl radicals: detection of 
    hydroxyl radicals by the hydroxylation of aromatic compounds.  
    Anal Biochem 118: 328-335, 1981

38. Rowley D, Gutteridge JMC, Blake D, Farr M, Halliwell B: Lipid 
    peroxidation in rheumatoid arthritis.  Thiobarbituric-acid-
    reactive material and catalytic iron salts in synovial fluid 
    from rheumatoid patients.  Slin Sci (in press) 1984

39. Rowley DA, Halliwell B: DNA damage by superoxide-generating 
    systems in relation to the mechanism of action of the anti-
    tumour antibiotic adriamycin.  Biochim Biophys Acta 761: 86-
    93, 1983

40. Sawyer DT, Gibian MT: The redox chemistry of superoxide ion.  
    Tetrahedron 35: 1471-1481, 1979

41. Sohal RS (ed): Age pigments.  Elsevier, Amsterdam 1981

42. Takahashi MA, Asada K: Superoxide anion permeability of 
    phospholipid membranes and chloroplast thylakoids.  Arch 
    Biochem Biophys 226: 558-566, 1983

43. Tauber AI, Borregaard N, Simons E, Wright J: Chronic 
    granulomatous disease: a syndrome of phagocyte oxidase 
    deficiencies.  Medicine 62: 286-309, 1983

44. Weiss SJ, Young J, LoBuglio AF, Slivka A, Nimeh NF: Role of 
    hydrogen peroxide in neutrophil-mediated destruction of 
    cultured endothelial cells.  J Clin Invest 68: 714-721, 1981

45. Westman NG, Marklund SL: Copper-and zinc-containing 
    superoxide dismutase and manganese-containing superoxide 
    dismutase in human tissues and human malignant tumours.  
    Cancer Res 41: 2962-2966, 1981

46. Weitburg AB, Weitzman SA, Destrempes M, Latt SA, Stossel TP: 
    Stimulated human phagocytes produce cytogenetic changes in 
    cultured mammalian cells.  N Engl J Med 308: 26-30, 1983

47. Weitzman SA, Stossel TP: Mutation caused by human phagocytes.  
    Science 212: 546-547, 1981

48. Williams MD, Leight JS, Chance B: Hydrogen peroxide in human 
    breath and its probable role in spontaneous breath 
    luminescence.  Ann NY Acad Sci 45: 478-483, 1983

49. Willson RL: Free radicals and tissue damage: mechanistic 
    evidence from radiation studies.  In: Biochemical mechanism 
    of liver injury.  Ed. T. F. Slater. Academic Press, London 
    1978

50. Winterbourn CC: Lactoferrin-catalysed hydroxyl radical 
    formation.  Additional requirement for a chelating agent.  
    Biochem J 210: 15-19, 1983

Address:   B. Halliwell
           Department of Biochemistry, University of London
           King's College
           Strand, London
           UK

