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What Is Oxidative Stress?

D. J o h n B e t t e r i d g e

Oxidative stress, defined as a disturbance in the balance between the production of reactive oxygen species (free radicals) and
antioxidant defenses, is discussed in relation to its possible role in the production of tissue damage in diabetes mellitus.
Important free radicals are described and biological sources of origin discussed, together with the major antioxidant defense
mechanisms. Examples of the possible consequences of free radical damage are provided with special emphasis on lipid
peroxidation. Finally, the question of whether oxidative stress is increased in diabetes mellitus is discussed.
Copyright © 2000 by W.B. Saunders Company

HE PURPOSE OF THIS short review is to provide the anion (Oz-), transition metals such as iron and copper, nitric
T reader with a simple introduction to the concept of
oxidative stress as a potentially important contributor to tissue
oxide (NO), and peroxynitrite (ONOO - ).
The hydroxyl radical, the most potent oxidant known, has an
damage. The role of oxidative stress has been hypothesized in extremely short half-life, reacting at the site of its formation
many clinical situations, but here attention wilt be focused on its through its ability to attack most biological molecules resulting
possible role in the microvascular and macrovascular complica- in the propagation of free radical chain reactions. Superoxide is
tions of diabetes. formed when oxygen accepts an electron and is not in itself
Oxidative stress will be defined along with a description of particularly reactive. It can act as a weak oxidizing agent, but is
the origin of reactive oxygen species and other free radicals. much stronger as a reducing agent of iron complexes such as
Because of the potential for catastrophic damage as a result of cytochrome C. It is likely to be more important as a source of
free radical attack, complex antioxidant defense mechanisms hydroxyl radicals and hydrogen peroxide. Nitric oxide, an
have evolved to protect body tissues. These processes will be example of a physiological radical, is of considerable interest
discussed together with reference to their possible deficiencies through its rote as a mediator of vascular tone)
in the diabetic state. Examples will be provided of the potential
for free radical attack on important cellular components, with WHERE DO FREE RADICALS COME FROM?
particular emphasis on lipid peroxidation. Free radicals can be produced by several different biochemi-
Generally, the chemical reactions involved in free radical cal processes within the body including: reduction of molecular
damage occur almost instantaneously so that direct measure- oxygen during aerobic respiration yielding superoxide and
ment is a major problem. Some of the various methods that have hydroxyl radicals; by-products of chemistry such as oxidation
been developed to assess oxidative stress that concentrate of catecholamines and activation of the arachidonic acid
mainly on the detection of the consequences of tree radical cascade product electrons, which can reduce molecular oxygen
attack will be described. Finally, the potential role for oxidative to superoxide; production of snperoxide and hypochlorous acid
stress in diabetes will be introduced to provide a background to (HOC1), a powerful oxidant, by activated phagocytes; nitric
the reviews that follow. The close interaction between glycation oxide production by vascular endothelium and other cells. In
and oxidative stress is likely to be important in the etiology of addition, free radicals can be produced in response to external
diabetic tissue damage. The reader is referred to recent reviews electromagnetic radiation, such as gamma rays, which can split
for more detailed descriptions of the biochemical processes water to produce hydroxyl radicals (Fig 1).
involved in oxidative stress.l-6
THE POSSIBLE CONSEQUENCES OF FREE
WHAT IS OXIDATIVE STRESS? RADICAL-MEDIATED TISSUE DAMAGE

Oxidative stress has been defined as a disturbance in the Free radicals are inherently unstable molecules because of
balance between the production of reactive oxygen species (free the presence of unpaired electrons. As a result, they can be
radicals) and antioxidant defenses, which may lead to tissue highly reactive, although this varies from radical to radical,
injury. 1 reacting locally to accept or donate electrons to other molecules
Free radicals are formed in large amounts as an unavoidable to achieve a more stable state. As most molecules are not free
by-product of many biochemical processes and in some in- radicals, the majority of reactions will involve nonradicats.
stances, deliberately, such as in activated neutrophils. In addi- Reaction of a radical with a nonradical (all biological macromol-
tion, free radicals may be generated in the body in response to ecules, lipids, proteins, nucleic acids, and carbohydrates are
electromagnetic radiation from the environment and acquired possible targets) produces a free radical chain reaction with the
directly as oxidizing pollutants such as ozone and nitrogen formation of new radicals, which in turn can react with further
dioxide. If antioxidant defenses are deficient then damage may
occur in a variety of tissues.
From the Department of Medicine, Sip" Jules Thon7 hzstitute, The
WHAT ARE FREE RADICALS? Middlesex Hospital, London, UK.
Address reprint requests to D. John Betteridge, BSc, PhD, MD,
Free radicals can be defined as any chemical species that FRCP, Department of Medicine, University College London, The
contains unpaired electrons. Unpaired electrons increase the Middlesex Hospital, Mortimer St, London WIN 8AA, UK.
chemical reactivity of an atom or molecule. Common examples Copyright © 2000 by W.B. Saunders Company
of tree radicals include the hydroxyl radical (OH), superoxide 0026-0495/00/4902-1002510.00/0

Metabolism, Vol 49, No 2, Suppl 1 (February), 2000: pp 3-8 3


4 D. JOHN BETTERIDGE

a) Reduction of molecular oxygen chain-breaking antioxidant such as vitamin E. The resulting


02 4-e- + H÷ ~ HO2 lipid peroxides are reasonably stable compounds, but their
decomposition can be catalyzed by transition metals and metal
HO2 ~' H + + O2-
complexes producing alkoxyl and peroxyl radicals, which can
0~-+ 2H + + e- ~ H202
stimulate further lipid peroxidation. It is of interest that
H202 + e- ~ OH- +'OH
disrupted tissue is more susceptible to lipid peroxidation. Lipid
"OH + e- + H + ~ H20 peroxidation can have profound effects on cellular function.
HO~ = hydroperoxylradical Extensive peroxidation in cell membranes will result in changes
O~- = superoxideradical
H202 = hydrogen peroxide in fluidity, increased permeability, a decrease in membrane
"OH = hydroxylradical potential, and eventually membrane rupture.
b) Production of hypochlorous acid from hydrogen peroxide Over recent years, it has become clear that oxidation of
low-density lipoprotein (LDL) is central to many of the
H202 + CI- + H + .............~ HOCI + H20
processes of atherogenesis. 12J3 LDL cholesterol explains the
HOCI = hypochlorous acid
strong, positive, independent relationship between total plasma
c) Oxidation of catecholamines cholesterol and atherosclerosis-related disease particularly coro-
HO O nary heart disease. Steinberg et a114 have hypothesized that
oxidatively-modified LDL is the atherogenic particle. Important
HO - - ~ - - R I~ 0 ~ - - R + 2e- + 2H + cells in the atherogenic process, arterial endothelial cells,
smooth muscle cells, and macrophages are all able to oxidize
native LDL. Oxidized LDL can be taken up by monocyte!
macrophages through a scavenger receptor. This receptor up-
d) Activation of the arachidonic acid cascade o;- take mechanism, unlike the classical LDL receptor, is not
Arachidonic acid downregulated by increased cellular cholesterol ester content
such that lipid-laden foam cells are formed. Accumulation of
these cells (the fatty streak) in the subendothelial space
Prostag~andin G 2
! Lipid peroxidation

Prostaglandin H2 + e-
Fattyacid

O; Hydrogen
- H" abstraction
Fig 1. Examples of the production of free radicals by different
biochemical processes within the body.

macromolecules. Important examples are lipid peroxidation and Molecular


protein damage, eg, addition of carbonyl groups or crosslinking rearrangement
or fragmentation. Carbonyl derivatives of amino acid residues
render the protein susceptible to proteolysis, 8 DNA base (eg, / Conjugated diene
conversion of guanine into 8-hydroxyguanine and other prod-
ucts) single- and double-strand breaks, and protein/DNA
0 Oxygen
crosslinks. 9-11 Lipid peroxidation is perhaps the most exten- uptake
sively studied consequence of free radical attack and is of
Peroxylradical:abstractionof
potential importance in diabetic vascular damage. For these H"from anotherfatty-acid-
reasons, lipid peroxidation will be described in some detail producing chain reaction

(Fig 2). O
I
Reactive free radicals, eg, the hydroxyl radical, have the
capacity to abstract a hydrogen atom (H) from a methylene
o jH"
group ( - - C H , - - ) of fatty acids, leaving behind an unpaired
electron on the carbon ( - - C H - - ) . Polyunsaturated fatty acids
Lipid hydroperoxide ~ Fragmentation
are particularly prone to free radical attack because the presence J. to aldehyde
Cyclic peroxide and
of a double bond weakens the carbon-hydrogen bond at the ROOH" 1 polymerization
adjacent carbon atom. The remaining carbon-centered radical O Cyclic endoperoxide products
undergoes molecular rearrangement resulting in a conjugated I
O
diene. Conjugated dienes can combine with oxygen forming a I
peroxyl radical. This is itself able to abstract a further hydrogen H
atom and begin a chain reaction that continues either until the Fig 2. Lipid peroxidation as an example of free radical-mediated
substrate is consumed or the reaction is terminated by a tissue damage. Reprinted by Gutteridge. 3
OXIDATIVE STRESS 5

damages the overlying endothelium, which allows platelets to are present within cell membranes. The lipophilic vitamin E
aggregate and release powerful mitogens that contribute to the (et-tocopherol) is a highly effective antioxidant when incorpo-
further development of the lesion. rated in the lipid core of cell membranes. It has the ability to
LDL oxidation results in extensive fragmentation of LDL scavenge intermediate peroxyl radicals and, therefore, interrupt
polyunsaturated fatty acids with conjugation of the fragments to the chain reaction of lipid peroxidation. For this reason, vitamin
LDL apoprotein B and phospholipid. Lecithin is converted to E is described as a chain-breaking antioxidant. 17
lysolecithin by LDL-associated phospholipase A2. At a later
stage, there is fragmentation of LDL apoprotein B forming a 02 O2H
new configuration, which is recognized by the scavenger
I I
receptor. Oxidative/y-modified LDL also has additional proper-
ties that render it more atherogenic than native LDL including: -C- + TH ~ -C- + T"
direct cytotoxicity, induction of adhesion and chemoattractant (c~-tocopherol)
molecules, inhibition of endothelium-dependentrelaxation fac-
tor and increased expression of tissue factor, activation of The tocopherol radical (T') is much less reactive and is
platelets and T cells, stimulation of smooth muscle cell growth, converted back to ec-tocopherol by vitamin C. In addition to the
stimulation of plasminogen activator inhibitor-1 and endothelin presence of antioxidants, membrane structure with appropriate
from endothelial cells, and immunogenicity.15 proportions of cholesterol and phospholipids is important in
resistance to free radical attack.
Major extracellular antioxidant defenses include the metal-
WHAT ARE THE TISSUE DEFENSES AGAINST FREE
binding proteins. As discussed earlier, the free metals iron and
RADICAL ATTACK?
copper can promote free radical damage, accelerating lipid
Given the potential for tissue damage, it is perhaps not peroxidation and catalyzing hydroxyl radical formation. The
surprising that the body has evolved major antioxidant defense body is protected against these potentially adverse effects by
mechanisms to protect it from free radical attack. These binding proteins (transferrin, lactoferrin, and ceruloplasmin),
defenses can be convenientlyconsidered as cellular, membrane, which ensure that these metals are maintained in a nonreactive
and extracellular mechanisms. Gutteridge 3 has defined an state. Similarly, haptoglobins, hemopexin, and albumin bind
antioxidant as any substance that, when present at low concen- hemoglobin and heme, which can also accelerate lipid peroxida-
trations, compared with those of the oxidizable substrate, tion. In addition to the major protective role of the metal-
considerably delays or inhibits oxidation of the substrate. binding proteins, various low-molecular-weightmolecules such
Cellular antioxidant defenses include the dismutase, peroxi- as bilirubin, vitamin C, and urate have antioxidant proper-
dase, and catalase enzymes. In addition, the potential for tiesJ 8,~9 Furthermore, distinct, extracellular forms of glutathi-
intracellular free radical production is greatly diminished by the one peroxidases and superoxide dismutases have been de-
ability of mitochondrial cytochrome oxidase to function catalyti- scribed. 2o,2~
cally in the electron transport chain without releasing reactive
oxygen species. 16 Superoxide dismutases (containing copper HOW IS OXIDATIVE STRESS MEASURED?
and zinc at the native site) in cytosol and in mitochondria
(containing manganese) catalyze the dismutation of snperoxide As described earlier, it is currently not possible to measure
to hydrogen peroxide and oxygen: reactive oxygen species directly so attempts at the evaluation of
free radical production and the pro-oxidantJantioxidant balance
20~- + 2H + ~ H 2 0 2 q- O 2 is based on markers of oxidative reactions often in in vitro
experiments. It is important to bear in mind that differing
The product of this reaction, hydrogen peroxide, is a weak experimental conditions, for example, the type of substrate
oxidant and is relatively stable. However, unlike superoxide, employed and the nature of oxidative conditions applied, will
hydrogen peroxide can rapidly diffuse across cell membranes, result in the generation of different products.
and in the presence of transition metal ions it can be converted In general terms, oxidative stress can be assessed by measure-
to hydroxyl radicals via Fenton chemistry: ment of reaction products of oxidative damage, for example,
lipid peroxidation, DNA oxidation, and protein oxidation. A
Fe2+ + H20 e ---*Fe3+ + "OH + OH
complementary approach is the measurement of depletion of
Two enzyme systems can break down hydrogen peroxide, antioxidants such as ~x-tocopherol, vitamin C, and thiol groups.
Glntathione peroxidases present in cytosol and mitochondria Lipid peroxidation is the most studied with a variety of
have a major role in removing hydrogen peroxide generated by techniques employed including thiobarbituric acid-reactive ma-
superoxide dismutase with the oxidation of glutathione (GSH): terial assays, conjugated dienes, hydroperoxides, F2 isopros-
tanes, and nitroxides. 22 Recently, Visioli and Galli23 have
2GSH + H202 ----,GSSG + 2H20 provided an overview of the evaluation of oxidative processes
Catalases that are present in peroxisomes in many tissues in relation to cardiovascular disease.
remove hydrogen peroxide when present in high concentra- Although oxidative stress has been implicated in cardiovascu-
tions: lar disease and many other conditions, it is important to bear in
2H, O2 ---*02 + 2H20 mind, as Halliwell~ has emphasized, that implicated does not
mean important, and the relationship between oxidative stress
Antioxidants such as vitamin E, [3-carotene, and coenzyme Q and the onset and progression of disease processes is not fully
6 D. JOHN BETTERIDGE

established. Furthermore, oxidative damage may be a conse- cations. A significant relationship was observed between the
quence of a particular pathological process rather than its cause. ratio of lipid hydroperoxide to cholesterol standardized a-tocoph-
Hopefully, with improved techniques such as electron spin erol and fasting blood glucose in the diabetics but not with
resonance 24 discussed elsewhere in this volume, further insight hemoglobin Ab plasma cholesterol, or triglyceride. These data
into the role of oxidative stress will be obtained. suggest that the elevated plasma hydroperoxides are associated
with diabetes itself rather than the presence of complications,
IS OXIDATIVE STRESS INCREASED and perhaps that acute hyperglycemia is a more significant
IN DIABETES MELLITUS? determinant than chronic glycemia as reflected by the hemoglo-
There is considerable evidence that oxidative stress is bin AI. Recently, plasma hydroperoxides measured by the
implicated in the development of diabetic complications. 25 techniques previously described have been shown to decrease
Furthermore, the close interrelationships between the processes with continuous subcutaneous insulin infusion compared to
of glycation and advanced glycation end product (AGE) conventional insulin therapy in type 1 diabetic patients. 48 In this
production, a major pathogenetic mechanism for diabetic study, plasma hydroperoxides did correlate significantly with
complications, 26-% and oxidative stress have been emphasized. glycated hemoglobin levels during the study and with the level
The term "glycoxidation" has been coined to describe this of microalbuminuria.
concept. ~-9It is beyond the scope of this article to describe these A further approach adopted in our own studies has been the
processes in detail and the reader is referred to the comprehen- measurement of products formed during peroxidation. The
sive reviews previously cited. F2-isoprostanes are a series of prostaglandin F2-1ike compounds
Several factors may contribute to increased potential for formed during arachidonic acid peroxidation independent of the
production of free radicals in diabetes including glucose cyclooxygenase pathway. 49 One of these compounds, 8-epi-
auto-oxidation, 3° redox imbalance, 31 and AGE/receptor interac- PGF2~ has received considerable interest because of its role as a
tions. 32 In addition, free radical defense mechanisms may be potent vasoactive mediator. 5° Patients with type 2 diabetes were
reduced. As a consequence of glycation, proteins containing transi- found to have significantly higher plasma 8-epi-PGF~,~ concen-
tion metals may undergo conformafional change with the potential trations measured by gas chromatography-mass spectrometry/
for increased availability of metal ions to catalyze.33,34 [n addition, negative ion chemical ionization compared to controls. 51 In this
Cu-Zn superoxide dismutase is inactivated by glycation,35 and study, no correlations were observed between concentrations of
anfioxidants (eg, vitamin E, vitamin C, catalase, and reduced 8-epi-PGF2~ and glycated hemoglobin A1, plasma glucose,
glutathione) have been reported to be decreased in diabetes. 36,37 plasma cholesterol, or triglyceride.
The interaction of glycation and oxidant processes is well-
illustrated by LDL modification, which is central to atherogen- CONCLUSION
esis. Recently, the possible mechanisms by which LDL oxida- A concise, simplified introduction to the concept of oxidative
tion is promoted in the diabetic state have been reviewed. 38,39 stress has been provided in this short review together with its
Glycoxidation, the myeloperoxidase and reactive nitrogen path- relevance to diabetes and diabetic complications. The close
ways, and other mechanisms including the action of lipoxyge- interaction between glycation and oxidative processes has been
nase together with reduced antioxidant mechanisms are all discussed. It should be apparent that this is a complex area and
likely to contribute to increased LDL oxidation. Furthermore, much is still to be learned. Indeed, although there is consider-
the change in LDL subfraction distribution towards smaller, able evidence for increased oxidative stress in diabetes, this is
denser particles, which is well-described in type 2 diabetes, 4°,41 not supported by some studies. 52,53 For instance, the level of
increases susceptibility to oxidation. 42 advanced glycation end-products carboxymethyl lysine and
As discussed previously, the measurement of oxidative stress pentosidine present on collagen in diabetes could be explained
in vivo is difficult.43 In our laboratory, this problem was by mechanisms other than oxidative stress. 52 Furthermore,
approached using the ferrous oxidation with xylenol orange oxidized amino acids, methionine sulfoxide, and ortho-tyrosine
assay coupled with the selective hydroperoxide reductant in skin collagen were not increased in diabetes. 53 However,
triphenylphosphine to determine levels of lipid hydroperoxides these observations certainly do not exclude more localized
in type 2 diabetic patients. 44 47 Plasma lipid hydroperoxides tissue-specific oxidative stress.
were significantly higher in the diabetics compared to controls. Hopefully this review will provide the basis for the subse-
The ratio of lipid hydroperoxides to cholesterol standardized quent articles in this volume that will explore further some of
e~-tocopherol was also significantly higher in diabetic patients. the important concepts raised, along with a discussion of the
No differences were observed in lipid hydroperoxides or potential beneficial effects of the sulphonylurea gliclazide on
c~-tocopherol between diabetics with or without clinical compli- aspects of oxidative stress.

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