Asia Pacific J Clin Nutr (1999) 8(1): 53–63 53

Review Article OA 36 EN

Free radicals, antioxidants and international nutrition*

Okezie I Aruoma PhD, DSc, MBA, FRSC

Drug, Antioxidant and Nutrient Research Centre, Faculty of Pharmaceutical Sciences, University of Sao Paulo at
Ribeirao Preto, Ribeirao Preto-Sao Paulo, Brazil

The oxidative degradation of polyunsaturated fatty acids is the primary factor in limiting the shelf-life of most
manufactured foods. Free radical mechanisms are implicated in the pathogenesis of human diseases and in the
process of ageing. This has led to the suggestion that antioxidants, and plant diet-derived antioxidants in
particular, might have health benefits as prophylactic agents. Delineating the in vivo contribution of plant
extracts and/or plant-derived antioxidants (the pure active principles in plant extracts with antioxidant
indications) to the modulation of the pathological consequences of oxidative stress in the human body is
complicated by the fact that antioxidant actions may be achieved through more than one mechanism. The
interest in the health promoting qualities of plant foods may be ascribed to the observation that various
compounds present in these foods possess antioxidant properties in vitro. From a food stability perspective, one
would be interested in the integrity of the food and the effects of storage on the molecular components of the
food. For humans, the emphasis is on the importance of nutritional antioxidants in health and disease
management.

Key words: free radicals, antioxidants, flavonoids, DNA damage, isoprostanes, phytochemicals, protein damage,
atherosclerosis, lipid peroxidation, oxidative stress.

Free radical chemistry: An introduction Suggestions that oxidative stress play a role in human
A free radical is any chemical species capable of independent diseases have led to the proposal that health might be
existence and possessing one or more unpaired electron, an improved by increased dietary intake of antioxidants.9–13
unpaired electron being one that is alone in an orbital. Radi- Drugs (prescription only medicines and over the counter
cals, often denoted by the insertion of the superscript dot (.), medicines) with antioxidant indications, have a functional
are generally less stable than non-radicals, although their relationship between health status and disease state.14–15 The
reactivities vary. The rate and selectivity of reactions of this role that food and drugs might play in the management of
type depends on high concentrations of the radicals, delocal- health is shown in Fig. 1.
ization of the single electron of the radical (thus increasing its
life time), and on the absence of weak bonds in any other
molecules present with which the radical could interact. Most
biological molecules, however, are non-radicals containing
only paired electrons. Much of the work in physical and
organic chemistry1–3 relating to free radicals gathered
momentum following the demonstration of the existence of
the triphenylmethyl radical (Ph3C.).4 Gerschman et al pro-
posed ‘that oxygen poisoning and radiation injury have at
least one common basis of action, possibly the formation of
oxidizing free radicals’.5 This pioneering idea soon began to
capture the imagination of scientists. In the early 1960s,
superoxide was found to be associated with a number of
enzymes, including xanthine oxidase. In 1968 it was discov-
ered that superoxide was secreted into solution, allowing
superoxide to mediate cellular toxicity.6–7 Figure 1. Functional relationships between health status and disease
From an environmental perspective, photochemical state and the role that food and drugs might play in the management of
reactions involving reactive oxygen species are attractive for health.
cleaning up pollution given that many ‘self-repair’ processes
in the atmosphere and natural waters are driven by light.8 Correspondence address: Professor Okezie I Aruoma, Drug,
Because electronically excited states of molecules may be Antioxidant and Nutrient Research Centre, Faculty of
both better oxidizing and/or reducing agents compared with Pharmaceutical Sciences, University of Sao Paulo at Ribeirao Preto,
their ground state counterparts, electron transfer processes Via do cafe s/n°, Cep 14040-903, Ribeirao Preto-Sao Paulo, Brazil.
can generate highly reactive species, which can be used to Tel: 55 16 602 4294; Fax: 55 16 602 4163.
chemically decompose a pollutant into harmless end *1997 Goodman Fielder Oration in International Nutrition, Monash
products. University Medical Center, Melbourne, Australia.
54 OI Aruoma

Reactive oxygen species antioxidant’s protective action might be even greater in vivo
Free radicals of importance in living organisms include depending, of course, on the precise location of the anti-
hydroxyl (OH.), superoxide (O2.–), nitric oxide (NO.) and oxidant in relation to the site of ONOO– generation.
peroxyl (RO2.). Peroxynitrite (ONOO–), hypochlorous acid Although inactivation of α1AP may involve a direct attack on
(HOCl), hydrogen peroxide (H2O2), singlet oxygen1∆g (often methionine residues by ONOO–, nitration of tyrosine by
written as 1O2) and ozone (O3) are not free radicals but can ONOO– is a complex reaction that may involve such species
easily lead to free radical reactions in living organisms. The as NO2., NO2+ and CO2. Activated human polymorpho-
term ‘reactive oxygen species’ (ROS) is often used to include nuclear neutrophils have been shown to convert NO– into
both the radical and non-radical species. Oxidative stress is NO2Cl and .NO2 through myeloperoxidase pathway, a reac-
the term referring to the imbalance between the generation of tion that may contribute to cellular dysfunction.39 The reac-
reactive oxygen species and the activity of the antioxidant tion of peroxynitrite with tyrosine (in proteins) and phenolic
defenses. Severe oxidative stress can cause cell damage and ‘antioxidant’ compounds and its inactivation of the prote-
death. It has been implicated in numerous human diseases olytic inhibitor α1-antiproteinase are good assays for deter-
including cancer, atherosclerosis, iron overload, rheumatoid mining putative antioxidant activity.22,33–37,40–42
arthritis, Parkinson’s disease, motor neurone disease, dia-
betes, malaria, and in HIV infection and AIDS.16–19 The Peroxyl radicals
importance of oxidative stress injury is dependent on the These are formed during lipid oxidation chain reactions, such
molecular target, the severity of the stress and the mechanism as the oxidation of polyunsaturated fats resulting in deterio-
by which the oxidative stress is imposed, that is, drug ration of lipid-containing foods. Lipid peroxidation may be
induced, Fenton chemistry, trauma, enzyme activation (e.g., initiated by any species that has sufficient reactivity to
nitric oxide synthase activity) and the cellular transduction abstract hydrogen from a polyunsaturated fatty acid side
mechanisms which may affect the expression of certain pro- chain (e.g. those of arachidonic acid and linolenic acid) in
teins including the DNA repair enzymes.20,21 Brief comments membrane lipids. End-products of lipid peroxidation could
on nitric oxide, peroxyl radicals, hydroxyl radicals and also have profound effects on vascular function because of
hypochlorous acid will be made to illustrate the complexity their ability to mimic or antagonize the actions of some of the
of the mechanism of reactions involving ROS. stereospecific products formed by cyclooxygenase and
lipoxygenase enzymes. For example, the F2-isoprostanes are
Nitric oxide generated by the peroxidation of arachidonic acid via the
Nitric oxide plays a significant role in the regulation of cell generation of peroxyl radical isomers which undergo endo-
function and tissue viability: this includes the recognized cyclization to prostaglandin-like compounds. Their forma-
ability to mediate signal transduction via stimulation of tion in vivo appears to be enhanced under conditions of
guanylate cyclase-mediated cGMP synthesis.22–27 The role of oxidative stress, such as smoking or exposure to xenobiotics,
NO. has been demonstrated with relation to malaria, whereby and under pathological conditions associated with inflamma-
NO. appears to be partially involved in resistance to malaria tion. The mechanism of LDL oxidation possesses the general
infection, in cardiovascular disease, acute inflammation, can- characteristics of the free radical reaction of lipid peroxida-
cer, neurodegenerative diseases and diabetes.23 The reaction tion.43–45
between NO. and O2.– leads to DNA oxidative damage due to
the formation of peroxynitrite, which may have OH.-like Hypochlorous acid
potential leading to the formation of nitroguanine and other Hypochlorous acid is produced by the neutrophil-derived
loose products.28–32 It has been suggested that peroxynitrite enzyme myeloperoxidase at sites of inflammation and when
formed by the reaction between NO. and O2.– mediates NO. activated neutrophils infiltrate reoxygenated tissue. The
dependent toxicity. In addition to the DNA base nitration enzyme oxidizes chloride (Cl–) ions in the presence of
mentioned above, ONOO– potentiates endothelial-dependent H2O2.46–49 Hypochlorous acid is a potent chlorinating and
activation of guanylate cyclase, bactericidal activity, try- oxidizing agent.50 Cholesterol forms chlorohydrins that
panocidal activity, conversion of low density lipoprotein could disrupt cell membranes, leading to cell lysis and
(LDL) to a form that may be recognized by the macrophage death.51 Cholesterol chlorohydrins may become potential
scavenger receptor, induction of peroxidation of lipids, oxi- biomarkers for oxidative damage associated with neu-
dation of methionine and SH residues in proteins, depletion trophil/monocyte activation. Hypochlorous acid can attack
of antioxidants (e.g. ascorbate, glutathione), nitration of tyro- many other biological molecules. For example, the prote-
sine residues and inactivation of α1-antiproteinase (a major olytic inhibitor α1AP is the major inhibitor in human plasma
inhibitor of serine proteases in vivo). of proteolytic enzymes such as elastase. The α1AP protein
accounts for approximately 90% of the elastase-inhibitory
Reaction of peroxynitrite with antioxidants capacity of the human serum.52 Thus, its inactivation by
The addition of ONOO– to biological fluids leads to the nitra- HOCl might greatly potentiate tissue damage because elas-
tion of tyrosine residues: the presence of these appears to be tase is also released from activated neutrophils. Hypochlor-
a ‘marker’ of ONOO–-dependent damage in vivo. Peroxyni- ous acid attacks primary amines and sulfhydryl (SH) groups
trite inactivates α1-antiproteinase, the major inhibitor of ser- in proteins, and chlorinates purine bases in DNA.50,53,54
ine proteases such as elastase, in human body fluids. Thus, Physiological levels of HOCl can cause protein fragmenta-
ONOO– generation in vivo can facilitate both oxidative and tion of collagenase and prevent collagen gelation.55 Reac-
proteolytic damage.22,38 The protein α1-antiproteinase tions of HOCl/OCl– react with endogenous amines to form
(α1AP) is an especially sensitive target of damage, so the N-chloramines, which exhibit a lower oxidizing potential
 Free radicals, antioxidants and international nutrition 55

than HOCl.50,56 Hypochlorous acid reacts with substituted Protection against ROS-induced damage
aryl amine-aniline, 1-naphthylamine and 1-naphthol to form The phagocytes (i.e. neutrophils, monocytes, macrophages,
long-lived products that bind DNA and that are suggested to eosinophils) provide protection against foreign organisms.
be genotoxic to human cells57 They generate O2.–, H2O2 and, in the case of neutrophils,
HOCl as one of their mechanisms for killing foreign organ-
Reactions of antioxidants with hypochlorous acid isms.46,47 This essential defence mechanism, however, can go
Assays for hypochlorous acid that could be performed with wrong; several diseases, such as rheumatoid arthritis and
ease to test the ability of an antioxidant to react with the inflammatory bowel disease, are accompanied by excessive
molecule include the elastase assay;58,59 assay with cata- phagocyte activation and resulting tissue damage, to which
ROS contribute. The interrelationship between ROS and
lase;60 inhibition of taurine-chloramine formation;61 and the
antioxidants in humans is very complex.15 Of the known anti-
oxidation of 5-thio-2-nitrobenzoic acid (TNB).62 An anti-
oxidant enzymes, superoxide dismutases (SOD)7,76 remove
oxidant protecting against damage by HOCl might do so not
the superoxide radical (O2.–) by accelerating its conversion to
only by scavenging HOCl but also by inhibiting myeloper- H2O2. Human cells have a SOD enzyme which contains man-
oxidase. Thiols that are good scavengers of HOCl might also ganese at its active site (MnSOD) in the mitochondria. A
act as competing substrates for myeloperoxidase enzyme and SOD with copper and zinc at the active site (Cu,Zn-SOD) is
therefore slow down HOCl formation.63–65 Interestingly, sev- also present but largely in the cytosol. Mutations to the SOD-
eral phenolic compounds including flavonoids react quickly 1 have been associated with the pathology of the degenera-
with HOCl and can protect α1-antiproteinase and other sus- tive disease amyotrophic lateral sclerosis (ALS).77 Catalases
ceptible targets against damage in vitro.66,67 in the peroxisomes convert H2O2 into water and O2 and help
to dispose of H2O2 generated by the action of oxidase
Hydroxyl radicals enzymes located in these organelles. However, the most
The hydroxyl radical (OH.) is a highly reactive oxygen- important H2O2-removing enzymes in human cells are glu-
centred radical with an estimated half life in cells of only 10–9 tathione peroxidases (GSHPX).78 These enzymes require
s. One feature of the hydroxyl radical is that it begets another selenium, as selenocysteine at the active site, for their action.
radical when it reacts with a molecule: the result is the for- Glutathione peroxidases enzymes remove H2O2 by using it to
mation of another radical species. The resulting species oxidize reduced glutathione (GSH) to oxidized glutathione
usually has lower reactivity than the OH.. Hydroxyl radical (GSSG). Glutathione reductase, an FAD-containing enzyme,
attacks all proteins, DNA, polyunsaturated fatty acids in regenerates GSH from GSSG, with NADPH as a source of
reducing power. A variety of radical-scavenging antioxidants,
membranes and almost any biological molecule it touches. In
including GSH, uric acid, α-tocopherol (vitamin E) (Fig. 2)
the case of OH. generation by Fenton-type chemistry,68,69 the
and ascorbic acid (vitamin C) exist. α-Tocopherol delays
extent of OH. formation is largely determined by the avail-
lipid peroxidation by reacting with chain-propagating per-
ability and location of the metal ion catalyst.
oxyl radicals faster than these radicals can react with proteins
Copper ions are more reactive in causing DNA damage in or fatty acid side-chains.79 In theory, β-carotene (Fig. 3) has
the presence of H2O2 compared with equimolar iron ions in remarkable antioxidant chemistry, a function that has been
vitro.70 Metal-dependent carcinogenesis is widely discussed difficult to demonstrate in a beneficial manner in biological
in the literature.71 Iron ions are absorbed from the gut and systems. Excellent accounts on vitamin E and β-carotene
transported to iron requiring cells by the protein transferrin. may be found in several reports.79–84 Thus, scavenging
Iron specifically bound to transferrin does not participate in enzymes and antioxidants can inhibit free radical production
free radical reactions.72 Excess iron is stored as ferritin and by chelating the transition metal catalysts, breaking chain
haemosiderin in an attempt to keep the iron pool as small as reactions, reducing concentrations of ROS, and by scaveng-
possible. Hydroxyl radical generation can take place when ing initiating radicals.
the homeostasis is altered. For example, copper and iron ions That ascorbate (vitamin C) may serve as an important
released into perfusates can cause ischemia–reperfusion antioxidant in vivo is widely claimed.85 Ascorbic acid and its
injury.73 Tissue injury can itself cause ROS generation (e.g. derivatives have useful functions in the food industry, where
by causing activation of phagocytes or releasing transition they are used during processing to enhance food stability.86
metal ions from damaged cells), which may, or may not The ability of ascorbic acid to show antioxidant properties is
depending on the situation, contribute to a worsening of the related to the fact that the dehydroascorbate radical is less
reactive than are many of the radicals that can be scavenged
injury. Traumatic brain injury and stroke may involve a
by ascorbate.87 Intracellular enzymic systems exist in vivo to
postinjury stimulation of iron ion-dependent free radical
reduce this radical back to ascorbate using NADH (the
reactions. Parkinson’s disease is caused by the death of cells
NADH-semidehydroascorbate reductase enzyme) or GSH
in the substantia nigra. Lysis of dead cells could cause iron (the dehydroascorbate reductase enzyme) as sources of
ion release. Thus, patients with Parkinson’s disease may be reducing power. Ascorbic acid is often rapidly depleted in
under oxidative stress and free radical reactions are probably human extracellular fluids under conditions of oxidative
contributing to the degeneration of the substantia nigra.74 stress.88
Data from the assessment of oxidative DNA damage in the Evaluating the role of free radicals in disease pathology
brain have shown that DNA damage is higher in the tempo- and establishing a logical basis for the therapeutic use of anti-
ral lobe compared with other brain regions in Alzheimer’s oxidants requires the use of validated biomarkers (Table 1).
disease.75 Numerous antioxidant supplementation studies for the
56 OI Aruoma

Figure 3. Typical carotenoid structures, including xanthophylls (oxy-

genated carotenoids) and carotenes (hydrocarbons).

Table 1. Measurement of oxidative damage in humans.

There are several indicators of the extent of oxidative damage in
humans. Some of the most common include measuring:
Oxidative DNA damage
GC/MS/SIM detection of oxidized base products
Figure 2. Chemical structure of tocopherols. HPLC-based assays for oxidized base products
Single gel electrophoresis assay (Comet assay)
Oxidative damage to lipids
primary prevention of chronic diseases have been under- Measurement of conjugated dienes
taken. In each case, the principal endpoint has been ‘inci- Measurement of isoprostanes
dence’ of the respective disease, that is, incidence of cancer Measurement of hydroperoxides
or cardiovascular disease. In a departure from this, the extent Measurement of thiobarbituric acid reactive materials by HPLC
of oxidative DNA damage in Scottish men aged between 50 Assessment of the levels of antioxidant enzymes
and 59 years was investigated by Duthie et al.89 Their result Catalase, superoxide dismutase and glutathione peroxidase
Assessment of protein damage
suggests that long-term antioxidant supplementation can
Steady state protein damage can be quantified in terms of the
decrease both endogenous and exogenous oxidative DNA numbers of protein carbonyls and modified tyrosine residues.
damage in lymphocytes. Future supplementation studies in Total ongoing (repaired) protein damage can be indicated by
order to evaluate the pharmacology of antioxidants (drug- the concentration of modified tyrosines and fluorescent
derived or plant-derived antioxidants) should balance the use bityrosines in the urine.
of in vivo biomarkers with the choice of population, formu- Assessment of levels of low molecular weight antioxidants and
lation and dose of antioxidants being used, the expected out- vitamins
come variables, and the pathologic viables. Figure 4 suggests Uric acid/allantoin, glutathione, flavonoids, vitamin E and C,
one such rationale. The global direction would be for food β-carotene
and drug antioxidants to be evaluated for their inherent prop-
erties using in vitro models. Assessment of their protective Measurement of oxidative DNA damage
effects in human health and disease should then consider how Oxidative damage to DNA appears to occur continuously in
the steady state levels of markers of oxidative damage are vivo, in that low levels (presumably a ‘steady state’ balance
affected by the antioxidants. between DNA damage and repair) have been detected in
 Free radicals, antioxidants and international nutrition 57

Lipid oxidation and its measurement

Lipid peroxidation is important in vivo and for the stability of
processed foods. It contributes to the development of cardio-
vascular diseases such as pre-eclampsia and atherosclerosis,
and the end-products of this process, particularly cytotoxic
aldehydes such as malondialdehyde (MDA) and 4-hydroxy-
nonenal (HNE), can cause damage to proteins and to DNA.
Peroxidation causes impairment of biological membrane
functioning: for example, it decreases fluidity, inactivates
membrane bound enzymes and receptors, and it may change
non-specific calcium ion permeability.100,101 The more unsat-
urated a fatty acid side-chain, the greater its propensity to
undergo lipid peroxidation.
The ability to measure levels of isoprostanes and
hydroxyeicosatetraenoic acids (HETE) represent important
developments in attempts to measure clinically relevant
oxidative lipid damage. The hydroperoxides HETE and iso-
prostanes are biologically active. Hydroxyeicosatetraenoic
acids are chemotactic for neutrophils and have been shown to
facilitate calcium uptake and protein kinase C mobiliza-
tion.102,103 The 12-HETE are involved in adrenocorticotropin
and parathyroid secretion, modulation of mitogenic pro-
cesses and lymphocyte function,104–106 while the 15-HETE
may inhibit neutrophil migration across cytokine-activated
endothelium.107
Isoprostanes are a series of prostaglandin-like compounds
formed during peroxidation of arachidonic acid.108 Similar
products are probably formed from other polyunsaturated
fatty acids (PUFA) as they are structurally similar to
Figure 4. Human antioxidant strategy.
prostaglandin F2α, the compounds are collectively referred to
as F2-isoprostanes. The majority of plasma isoprostanes are
esterified to phospholipids, but some are ‘free’. One of the
isoprostanes, 8-iso PGF2α is a powerful renal vasoconstrictor
DNA isolated from human cells and tissues.32,90–92 Back- which is known to cause decreased kidney blood flow and
ground radiation may be one source but radiation-generated glomerular filtration rate at low nanomolar concentrations.108
OH. is formed over the whole cell and only a small fraction Elevated circulating concentrations of F2-isoprostanes may
hits DNA.93 Other potential sources of OH. or OH.-like contribute to the pathology of hepatorenal syndrome, an
species include the decomposition of ONOO–, the reaction of almost uniformly fatal disorder characterized by the develop-
O2.– with HOCl, and HOCl itself, which can attack DNA ment of kidney failure in patients with severe liver disease.
Urinary excretion of isoprostanes is elevated in patients with
bases generating chlorinated products. The greatest interest
scleroderma and in smokers. Isoprostanes and their metabo-
has been in reactions of transition metal ions with H2O2 as a
lites can be measured in human urine and this may prove to
source of OH..70,71,94–96 Oxidative stress and cell death can be a valuable assay of whole body lipid peroxidation if a con-
liberate metal ions from their normal sequestration sites and founding effect of diet can be ruled out. These developments
they might then bind to DNA, a powerful metal ion chelator. are discussed in a number of studies.43,108–114
Several DNA base damage products are excreted in human In a study by Bachi et al., the levels of 8-epi-PGF2a excre-
urine, including the nucleoside 8-hydroxydeoxyguanosine tion in non-smokers tended to be constant, with relatively
(8-OHdG), 8-hydroxyadenine and 7-methyl-8-hydroxy- low interindividual variations, suggesting that individual
guanine, but the one most exploited is 8-OHdG, which is ‘normal level’ in the absence of oxidant injury such as smok-
usually measured by a method involving HPLC with electro- ing may also result from physiological or biochemical
chemical detection. The level of 8-OHdG in urine is proba- event(s) occurring at a constant rate with by-production of
bly not affected by the diet since nucleosides are not free radicals and oxidants (see Fig. 5).115 Given that basal 8-
epi-PGF2a production in vivo may result from lipid peroxida-
absorbed from the gut. It is also possible that some or all of
tion triggered by a chain of chemical events starting with the
the 8-OHdG excreted in urine may arise not from DNA but
reaction of endothelium-derived peroxynitrite, measurement
from deoxyGTP (dGTP) in the DNA precursor pool of of isoprostanes may provide a supperior novel approach to
nucleotides. An enzyme has been described which hydro- assessing lipid peroxidation in vivo. In Fig. 5 we can imagine
lyzes dGTP containing oxidized guanine to prevent its incor- that the plots for non-smokers and smokers represent normal
poration into DNA.97,98 The need to validate measurements and disease states. The efficacy of antioxidants in helping to
of DNA base products as markers of oxidative damage in maintain the baseline ‘healthy’ levels of endogenous oxida-
vivo is critical.99 tive lipid damage could easily be monitored by comparing
58 OI Aruoma

Figure 5. Levels of 8-epiPGF2α in smokers.

Reprinted with permission from reference 116,
Elsevier Science Inc.

the rise and fall of the levels of isoprotanes. Alternatively, leading to fragmentation. Oxidized LDL inhibits endo-
reducing the tendency of a given disease condition to worsen thelium derived relaxing factor (EDRF) and may activate T-
and bring patients to the state of normal health is funda- lymphocytes in atherosclerotic lesions and stimulate
mental in disease management (Fig. 1). Thus, strategies to proliferation of smooth muscle cells. The induction of the
reduce observed increases in isoprostane level can be used to expression of the gene coding for the A-chain of platelet
monitor the efficacy of antioxidant prophylactic agents. Cur- derived growth factor may cause disturbances of eicosanoid
rently, the lack of validated assays for isoprostane other than homeostasis and aggregation of platelets in support of the
those that make use of mass spectrometry has greatly cur- atherogenicity of oxidized LDL.118,119
tailed the general use of this approach to assess oxidant
injury. There is, therefore, a need to develop an inexpensive Protein oxidation and its measurement
assay for isoprostanes. The availability of a simpler (e.g. Oxidative damage to proteins in vivo may affect the function
immunoassays) and more reliable method for the measure- of receptors, enzymes, transport proteins, etc., and perhaps
ment of isoprostanes could provide new and exciting insights generate new antigens that provoke immune responses. Prod-
into the role of free radicals and lipid peroxidation in human ucts of oxidative protein damage can contribute to secondary
diseases. damage to other biomolecules, for example, inactivation of
Oxidation of LDL is a free-radical-mediated process that DNA repair enzymes and loss of fidelity of DNA polymer-
results in numerous structural and functional changes. The ases in replicating DNA. The chemical reactions resulting
initiation of LDL oxidation occurs by the peroxidation of the from attack of ROS upon proteins are complex. Free radical
polyunsaturated fatty acids (PUFA) in LDL. Oxidation of attack upon proteins generates radicals from amino acid
LDL is initiated by hydrogen abstraction from a double bond residues, and electrons can be transferred between different
in PUFA, followed by molecular rearrangement that leads to amino acids. The levels of any one or, preferably, of more
the formation of conjugated dienes (CD). During this than one of these products in proteins could in principle be
process, the rate of oxidation is dependent on endogenous used to assess the balance between oxidative protein damage
antioxidants in LDL, accounting for the lag phase of oxida- and the repair of, or more likely the hydrolytic removal of,
tion. The lag phase is followed by a rapid propagation phase damaged proteins. The molecular biology of free radical
that occurs after depletion of endogenous antioxidants and attack on proteins and its implications in certain pathology is
involves abstraction of another H. by a PUFA-peroxyl radical extensively reviewed in Dean et al.120 and Fu et al.121
(LOO.) from another PUFA, resulting in the formation of
lipid peroxides.44,45 The propagation phase is followed by a Characterization of antioxidant actions
decomposition or degradation phase in which there is cleav- An antioxidant may be defined in a number of ways. For
age of double bonds, resulting in the formation of aldhydes, example, as a substance which, when present at low concen-
such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) trations compared with those of an oxidizable substrate, such
and hexanal, that can crosslink with amino groups on apo B- as fats, proteins, carbohydrates or DNA, significantly delays
100. Monoclonal antibodies for the detection of 4-hydroxy- or prevents the oxidation of the substrate.122 Acidic com-
nonenal modified proteins, which is selective for HNE bound pounds (including phenols) usable in foods which can readily
to histidine with some cross reaction to HNE bound to lysine donate an electron or a hydrogen atom to a peroxyl or alkoxy
and cysteine, have been described by the late Hermann Ester- radical to terminate a lipid peroxidation chain reaction or to
bauer and his group at the University of Graz, Austria (Waeg regenerate a phenolic compound, or which can effectively
et al.116). Changes in the protein moiety of LDL also occur chelate a pro-oxidant transition metal,123,124 are also anti-
during oxidation.117 Oxidation is followed by an increase in oxidants. A pro-oxidant is a chemical species capable of
the negative charge on the LDL, possibly due to derivatiza- increasing the oxidative burden of a system by increasing the
tion of positively charged amino groups through the forma- synthesis of reactive oxygen, nitrogen, chlorine and sulfur
tion of Schiff base with aldehydes. Furthermore, following species and by modulating the gene expression of the anti-
oxidation the apo-B protein undergoes oxidative scission oxidant enzymes. A compound might exert antioxidant
 Free radicals, antioxidants and international nutrition 59

actions in vivo or in food by inhibiting generation of ROS, or Future perspective

by directly scavenging free radicals. Additionally, in vivo an When endogenous antioxidant defenses are inadequate for
antioxidant might act by raising the levels of endogenous the purpose of scavenging the ROS completely, ongoing
antioxidant defenses (e.g. by upregulating expression of the oxidative damage to DNA, lipids, proteins and other mole-
genes encoding SOD, catalase or glutathione peroxidase). cules can be demonstrated. Although ongoing oxidative dam-
It is increasingly being suggested that natural anti- age in vivo is implicated in the pathology of several human
oxidants from plants may be applied in the preservation of diseases, it is important to appreciate that they may not be the
food materials. Simple experiments (Fig. 6) can be per- primary cause of these diseases. Although diet-derived and
formed to examine direct antioxidant ability in vitro and to drug-derived antioxidants may be particularly important in
test for possible pro-oxidant effects on different molecular protecting against a number of human diseases, and assum-
targets. This ‘screening’ approach can be used to rule out ing that the active components become bioavailable, the
direct antioxidant activity in vivo: a compound that is poorly physiological relevance of the compounds, their ability to
effective in vitro will not be any better in vivo. Measurements upregulate defense antioxidants and to modulate gene
of lipid peroxidation should be the first line of tests to estab- expression (with respect to synthesis of DNA repair enzymes),
lish the potential antioxidant action of dietary antioxidant affect the cellular transduction mechanisms. With respect to
compounds. An antioxidant index based on the ability to free radical research, how the in vivo markers of oxidative
scavenge peroxyl radicals may then provide support for anti- stress are affected by antioxidants still needs to be fully
oxidant efficacy in in vitro systems.124–126 Antioxidants that evaluated.
protect lipids against free radical damage may actually accel- In order to exploit the significance of antioxidants, it is
erate damage to other molecules such as DNA, carbohydrates critical to understand the features of the molecular compo-
and proteins under certain conditions. This points to the need
nents of free radical biology and the interrelationships of
to examine suggested antioxidant activity or the activity of
these components in mediating tissue injury. Free radicals
components with a proposed ‘antioxidant cocktail’ using
and oxidants are part of normal human metabolism but when
assays involving a variety of substrates. The deoxyribose
produced in excess, they can cause tissue injury. Tissue
assay allows the determination of rate constants of reactions
injury can itself cause ROS generation, for example, by caus-
with OH. radicals, the assessment of abilities to exert pro-
ing activation of phagocytes or releasing transition metal ions
oxidant action, and the assessment of abilities to chelate
from damaged cells, which may (or may not, depending on
metal iron. Assays involving DNA damage have also been
the situation) contribute to a worsening of the injury. One
developed for assessing pro-oxidant actions. These assays
have unique features. The positive pro-oxidant actions in the could argue that careful use of a range of antioxidants, plant-
deoxyribose system rely on the ability of the compounds to extracts, plant-derived antioxidants, drug-derived anti-
interact with metal ions (i.e. to promote reduction of Fe3+ to oxidants, and/or antioxidant vitamins (β-carotene, vitamins E
Fe2+ chelates) and hence, to promote OH. formation in the and C) supplements, combined with new methods for mea-
presence of H2O2. The assays involving DNA rely on the suring oxidant generation in humans, would enable the
ability to reduce the metal ions in the iron-bleomycin-DNA unequivocal delineation of the exact contribution of ROS
or the copper-1,10-phenanthroline-DNA complex.124 generation to disease pathology and to the maintenance of
During in vitro testing, it is essential to examine the health.9 Although the global recommendation of the exact
action of a compound over a concentration range that is rele- amount of vitamin E, C, β-carotene and/or flavonoids
vant to its intended use. For example, if the compound is pre- derived from the human diet (rich in fruits and vegetable or
sent in vivo at low concentrations (less than 1 µmol), its from supplements) required for optimal health must await the
ability to inhibit lipid peroxidation only at high millimolar results of scientific endeavours directed to the issues. In this
concentrations is irrelevant unless there is good reason to context, the ultimate goal in the 21st century is to maintain
suspect that it concentrates at a particular site in vivo. The health through nutrition.
same is true if the compound exerts a pro-oxidant effect at Acknowledgements. I am grateful to Goodman Fielder for their leader-
high concentrations in vitro and is only present at low con- ship in recognizing that food industries have a major role to play in the
centrations and exerts antioxidant action to different species. promotion of health through nutrition.

Figure 6. Experimental approaches for the

characterization of potential antioxidant and
pro-oxidant actions.
60 OI Aruoma

Free radicals, antioxidants and international nutrition

Okezie I Aruoma
Asia Pacific Journal of Clinical Nutrition (1999) Volume 8, Number 1: 53–63

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