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Cardiovascular Research 47 (2000) 410418

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Review
Lipid peroxidation, antioxidants and cardiovascular disease: how should we
move forward?
Barry Halliwell
Department of Biochemistry, National University of Singapore, Kent Ridge Crescent, Singapore 119260, Singapore
Received 27 January 2000; accepted 28 March 2000
Keywords: Coronary disease; Free radicals
1. Introduction antioxidants, or combinations of a few antioxidants. For
example the intervention trials with b-carotene convincing-
It is widely agreed that increased consumption of fruits, ly demolished the concept that this carotenoid is an
grains and vegetables, decreased intake of saturated fats, a important anti-cancer agent in humans, at least in smokers
moderate degree of exercise and perhaps judicious con- [30]. Hence high plasma levels of b-carotene are nega-
sumption of red wine or other alcoholic beverages (or even tively associated with cancer incidence because both are a
tea) would improve the cardiovascular health of the consequence of eating a good diet; b-carotene is not the
populations in most developed and near-developed coun- protective agent in that diet [30]. Indeed, the antioxidant
tries [18]. Fruits, grains, teas, vegetables and red wines hypothesis might have predicted this: by comparison with
are rich in antioxidants (ascorbate, tocopherols, tocot- avonoids, tocopherols and ascorbate, b-carotene is a poor
rienols, avonoids, other phenols and carotenoids are antioxidant even in vitro and probably unlikely to be any
among the antioxidants found in various plants consumed better in vivo [31]. This is further illustrated by the
by humans; reviewed in [9]), and so it is widely thought observation that increased vegetable and fruit consump-
that antioxidants make an important contribution to this tion, and (under some circumstances) physical exercise,
cardiovascular protective effect [914]. This assumption is can decrease levels of oxidative DNA damage (a putative
logical, because there is good evidence that oxidative risk factor for cancer development) in humans and other
damage contributes to the pathology of atherosclerosis and animals, and high-fat diets can increase these levels, but
vascular dysfunction generally, and that free radicals are b-carotene supplements have little or no effect on levels of
involved in myocardial ischemia-reperfusion injury [9,15 oxidative DNA damage in the human body [3142].
20]. However, intervention trials with vitamin E that assess Is the situation any different for cardiovascular disease?
clinical end-points are giving a confused picture [2123]. There is little evidence that b-carotene has any direct
Indeed, foods and beverages derived from plants are effect on incidence of cardiovascular disease or diabetes
chemically complex, and cardiovascular protective effects [4346]. Data on the relation of vitamin C intake to
could also arise from many other components or mixtures cardiovascular disease are conicting, but no consensus
of components present, including bre, immunostimulatory has emerged for a cardiovascular protective effect
agents, monounsaturated fatty acids, agents that modulate [43,44,47], although the suggested association of low
cholesterol synthesis, B-vitamins, folic acid, agents modu- ascorbate intake with hypertension is intriguing [48,49].
lating nitric oxide production, and even the humble ethanol Improved vascular function in some subjects has been
molecule itself [2,3,5,6,14,2429]. reported after consumption of gram quantities of ascorbate
One obvious way to assess the contribution of anti- [5052], but it is uncertain as to whether antioxidant or
oxidants to the cardiovascular protective effects of the other properties of ascorbate are responsible [53]. In
above diets is to conduct intervention trials with single relation to cardiovascular disease prevention, vitamin E is
E-mail address: bchbh@nus.edu.sg (B. Halliwell). Time for primary review 27 days.
0008-6363/ 00/ $ see front matter 2000 Elsevier Science B.V. All rights reserved.
PI I : S0008- 6363( 00) 00097- 3

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B. Halliwell / Cardiovascular Research 47 (2000) 410418 411
perhaps the most controversial antioxidant, with results of lated to their antioxidant activity. Examples include puta-
supplementation trials ranging from dramatic protective tive effects on blood pressure, cell proliferation, monocyte
actions to absence of effect [2123,46,54,55]. function, vascular responsiveness and blood coagulation
[4852,6064].
2. Are we asking the correct questions?
3. Where are we now?
Why this confusion? Let us step back for a moment to
examine fundamentals. The starting hypothesis is that At what stage are we now in understanding the impact
oxidative damage, especially lipid peroxidation, is a key of diet on oxidative damage in cardiovascular disease?
contributor to the progression, and perhaps to the origin of, Several studies have addressed the effects of diet (as
cardiovascular disease [9,15,16]. We know that diets rich opposed to antioxidant supplements) on lipid peroxidation
in plant products, which in turn are rich in antioxidants, in the human body. For example, diets rich in fruits and
have a cardiovascular protective effect. Key questions vegetables were reported to diminish ethane exhalation
must then surely be: [65,66], urinary excretion of the isoprostane 8-epi-PGF
2a
(1) Is the protective effect of such diets due, in whole or (but not of malondialdehyde) [39] and to decrease the
in part, to antioxidant mechanisms? In other words, do susceptibility of low-density lipoproteins (LDL) to ex vivo
such diets decrease oxidative damage in vivo, especially peroxidation [66]. Patients participating in a programme of
lipid peroxidation, to an extent that could explain their exercise plus a low-fat diet also had LDL with decreased
protective effects? susceptibility to peroxidation [67]. But were the indices of
(2) If the diets do decrease oxidative damage, which lipid peroxidation used in these studies valid? What is the
component(s) are responsible? Is it vitamin E, vitamin C, best way to measure lipid peroxidation in human interven-
avonoids, carotenoids etc.? Or do we need a mixture of tion studies?
components that act synergistically [56] or is the anti-
oxidant effect due to previously unsuspected molecules?
(3) In carrying out an intervention trial with a putative 4. Assaying lipid peroxidation
antioxidant, have we succeeded in decreasing oxidative
damage in the test subjects? If we have not, a negative The criteria that should be met by the ideal assay of
result is predictable from our initial hypothesis, i.e. car- lipid peroxidation are summarised in Table 1. No existing
diovascular disease incidence will not drop because we assay meets all these criteria, but some are better than
have not inhibited the oxidative damage that contributes to others. First, lipid peroxidation should not be measured in
it [57]. For example, a recent comprehensive review [58] human tissues or body uids by simple diene conjugation
concluded that there was little convincing evidence that methods or determinations of thiobarbituric acid-reactive
vitamin C supplements had lowered levels of oxidative material (TBARS), except where they have been previous-
damage in human intervention studies. One reason may be ly calibrated against more sophisticated assays. The simple
that many such studies have been carried out on subjects TBA test and diene conjugation methods are awed
who already had sufcient tissue and body uid levels of (reviewed in [9,68]).
vitamin C to achieve the maximal antioxidant effect. There The peroxidation that contributes to atherosclerosis is
is evidence from studies of oxidative DNA damage that thought to occur within blood vessel walls and not (or at
intakes of ascorbate of 100200 mg per day maximize its least not to a large extent) in LDL circulating in the blood
protective effects, and higher intakes may even increase [15,16]. Just as LDL can enter vessel walls, minimally
oxidative DNA damage (reviewed in [59]). If the putative modied LDL (i.e. LDL that has undergone some oxida-
protective effect of ascorbate against cancer development tion, but insufcient for recognition by scavenger re-
is due to its antioxidant properties in protecting DNA ceptors) may escape back into the circulation. Therefore,
against oxidative damage, supplementation carried out on one potentially useful biomarker perhaps indicative of
populations who already had intakes of 100200 mg per peroxidation in blood vessels may be the susceptibility of
day would be expected to reveal no effect. As I have circulating LDL to peroxidation. Indeed, this assay is
stressed recently [57], it is pointless to do large interven- widely used to show that antioxidants that inhibit lipid
tion trials with antioxidants in humans without preceding peroxidation in vitro might exert effects in vivo [69,70].
and accompanying studies to show that the antioxidant at The dietary manipulation [66,67] or antioxidant supple-
the dose to be given is capable of decreasing oxidative ment is administered to the subject and LDL is subsequent-
damage signicantly in the population in question. Unless ly isolated and subjected to prooxidant challenge. This
this is shown, it will be impossible to interpret the trial technique has been used by the group of Esterbauer and
results in relation to the initial hypothesis. others [69,70] to investigate the optimal intake of vitamin
A parallel question is whether vitamins E, C and E to make LDL most resistant to peroxidation. That intake
carotenoids exert cardiovascular protective effects unre- turns out to be higher (.150 IU of RRR-a-tocopherol)

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412 B. Halliwell / Cardiovascular Research 47 (2000) 410418
Table 1
a
The ideal assay of lipid peroxidation
(1) It should quantitate a major product(s) of the peroxidation process.
(2) The coefcient of variation between multiple runs of the same sample should be very small
in comparison with the differences between subjects.
(3) It must not be subject to interference by other biomolecules.
(4) It must employ chemically robust measurement methods (e.g. mass spectrometry, or HPLC
with good identication methods such as diode array or coulometric detectors) or be validated
by such methods (e.g. an immunoassay for isoprostanes that is calibrated against,
and gives the same results as, mass spectrometry).
b
(5) It must not be confounded by uptake of oxidized lipids present within foods.
(6) It should be able to assess both steady-state levels of peroxidation products (i.e. the balance between
rates of peroxidation and rates of metabolism/ clearance of peroxidation products)
and total rates of ongoing lipid peroxidation.
(7) The parameter measured should be stable on storage (essential for human epidemiological studies)
and should not be formed artifactually in stored samples.
a
Adapted from [134].
b
If plasma peroxides are cytotoxic and contribute to atherosclerosis, it does not perhaps matter in terms of cardiovascular damage if they originate from
diet or endogenously. However, when assessing effects of dietary changes on lipid peroxidation in vivo spurious answers can be obtained if the peroxide
load of the diet is changed by the intervention, and it is better to use an assay that is not confounded by this.
than can be achieved by relying on foods as a source of A related approach is to measure the levels of circulat-
vitamin E. This is consistent with the epidemiological data; ing antibodies against LDL, which is presumably related to
although there is considerable disagreement, the results of the extent of LDL oxidation in vivo [87,88]. It should be
intervention trials suggest that the intakes of vitamin E that noted, however, that the precise relationship of LDL
exert cardiovascular benet in some studies are higher than oxidation in the vessel wall to LDL oxidation in plasma
can be obtained from diet alone [22,55,71]. The ex vivo remains to be clearly elucidated.
LDL peroxidation method has also been used to examine
the effects of consumption of wines and teas on LDL 4.1. Direct assays of lipid peroxidation
peroxidisability in humans: several papers suggest that tea
has no effect [7174] but reports for red wine are In principle, a simple approach would be to measure
conicting [7579], although on balance suggestive of products of lipid peroxidation in plasma or urine. Multiple
benet. methods have, for example, been used to measure lipid
An important question in studies of ex vivo LDL peroxide levels in human plasma. Simple colorimetric
peroxidation is how to initiate the peroxidation. This is assays give values for plasma peroxides in the mM range
21
usually achieved by adding Cu ions. There is evidence (Table 2). Similar values are often given by TBA tests
that copper plays a role in LDL oxidation in vivo [80,81], linked to HPLC to remove interfering chromogens [9,68].
but peroxynitrite, reactive chlorine species, haem proteins, By contrast, methods based on direct HPLC analysis of
hydroxyl radicals, and lipoxygenases could also be in- plasma peroxides produce lower values, usually ,40 nM
volved [8186] and the predominant oxidizing species for the total of phospholipid and cholesterol ester hy-
may differ from lesion to lesion or even in different droperoxides assayed by HPLC with coulometric or
regions of the same lesion. It is sensible to check several luminescence detection [89,90]. Is it that the simpler
different methods (including incubating the LDL with methods (Table 2) are non-specic, measuring other
endothelial and other cell types found in the vessel wall) of oxidizing species (e.g. protein peroxides [82]), or is it that
oxidising LDL in ex vivo studies before forming conclu- the more complex methods degrade peroxides or otherwise
sions about the efcacy of administered antioxidants. For fail to measure some of them during analysis [91]?
21
example, an antioxidant that inhibits Cu -dependent LDL The GCMS-based analysis of specic peroxides after
21
oxidation ex vivo by chelating Cu may not be effective reduction to alcohols is a chemically robust technology
in lesions in which the predominant LDL oxidation is (Table 1) and gives information about the peroxidation of
achieved by other mechanisms. Another problem is that individual fatty acid residues [92,93]. For example con-
certain amphipathic antioxidants may protect LDL in the centrations of C18 hydroxyacid in plasma of healthy men
plasma environment but wash out during the prolonged and women were approximately 1 mM, whereas C20
procedures often used to purify LDL, so that they could hydroxyacid concentrations were 100150 nM [92,93].
exert protection in vivo but appear to exert no effect in ex However, these methods will measure hydroxyacids (lipid
vivo studies. This could conceivably happen with many alcohols) originally present in the biological material, in
plant phenolics, which have some degree of solubility in addition to those generated from the peroxides present by
aqueous media. the reduction step prior to analysis. Other chemically

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B. Halliwell / Cardiovascular Research 47 (2000) 410418 413
Table 2
Levels of lipid peroxides in human plasma or serum from healthy subjects: simple colorimetric assays
Assay Typical level Comment / reference
Iodine liberation 2.14.6 mM Widely used in the food industry;
Anal Biochem 1989;176:300;
Anal Biochem 1989;176:353;
Atherosclerosis 1996;121:193.
Can detect any species
2
capable of oxidizing I to I .
2
Ferrous Oxidation Xylenol Orange (FOX) 3.04.0 mM Levels elevated in diabetes. Can detect
(higher in some papers) any species (including H O ) that can
2 2
21 31
oxidize Fe to Fe ;
Anal Biochem 1994;220:403;
Meth Enzymol 1999;300:58
Glutathione peroxidase-based assays |1.0 mM Chem Res Tox 1989;2:295
Activation of cyclooxygenase |0.5 mM Relates the presence of peroxides to one
of their potential biological actions,
stimulation of eicosanoid synthesis;
Anal Biochem 1985;145:192 and 1991;193:55
Methylene blue method 8.665.8 mM Atherosclerosis 1996;121:193;
Exp Gerontol 1987;22:103
robust methods include analysis of end-products of perox- currently available biomarkers of lipid peroxidation in the
ide breakdown, such as malondialdehyde (MDA) and 4- human body is the isoprostanes, which are specic prod-
hydroxynonenal (HNE) by mass spectrometry [94,95]. ucts arising from the peroxidation of unsaturated fatty acid
Levels of 25140 nM MDA have been reported in human residues in lipids [19,20,101,105120]. Some isoprostanes
plasma [94,96], whereas levels of HNE vary over a wide exist free in human plasma, but most are esteried to
range and have sometimes been claimed to approach 1 mM lipids. Isoprostanes can be accurately and sensitively
(reviewed in [9,95]). These aldehydes result from break- measured by mass spectrometric techniques, so that
down of peroxides, and thus their levels will be inuenced steady-state levels in human body uids can easily be
by rates of peroxide breakdown (which are slow at 378C detected [105,106,119,120]. Isoprostanes appear to turn
unless transition metal ions are present [9]), as well as by over rapidly, being both metabolised and excreted
rates of metabolism of both peroxides and the aldehydes. [105,106,116]. Detection of them and their metabolites in
There is evidence that peroxides and aldehydes in food can urine may therefore be a useful assay of whole-body lipid
be absorbed through the gut to a limited extent and can peroxidation [116,120]. As a biomarker of lipid peroxida-
potentially confound measures of MDA (especially in tion, isoprostane analysis seems to full many of the
urine), TBARS, and perhaps of plasma peroxides [97 criteria listed in Table 2. One exception is that the
101]. isoprostanes usually measured (often 8-epi-PGF ) are
2a
The discrepancy between the different levels of lipid only minor products of the peroxidation process. Several
peroxidation products measured by different assays, and methodological questions relating to isoprostane analysis
the question of confounding by diet, need resolution before remain to be resolved (reviewed in [105]). The question of
any of the above methods can be recommended for studies confounding by oxidized lipids in the diet has been
of the effect of diet on lipid peroxidation rates in vivo. One examined experimentally, and there is no evidence as yet
approach to determination of whole-body lipid peroxida- that measurements of plasma isoprostanes in humans are
tion has been measurement of exhaled hydrocarbons, so confounded [101,121,122].
especially ethane [102104]. Hydrocarbon gases are, Most analyses of isoprostanes to date have focused on
however, minor end-products of peroxidation and their measurement of some of the F -isoprostanes, which arise
2
formation, like that of aldehydes, depends on the decompo- from the peroxidation of arachidonic acid residues [106].
sition of peroxides, e.g. by transition metal ions [9]. Levels of certain F -isoprostanes in human body uids
2
Nevertheless, this assay has given some interesting results have been shown to be elevated in conditions that pre-
(e.g. Ref. [65]) and merits further exploration for use in dispose to accelerated development of cardiovascular
intervention studies. disease: diabetes [114,115], hypercholesterolaemia
[107,109] hyperhomocysteinaemia [123] and cigarette
4.2. Isoprostanes smoking [108,110,112]. F -isoprostane levels in humans
2
are responsive to dietary antioxidants (Table 3), although
There is a growing belief that the most valuable of the most studies have been on unhealthy subjects with

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414 B. Halliwell / Cardiovascular Research 47 (2000) 410418
Table 3
Effect of antioxidants on F -isoprostane levels in human body uids
2
Subjects Intervention Change Reference
Healthy volunteers 4 g/ day vitamin C, 37% fall in esteried F -Ips. Drug Metab Rev 1999;31:117
2
3200 IU/ day vitamin E,
300 mg/ day b-carotene,
all for 2 weeks.
400 IU a-tocopherol for 2 weeks 25% fall in unesteried F -Ips.
2
800 IU a-tocopherol for 2 weeks 37% fall in unesteried F -Ips.
2
400 IU per day a-tocopherol for 8 weeks, Both produced signicant falls in urinary Free Rad Biol Med 1999;27:1114
600 mg per day of lipoic acid for 8 weeks F -isoprostanes as measured by
2
commercial immunoassay kit
The combination was no better than each
antioxidant individually
Diabetic patients 600 mg/ day vitamin E for 14 days 37% decrease in urinary levels Circulation 1999;99:224
Smokers 100 or 800 U/ day Vitamin E, 29% fall in urinary levels with C; Circulation 1996;94:19
2 g/ day vitamin C, 22% fall with the combination;
both for 5 days no effect of E alone.
Hypercholesterolaemic patients 100 mg/ day vitamin E for 2 weeks 3436% fall in urinary levels. Arterio Thromb Vasc Biol 1997;17:3230
600 mg/ day vitamin E for 2 weeks 4758% fall in urinary levels
Patients with anti-phospholipid antibodies 900 IU/ day vitamin E, Signicant decrease in urinary levels Blood 1999;93:3401
2 g/ day vitamin C, both for 4 weeks
Patients with liver cirrhosis 300 mg vitamin E twice daily for 30 days 51% fall in urinary levels Blood 1999;93:2945
(see also J Invest Med 1998;46:51)
Patients with chronic alcoholic liver disease 2.5 g vitamin C for 10 days Approximately 50% fall in urinary levels J Clin Invest 1999;104:805
elevated F -isoprostane levels and more studies of anti- 5. Conclusion
2
oxidant effects on healthy volunteers need to be done;
preliminary data suggest that effects are smaller. Anti- Robust biomarkers of lipid peroxidation (of which at the
oxidants have also been shown to decrease isoprostane moment the best available seem to be the isoprostanes)
levels in animals [117,118,124127]. In studies on blood should be used to establish the effects of diet on lipid
samples from human volunteers, plasma levels of F - peroxidation in vivo, and in particular what contribution is
2
isoprostanes showed no correlation with levels of oxidative made to any effect of diet by the antioxidants present in
damage to DNA bases in white cells [128], suggesting that that diet (ascorbate, vitamin E, carotenoids, avonoids
these two parameters are not determined by the same etc.). Any cardiovascular disease intervention trial that
criteria. Indeed, whereas F -isoprostane levels appear does take place should be accompanied by measurements
2
responsive to dietary antioxidants (at least in subjects with of lipid peroxidation. This will help to assess whether any
elevated F -isoprostane levels), levels of oxidative DNA benecial effects observed on clinical end-points are
2
damage generally are not (see above). related to inhibition of lipid peroxidation, and/ or to other
Another potential advantage of the isoprostanes is that physiological effects [4852,6064] of antioxidants. Mea-
different families are formed from different PUFAs surements of lipid peroxidation should be carried out:
[105,129131], which gives a potential mechanism for
following the rates of peroxidation of different PUFAs in 1. Prior to the intervention study, to show (in short-term
vivo. It is well known that increasing unsaturation en- studies) that the intervention does actually decrease
hances the tendency of PUFAs to oxidize in vitro [9], but lipid peroxidation, preferably in a dose-dependent man-
the question of whether they peroxidize faster in vivo ner, in the subjects to be examined.
needs to be investigated using robust assays of lipid 2. At intervals during the study, to relate changes in the
peroxidation. Isoprostane biomarkers arising from differ- levels of lipid peroxidation to clinical end-points.
ent PUFAs should be helpful in investigating the conse-
quences of eating diets rich in various PUFAs, whether any One further point may be important. The steady-state
increased peroxidation that results can be ameliorated by levels of oxidative DNA damage and lipid peroxidation
antioxidants (e.g. vitamin E), and if so, how much anti- vary widely between healthy human subjects. Are those
oxidant is needed. with high levels of lipid peroxidation more at risk of

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B. Halliwell / Cardiovascular Research 47 (2000) 410418 415
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B):B211.
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1997;100:20282034.
of background peroxidation who respond minimally if at
[20] Mallat Z, Philip I, Lebret M et al. Elevated levels of 8-iso-
all to antioxidants [31,132,133]? The combination of
prostaglandin F in pericardial uid of patients with heart failure.
2a
excellent chemistry (robust and validated biomarkers of
Circulation 1998;97:15361539.
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455.
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