Advances in Bioscience and Biotechnology, 2013, 4, 48-61 ABB Published Online November 2013 (
Atherogenesis, the oxidative LDL modification
hypothesis revisited
Dov Lichtenberg*, Ilya Pinchuk
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv, Israel
Email: *
Received 23 September 2013; revised 25 November 2013; accepted 15 December 2013
Copyright © 2013 Dov Lichtenberg, Ilya Pinchuk. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The commonly-accepted “oxidized LDL hypothesis of
atherogenesis” is based on a large number of indi-
rect evidence that shows that oxidativ ely-modif ied LDL
plays a role in atherogenesis. Yet, the exact role is not
clear. Some researchers think that oxidatively modi-
fied biomolecules initiate atherogenesis; others be-
lieve that they “only” promote this multifactorial
process. Regardless of the exact mechanism responsi-
ble for the effect of peroxidation on atherogenesis, the
“oxidative theory of AS” is apparently inconsistent
with the results of meta-analysis, in which (the “ex-
pected”) significant correlation between CVD and
oxidative stress (OS) was found only when the OS was
evaluated on the basis of the plasma concentrations
of malondialdehyde (MDA), often based on the con-
centration of thiobarbituric acid reactive substances
(TBARS). Notably, even this association is question-
able due to 1) poor reliability of the laboratory assay
of MDA and 2) possible publication bias. Hence, it
appears that the commonly accepted paradigm re-
garding the role of oxidative damage in the patho-
genesis of CVD has been overestimated. Furthermore,
the hypothesis is apparently inconsistent with the
disappointing results of most of the clinical trials that
were designed to reduce OS by means of supplemen-
tation of antioxidants, mostly vitamin E. These ap-
parent inconsistencies do not contradict the oxidative
modification hypothesis of AS. The source of the ap-
parent contradictions is probably the oversimplified
considerations on which the predictions have been
based. Many reasonable arguments can be raised to
explain the apparent contradictions, which mea ns tha t
our current knowledge is insufficient to test the rela-
tionship of oxidative stress to cardiovascular disease.
Keywords: ROS; Free Radicals; Atherosclerosis;
Oxidative Stress; Antioxidants
Reactive oxygen species (ROS) and reactive nitrogen
species (RNS) play essential and diverse roles in humane
physiology [1-8]. They contribute to processes such as
apoptosis and mitogenesis, they help maintaining cell
number homeostasis and they play a central role in im-
mune responses and cell signaling, particularly at the
level of redox modulation. By contrast, excessive pro-
duction of these reactive species (RONS), particularly
free radicals, damages DNA, proteins and lipids, thus
being cytotoxic [9-11]. The concentration of RONS is
therefore a critical parameter in determining their ulti-
mate cellular response. ROS-induced stimulation of cells
has been shown to result in changes of the cellular redox
potential due to interruption of redox balance, which may
adversely affect cell function. Consequently, the de-
pendence of the response on the concentration of RONS
is unpredictable. In fact, the prevailing reputation of free
radicals is that they are the “bad guys” responsible for
aging [12-16] and for many diseases [17-19].
The oxidative modification hypothesis of atherogene-
sis, first proposed by Steinberg et al. in 1989, is that
atherogenesis is a consequence of oxidative modification
of LDL [20]. The physiological role of LDL is to carry
cholesterol from the liver to cells throughout the body.
Internalization of oxidized LDL into cells, including en-
dothelial cells and macrophages, differs from internaliza-
tion of native LDL. Whereas native LDL is recognized
and internalized into cells by way of the LDL receptor,
oxidized LDL is recognized by the so-called scavenger
receptors, which, unlike the LDL receptor, are not down-
regulated [21,22]. The liver is very rich in scavenger
receptors, so that soon after intravenous injection of oxi-
dized LDL it disappears from the blood, becoming ac-
cumulated in endothelial cells and macrophages. As a
result, these cells become “foam cells” and die, thus
*Corresponding author.
D. Lichtenberg, I. Pinchuk / Advances in Bioscience and Biotechnology 4 (2013) 48-61 49
forming atherotic plaques [23-25].
The peroxidation-induced atherogenesis hypothesis
[20] inspired the National Heart, Lung and Blood Insti-
tute to fund a workshop of a group of 30 distinguished
specialists in various aspects of the relevant fields to re-
view all of the evidence available up to that time, in-
cluding some epidemiologic data [26]. The panel found
that all the available knowledge is compatible with the
oxidative modification hypothesis. Hence, the panel’s
final recommendation was that studies utilizing natu-
rally-occurring antioxidant vitamins (e.g. vitamin E, B-
carotene and vitamin C) should proceed [27,28]. In view
of this conclusion, the oxidation hypothesis of athero-
sclerosis became the most commonly accepted paradigm.
Since the removal of oxidized LDL by the scavenger
receptors is rapid, minimally oxidized LDL disappears
from the circulation slower than more oxidized LDL.
Consequently, the level of oxidized LDL in the blood is
low and much of it is minimally oxidized LDL [29]. No-
tably, the autoantibody against oxidized LDL investi-
gated by Witztum et al. demonstrated that even minor
modifications in the structure of LDL make it immuno-
genic and that this specific antibody can be observed in
arterial lesions, in agreement with the notion that most of
the AS-relevant oxidation probably occurs in the artery
wall itself [29]. Notably, in the presence of copper, the
LDL-associated antioxidants become oxidized and only
then, rapid oxidation of LDL lipids (both fatty acids and
cholesterol) begins [30].
The obvious implication of this mechanism was that if
peroxidation of LDL is the cause of atherogenesis, anti-
oxidants should help prevent it. Unfortunately, the results
of clinical trials were disappointing. Specifically, al-
though several trials indicated that vitamin E supple-
mentation reduces the rate of both nonfatal MI and Non-
fatal Stroke, in other trials vitamin E had no significant
effect on different cardiac end points. The important ar-
gument of the supporters of vitamin E supplementation is
that some people benefit from it, whereas “if it does not
help, it does not hurt” [31]. In the words of William A.
Pryor: “In view of the very low risk of reasonable sup-
plementation with vitamin E, some supplementation ap-
pears prudent now” [31].
Being aware of the conflicting evidence, Witztum and
Steinberg recall that cardiovascular disease (CVD) is a
complex and multifactorial inflammatory disease associ-
ated with gradual progression of plaques. Furthermore,
they raised the possibility that the “Oxidative modifica-
tion Hypothesis” may not hold for humans and con-
cluded that we need to design clinical trails sensitive to
early lesions [29]. Moreover, in 2004, Stocker and Kea-
ney, in their review on the “Role of oxidative modifica-
tions in Atherogenesis”, specifically stated that it is not
clear whether oxidative events are a cause or a result of
AS [32]. At about the same time, two independent meta-
analyses concluded that vitamin E supplementation re-
sults in higher mortality [33,34], which raised the ques-
tion whether antioxidant supplementation is “Good in
Theory, but is the theory good?” [35].
The possibility that the theory is not good can not be
ruled out, but the apparent contradiction between the
predicted and observed effects of vitamin E can be ex-
plained differently. Specifically, the lack of beneficial
effect(s) of vitamin E supplementation may be due to at
least one of many processes discussed in this critical,
non-comprehensive review. For this discussion to be
understood, we must first briefly describe the physio-
logical roles of RONS and the evidence for their in-
volvement in atherogenesis. Next, we relate to the avail-
able data on the oxidative status in CVD patients in
comparison to matched controls. We then take a closer
look at the intervention studies, particularly vitamin E
supplementation trials. In an attempt to gain understand-
ing of the apparent inconsistencies between the hypothe-
sis and the experimental results, we relate to a variety of
possibilities with emphasis on the very complex (both
protective and atherogenic) role of HDL and of lipopro-
tein-associated enzymes on the peroxidation of LDL lip-
ids. Based on this critical review of available data, we
can only conclude that the specific roles of LDL oxida-
tive modification in atherosclerosis are in a state of un-
For many years, OS has been commonly regarded a
measure of a person’s probability to suffer from oxida-
tive damages. Thus, OS have been commonly blamed for
being involved in the pathogenesis of many diseases and
antioxidants predicted to be good to us [9,18]. Now we
realize that “Reactive oxygen species are ‘double-edged
swords’ in cellular processes: low-dose cell signaling
versus high-dose toxicity” [10,11]. Hence, “the dose re-
sponse curve is unpredictable” [10]. In other words, the
effects of “Free radicals and antioxidants in (both) nor-
mal physiological functions and human disease” [8] de-
pend upon their concentrations. At moderate concentra-
tions, both the production and metabolism of RONS are
tightly controlled [6] and cellular ‘redox homeostasis’ is
Under homeostatic conditions, ROS act as secondary
messengers in intracellular signaling cascades, including
the ROS-induced cell signaling and particularly the sig-
naling for skeletal muscle adaptation [3]. By that, ROS
contribute to the defense against infectious agents, as
well as to other immune reactions, apoptosis and mito-
genesis and anti-tumourigenic species. The claims made
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D. Lichtenberg, I. Pinchuk / Advances in Bioscience and Biotechnology 4 (2013) 48-61
by Ristow’s group [16] that “there is experimental basis
to question Harman's Free Radical Theory of Aging” [12]
and that “the existing data suggest that ROS act as essen-
tial signaling molecules to promote metabolic health and
longevity” [16], have yet to be evaluated but can not be
ruled out.
By contrast, under certain conditions free radicals may
induce and maintain the oncogenic phenotype of cancer
cells and this is merely one of many deleterious effects of
over produced ROS, including aging and human diseases,
particularly cancer, cardiovascular disease, atherosclero-
sis, hypertension, ischemia/reperfusion injury, diabetes
mellitus, Alzheimer’s disease and Parkinson’s disease
[5,18,19]. Admittedly, it is not clear whether excessive
formation of free radicals is the primary cause of any of
these diseases or merely a result of inflammation or/and
tissue injury. Yet, it is commonly believed that the ROS-
induced damage to cell components, including mem-
brane lipids, proteins and DNA contributes to all these
In short, the effects of RONS on both human function
and diseases depend in a complex, non-monotonic fash-
ion on their characteristics and concentrations in the
relevant compartment. These, in turn depend, in a com-
plex, interrelated fashion on a large number of factors.
Hence, at the present time we do not have a way to pre-
dict whether a given change of the redox status is likely
to promote or retard our health.
The “oxidative modification-hypothesis of AS” was origi-
nally based on five findings in Steinberg’s group [20,29]:
1) That “incubation of endothelial cells in culture with
high concentrations of LDL led to cell death, whereas the
LDL re-isolated from the medium of a cell culture after
incubation was markedly altered, such that it was taken
up by monocytes/macrophages in culture much faster
than native (normal) LDL [36].
2) That the major change that occurred during the in-
cubation did not take place when the medium in which
the cells were grown did not contain transition metal ions
3) That addition of antioxidants to cells in plasma–
containing cultures prevented the changes in the LDL.
Specifically, addition of vitamin E could completely
prevent oxidation of LDL induced by incubation with the
cells [37].
4) That oxidized LDL is a chemoattractant for blood
monocytes, which helps recruiting them into a develop-
ing lesion [24].
5) That oxidized LDL inhibits the motility of tissue
macrophages, which would tend to trap such cells in the
artery wall once they got there [25].
At that time, there were several preliminary evidences
that treatment of rabbits with several antioxidants re-
duced the rate of atherogenesis [38].
After the publication of the oxidative modification
hypothesis of AS, a large number of supporting, mainly
indirect lines of evidence appeared, including evidence
based on the composition of atherotic plaques [39] and
data on the effect of antioxidants on the rate of athero-
genesis in primates [28].
Today, we have many more lines of evidence for the
involvement of LDL oxidation in atherogenesis. First, we
know that there are at least ten different ways by which
different oxidation products of LDL can be atherogenic
via various mechanisms [4,29] and that the mode of LDL
oxidation affects the biological effects of the oxidation
products, as described by Levitan et al. in their compre-
hensive review [40]. Although these authors found “a
significant degree of specificity to different forms of
oxLDL”, they note in their concluding remarks that we
do not know “which of the oxLDL forms has the most
pronounced effect on the development of the AS lesions”
and that in spite of “the considerable progress made in
recent years…there are still unresolved issues”, which
are difficult to resolve because the studied oxLDL prepa-
rations are “poorly defined” [40].
We also know that other processes beside oxidation
can also lead to foam cell formation, irrespective of lipid
peroxidation, including formation of complexes of aggre-
gated [41,42] or slightly modified LDL particles with
antibodies against them [29]. Introduction of the latter
complexes into macrophages, by way of the immu-
noglobulins (FC) receptor, results in foam cell forma-
tion [43]. Foam cell formation via these and other per-
oxidation-independent mechanisms is presently a topic
of extensive investigations [44-46].
One of the most convincing lines of evidence for the
involvement of oxidized LDL in cardio vascular disease
is based on the clinically-validated association of various
attributes of cardiovascular pathologies with the levels of
oxidized phospholipids on apolipoprotein B (OxPL/apoB),
as assayed in-vitro by the murine monoclonal antibody
EO6 [47]. Using this assay, based on the specific binding
of the EO6 antibody to the phosphocholine head group of
oxidized phospholipids, it has been shown that the levels
of oxidized phospholipids on circulating lipoproteins in
the plasma:
Are independent of traditional risk factors and the
metabolic syndrome.
Enhance the risk prediction of the Framingham Risk
Predict the presence and progression of coronary,
femoral and carotid artery disease (particularly when
amplified by lipoprotein(a) (Lp(a)) and phospholi-
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D. Lichtenberg, I. Pinchuk / Advances in Bioscience and Biotechnology 4 (2013) 48-61 51
pases such as PLA2).
Increased following acute coronary syndromes and
percutaneous coronary intervention.
Reflect the biological activity of Lp(a) particles.
Provide diagnostic and prognostic information on the
presence and progression of cardiovascular disease
and clinically validated power to predict cardiovas-
cular events.
In more than one respect, the immunological assay is a
reliable tool to evaluate a well defined type of oxidative
stress, as discussed below.
4.1. Are CVD Patients under OS?
OS is an intuitively-defined term that describes a state of
an excess of pro-oxidative factors over antioxidative fac-
tors [19]. In our analysis of available information on the
OS, as evaluated on the basis of various biomarkers, we
found reasonable correlations between the OS, as meas-
ured by different methods when (and only when) the
biomarkers measured similar processes [48]. Thus, the
OS, as evaluated on the basis of different products of
lipid peroxidation correlated with each other, and the
same is true for different markers of DNA fragmentation
but the estimates of OS based on factors of two different
groups rarely correlate with each other. Based on this
analysis, we concluded that oxidative stress cannot be
defined by any universal index and proposed that these
results indicate that there are different types of OS.
We do not believe that it is possible to define a uni-
versal criterion for oxidative stress, which can enable
comparison of data from different laboratories using dif-
ferent methods. The term OS is ill-defined and covers a
range of different types of context-dependent OS. Rea-
sonable correlations have been observed between the OS
determined on the basis of different tests of chemically
similar factors. This justifies defining a criterion for each
of these types but even this possibility is questionable
because OS of any type may be local and either be re-
flected in available body fluids, or not.
Hence, comparison between the OS in one group of
subjects (e.g. CVD patients) and another group (e.g.
healthy people) is legitimate only in terms of the same
biomarker. Figure 1 depicts the results of meta-analyses
of the difference between the OS biomarkers in CVD
patients and healthy people, as evaluated on the basis of
different assays. The clear result is that the available data
exhibit significant differences only when OS was esti-
mated on the basis of the level of MDA [49]. Interest-
ingly, the MDA concentrations in patients with stable
8-OH-dG (1)
MDA (13)
LOOH (1)
Isoprostanes (2)
Vitamin E (7)
Vitamin C (5)
Carotenes (1)
Urate (2)
GSH (2)
SOD (3)
GPX (5)
CAT (1)
TEAC (2)
Lag time (4)
Max Ox Rate (1)
Figure 1. Indices of oxidative stress in cardiovas-
cular disease (adopted from [49]). The pooled stan-
dardized mean difference (SMD) and the 95% con-
fidence interval is given for each of the studied in-
dices. Number of studies is given in parenthesis.
Note that MDA is the only accepted index of oxida-
tive stress that shows a mean difference greater than
1 SMD.
angina pectoris (SAP) are not different from matched
controls, whereas unstable angina pectoris (UAP) pa-
tients have significantly higher MDA concentrations than
both healthy controls and patients with SAP [49].
The observed differences between the different groups
can not be taken as evidence that CVD patients are under
oxidative stress, particularly because even the data based
on MDA measurements is questionable due to: 1) the
(questionable) reliability of the laboratory assay of MDA
and 2) publication bias. This conclusion accords with
that of Verhoye et al. [50], who noted that at present
“published data linking oxLDL to cardiovascular disease
cannot be compared because of the difference in the used
assay protocol”. Furthermore, the ‘Asklepios Study’ re-
vealed that the results of oxLDL tests “show important
variability and must be interpreted with caution”. Taken
together, the results demonstrate the complexity of the
association between plasma oxLDL and CAD, to the
extent that the oxLDL level was “independently associ-
ated with femoral plaque, but not carotid atherosclero-
In spite of these uncertainties, the trend is that oxi-
dized phospholipids play a role in atherogenesis, as indi-
cated by the immunologic assays described above. The
role of oxidation in the pathogenesis of atherosclerosis,
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D. Lichtenberg, I. Pinchuk / Advances in Bioscience and Biotechnology 4 (2013) 48-61
as commonly described may appears to be overestimated
but this may reflect homeostatic mechanisms capable of
reducing the effects of various factors on the oxidative
stress, as described below.
4.2. The Effect of Low Molecular Weight
Antioxidants on Atherosclerosis
Based on the free radical hypothesis of both longevity
and atherosclerosis, supplementation of vitamin E could
have been expected to have positive effects on both the
rate of atherosclerosis and mortality. In fact, in animal
models of atherosclerosis, antioxidants markedly de-
crease the rate of progression of lesions [28,51], in ac-
cordance with the oxidative modification hypothesis. By
contrast, meta-analyses of the many published clinical
trials show, at least apparently, higher mortality in vita-
min E-treated people than in matched controls [33,34].
These meta-analyses induced serious critique (e.g. [52-
54]) and comprehensive responses to the critique [55,56].
The criticism related mostly to the following issues:
1) The choice of clinical studies to be included in the
2) The choice of mortality as the only end point.
3) The heterogeneity of the participants with respect to
both population and treatment.
4) The model used to analyze the data (hierarchical lo-
gistic regression model vs. the traditional meta regres-
In short, in their communication in 2007, Blumberg
and Frei described the “Clinical trials of vitamin E and
cardiovascular diseases” as possibly being “fatally flawed”
Decision Analysis [57,58] was designed to minimize
the critique, by addressing these specific comments of
the data as follows:
1) Inclusion of all the clinical trials quoted in the cri-
tique of the meta-analyses.
2) The use of quality-adjusted life years (QALY) [59]
as an endpoint reflects the effects of supplementation on
both mortality and morbidity.
3) Using Markov model-based Monte Carlo simula-
tions enable adjustment for heterogeneities (both for
population and treatment) on the basis of registries.
The main finding of this analysis was that vitamin E
reduces the average QALY by almost four months but it
also indicated that some people benefit from vitamin E
supplementation [58]. Hence, the challenge was (and still
is) to find criteria to differentiate between those who are
likely to benefit from vitamin E supplementation and
those who are not. Differentiation can be based either on
identification of criteria that can be assayed in the lab
or/and on data regarding the response of patients of spe-
cific diseases to supplementation (see below).
4.3. The Effects of Vitamin E on HDL
Oxidative modification of LDL is affected by HDL via
different mechanisms, including acceleration of LDL
peroxidation by HDL oxidation-products and inhibition
of LDL peroxidation by removal of LDL peroxida-
tion-products either by extraction from LDL to HDL
and/or by their hydrolysis, catalyzed by HDL-associated
enzymes. Hence, antioxidants can affect LDL peroxida-
tion by altering the peroxidation of HDL and/or by alter-
ing the effect of HDL on LDL peroxidation. In the fol-
lowing discussion we first describe the results of a recent
study that demonstrates the effect of vitamin E on HDL
oxidation before relating to the mutual effects of peroxi-
dation of HDL and LDL lipids.
The very complex effect of tocopherol on the suscep-
tibility of lipoproteins to oxidation is demonstrated in the
recent study of its effects on both in-vitro and ex-vivo
peroxidation of HDL2 & HDL3. Specifically, in their
recent investigation, Wade et al. [60] found that the ef-
fect of added tocopherol depends on whether its peroxi-
dation was tested before or after fractionation of the se-
1) When added after fractionation, tocopherol pro-
tected the fraction of HDL2&3 against peroxidation.
2) By contrast, when pre-incubated with non-fraction-
ated serum and tested after fractionation, tocopherol pro-
moted the peroxidation of HDL2&3.
3) In another experiment, the authors tested the sus-
ceptibility of the HDL2&3 fraction to peroxidation fol-
lowing 6-weeks of tocopherol supplementation. The main
result of this experiment was that the HDL2&3 fraction
of the tocopherol-treated people were more susceptible to
peroxidation than the same fraction of placebo-supple-
mented controls [60]. Interestingly, the increased sensi-
tivity to peroxidation was accompanied by a decrease of
the activity of both HDL2&3-PON-1 and HDL2-LCAT
(lecithin-cholesterol acyltransferase), probably because
in the presence of higher concentrations of Toc, lower
concentrations of the latter HDL-associated protective
proteins were required, as described below.
To conclude, tocopherol can either protect or promote
HDL oxidation, depending upon the timing of exposure
of serum to tocopherol. In turn, HDL (and its oxidation
products) affects LDL oxidation, thus altering the com-
plex effects of tocopherol on atherogenesis, as described
4.4. The Effect of HDL on LDL Peroxidation
HDL, the beneficial form of blood cholesterol, (com-
monly denoted “the good cholesterol”) is an established
predictor of cardiovascular health. Its protective effect of
the artery wall against atherosclerosis is well established.
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D. Lichtenberg, I. Pinchuk / Advances in Bioscience and Biotechnology 4 (2013) 48-61 53
Furthermore, even in individuals whose LDL level is low,
HDL remains a strong predictor of the risk of coronary
artery disease (CAD), indicating that HDL is an inde-
pendent risk factor [51,61]. The positive effects of HDL
are commonly attributed to its ability to evacuate choles-
terol from the periphery to the liver. In addition to this
“reverse” transport, HDL plays a modulatory role in in-
flammation [62], which may also contribute to its cardio-
protective effect. The importance of the antioxidative
effect of HDL is emphasized by the findings of associa-
tions between impaired antioxidant activity of HDL and
various diseases [63,64].
In view of these facts, it is apparently surprising that
under certain conditions, HDL oxidation accelerates LDL
oxidation. Under such conditions, tocopherol-induced
acceleration of HDL oxidation can be expected to pro-
mote LDL oxidation, thus promote atherogenesis. In
view of the latter possibility, much effort has been de-
voted to investigate the mutual relationship between the
oxidation of HDL and LDL, as described below.
4.4.1. Lipid Peroxidation in Mixtures of LDL and
Under many conditions, HDL is more susceptible to oxi-
dation than LDL [65,66]. This is not surprising in view
of the similar or higher content of Vitamin E and other
antioxidants, including beta-carotene, ubiquinol (coen-
zyme Q-10) and lycopene, in LDL. These antioxidants
can act as the first line of defense. Furthermore, the
higher surface /volume ratio in the smaller HDL particle
can also accelerate the peroxidation induced by transition
metal ions. It is also consistent with the finding that the
smaller LDL particles are more readily oxidized than the
larger ones [67]. However, the higher oxidizability of
HDL is apparently inconsistent with the cardio-protective
effect of HDL.
Systematic kinetic studies on the oxidation in mixtures
of HDL and LDL demonstrated the complexity of the
effect of HDL on LDL peroxidation (and vice versa).
Specifically, these mutual effects [68] depend in a com-
plex fashion on the composition and physical properties
of both these lipoproteins as well as on the inducer of
peroxidation and the concentrations of transition metal
ions and several serum proteins.
The main results of the latter study were that:
1) Oxidation of LDL induced either by AAPH or by
Myeloperoxidase (MPO) is inhibited by HDL under all
the studied conditions, whereas.
2) Copper-induced peroxidation of LDL is inhibited by
HDL at low copper/lipoprotein ratio but accelerated by
HDL at high copper/lipoprotein ratio.
These results indicate that the antioxidative effects of
HDL are only partially due to HDL-associated enzymes,
in agreement with the finding that reconstituted HDL,
containing no such enzymes, inhibits peroxidation in-
duced by low copper concentration [68]. Reduction of
the binding of copper to LDL by competitive binding to
the HDL can also contribute to the antioxidative effect of
The acceleration of copper-induced oxidation of LDL
by HDL, observed at high copper concentration, may be
attributed to the hydroperoxides formed in the “more
oxidizable” HDL, which can migrate to the “less oxidi-
zable” LDL and enhance the oxidation of the LDL lipids
induced by bound copper.
Given the sensitivity of the rate of oxidation to many
factors, this hypothesis does not necessarily contradict
previous results [69], observed under different conditions,
in which the migration of hydroperoxides between lipo-
protein particles was too slow to accelerate the peroxida-
tion in “native lipoprotein particles”. Notably, our inter-
pretation of the acceleration of copper-induced LDL
peroxidation by HDL is supported by the results of ex-
periments in which native LDL was added to oxidizing
lipoproteins at different time points. When the native
LDL was added prior to decomposition of the hydroper-
oxides in the oxidizing lipoprotein, the lag preceding
oxidation of the LDL was much shorter than the lag ob-
served when the native LDL was added at later stages
(Figure 2), after the level of hydroperoxides became
reduced due to their copper-catalyzed decomposition
Furthermore, this interpretation accords with the ac-
celeration of LDL peroxidation upon sequential exposure
to copper [70]. Altogether, the observed interrelationship
between the oxidation of HDL and LDL and its depend-
ence on the specific conditions should be considered in
future investigations regarding the oxidation of lipopro-
4.4.2. Mechanisms that Contribute to the Inhibition
of LDL Peroxidation by HDL
The oxidative damage caused by peroxidation products
can be reduced by their evacuation from the site of their
production, where they may accelerate the propagation
of free radicals chain reaction. Accordingly, the mecha-
nisms that can contribute to the net effect of HDL can be
divided to two categorize:
1) Mechanisms based on transporting peroxidation
products to the liver (here denoted “the physical mode”).
2) Mechanisms based on metabolism, particularly hy-
drolysis of short or/and oxidized chain phospholipids,
catalyzed by LDL-associated PAF acetyl hydrolase and
HDL-associated enzymes, including PON-1, PON-2
(here denoted “the chemical mode”).
In relating to the chemical mode, we recall that under
certain conditions the LDL-associated PAF-acetyl-hy-
drolase from human plasma prevented oxidative modi-
fication of LDL [71], whereas in other experiments,
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D. Lichtenberg, I. Pinchuk / Advances in Bioscience and Biotechnology 4 (2013) 48-61
Figure 2. Sequential exposure of lipoproteins to cop-
per-induced peroxidation (adopted from [68]). Accumu-
lation of hydroperoxides during copperinduced oxida-
tion at 37˚C is described as a function of time. In both
panels, the solid lines depict control peroxidation ex-
periments, conducted with native LDL. The dashed
lines in both panels depict sequential exposure experi-
ments, in which LDL was added at the time points in-
dicated by arrows, to a mixture of CuCl2 and either
LDL (panel A) or HDL (panel B). The cholesterol con-
centration in the pre-exposed lipoproteins was a fifth
of the concentration of the added LDL.
inhibition of this enzyme did not affect ex-vivo peroxide-
tion induced by either copper, Myeloperoxidase or AAPH
[72]. All that this apparent contradiction means is that the
effect of hydrolysis on the rate of peroxidation depends
on the experimental conditions via a not yet known me-
This is probably valid for other lipoprotein-associated
enzymes. Our results accord with those of Teiber et al.
[73], who showed that “Purified human serum PON1
does not protect LDL against oxidation in the in vitro
assays initiated with copper or AAPH”, in direct contrast
to the results of Fyrnys et al. [74] and others. Teiber et al.
attributed their results to the “removal of PON1 from its
natural environment”, which means that a method based
on the antioxidative properties of highly purified PON1
may be irrelevant to the antioxidative properties of the
HDL-associated enzymes. Although this interpretation
may hold for the antioxidative activity of purified PON1,
it does not explain our results regarding the effect of
LDL-associated PAF-AH on the peroxidation in unfrac-
tionated serum.
The role of PON has been extensively studied. In 1991
Mackness et al. [75] showed that PON is involved in the
protection of LDL phospholipids against oxidation dur-
ing the onset of the atherosclerosis process. Two years
later, these authors demonstrated that PON is a very
strong antioxidant, responsible for inhibition of HDL
peroxidation and for the effect of HDL on LDL peroxi-
dation [76]. These findings initiated extensive research of
both the HDL-associated enzyme PON1 and the intra-
cellular enzyme PON2, which is not carried via HDL.
Both these enzymes are antioxidants and anti-athero-
genic [77-81] probably due to prevention of the accumu-
lation of lipid peroxides in HDL and LDL by PON-
catalyzed metabolism of peroxidation products. Although
there is no general consensus, the level of PON has been
reported to be relatively low in many diseases, indicating
that reduced levels of PON may contribute to these dis-
eases. All we can conclude at this time is that we are far
from understanding in detail the role of PONs.
In relating to the physical mode, it is important to re-
call that the level of peroxidizable lipids in LDL is equal
or higher than in HDL. By contrast, the levels of both
early and late oxidation products are higher in HDL than
in LDL [61,82]. Thus, under conditions of continuing
production of peroxyl radicals, HDL accumulated more
lipid oxidation products than LDL. As mentioned above,
one contributing factor is the higher levels of lipid-solu-
ble antioxidants in LDL than in HDL. Notably, the effect
of the lipoprotein-associated antioxidants is also affected
by the concentration of water-soluble antioxidants due to
replenishment of the lipid-soluble antioxidants. As an
example, Vitamin C is soluble in water, but not in or-
ganic solvents or in lipids such as those found in LDL.
Hence, it cannot act within the LDL particle. Nonetheless,
vitamin C can reduce oxidized vitamin E at the wa-
ter-lipid interface, so that the molecule of vitamin E can
act once again as a protective agent. In this indirect way,
vitamin C “cycles” the vitamin E within the LDL particle
Another important factor is the relative rate of removal
of peroxidation-products from the two lipoproteins via
their enzymatic hydrolysis. This process is also depend-
ent in a complex fashion on the rate of transfer of per-
oxidation products (and their hydrolysis products) be-
tween HDL and LDL. Under certain conditions, the trans-
fer of lipid hydroperoxides appears to be too slow to
substantially influence the distribution of these com-
pounds in plasma [32], whereas under different condi-
tions the initially formed (in HDL) hydroperoxides ac-
celerate the peroxidation of LDL lipids [68]. The kinetic
profile of lipid peroxidation in mixtures of HDL and
LDL depends on the relative concentrations and suscep-
tibilities of the two lipoproteins. Specifically, under cer-
Copyright © 2013 SciRes. OPEN ACCESS
D. Lichtenberg, I. Pinchuk / Advances in Bioscience and Biotechnology 4 (2013) 48-61 55
tain conditions it may be possible to observe HDL per-
oxidation followed by peroxidation of LDL lipids. Under
different conditions, the different susceptibilities is not
sufficiently large to observe two time dependencies and
the time course of hydroperoxide accumulation appears
to be relatively broad and the lag being either independ-
ent of the less oxidizable LDL or an increasing function
of the LDL/HDL ratio [68].
4.5. The Relative Susceptibility of Serum
Lipoproteins to Peroxidation and Its
Association with the Concentrations of
Lipoprotein Fractions
An intriguing result of our previous study is the highly
significant positive correlation (p-value less than 0.001)
between the HDL levels in the sera of hypercholes-
terolemic patients and the susceptibility of the serum
lipids in unfractionated serum to copper-induced peroxi-
dation ex-vivo, as evaluated on the basis of kinetic assay
and expressed in terms of tmax [84] (Figure 3). We think
that the lag preceding the rapid interrelated peroxidation
of HDL and LDL reflects the ratio between the concen-
trations of the two lipoproteins. In other words, “HDL
particles contain high amounts of lipid hydroperoxides
[82] and thus, shorten the lag time and increase the
amount of oxidized PUFAs”, in agreement with the asso-
ciation of high HDL concentrations with LDL oxidation
[66] and with the high correlation between serum oxidi-
zability and HDL content found in the recent NMR study
of Tynkkynen et al. [85].
In view of the slow development of atherogenesis and
the possible protective role of HDL, we speculate that
the latter contra-intuitive result can be attributed to a yet
Figure 3. The relation between serum composition and oxida-
tion parameters (adopted from [84]). Correlation coefficients
are given between the serum concentrations of cholesterol (to-
tal), LDL cholesterol, HDL cholesterol, and total triglycerides
(TG), on one hand, and tmax, on the other. The black bars relate
to oxidation kinetics as recorded at 245 nm; the gray bars relate
to the kinetics as recorded at 268 nm. The symbol *corresponds
to a P-value smaller than 0.05; the symbol **corresponds to a
P-value smaller than 0.001.
unknown mechanism aimed at accelerating the removal
of oxidative products via “reverse transport”.
Similar to many other investigations, the general ap-
proach to the “Oxidative modification hypothesis” was to
test predictions based on the hypothesis. Specifically, the
hypothesis attributing atherogenesis to oxidative modifi-
cation led to the prediction that if the theory is correct,
antioxidants should be cardio-protective. In cellular sys-
tems, the experimental results appeared to be consistent
with this prediction. Furthermore, in cholesterol-fed ani-
mals, vitamin-E supplementation inhibited the athero-
genesis, as predicted. Unfortunately, clinical trials yielded
disappointing results, raising doubts regarding the causal
relationships between AS and OS.
As in other cases of inconsistencies between the pre-
dicted and experimental results, several studies ‘blamed
the experiments’; others questioned the hypothesis. The
question is often “which is wrong?” [86]. In fact, there is
another possibility, which is that the hypothesis is right,
the experiments are conducted correctly, but the predic-
tions based on the hypothesis suffer from over-simplifi-
cation. We think that the latter possibility is valid for the
present issue. Specifically, the predictions were based on
the “quench free radicals, prevent AS” approach, which
can not be expected to be valid for this multifactorial
process because it ignores many factors, including the
possible pro-oxidative effects of “antioxidants”, the pos-
sibility that the antioxidants may interfere with the syn-
thesis of antioxidative enzymes, the possible reduction of
the OS to levels lower than “normal”, the contribution of
oxidative modifications of LDL via non-radical mecha-
nism(s) (e.g. [87]) and the possibility that ROS induces
the process without being involved in later stages of
atherogenesis, which means that vitamin E supplementa-
tion after the initiation of the process is too late [88].
In other words, we think that although lipid peroxida-
tion is involved in atherogenesis, as indicated by many
experiments, a causal relationship could not have been
predicted on the basis of the limited available knowledge
regarding the complex, multi-factorial, interdependent
dependencies of atherogenesis on the “oxidative stress”
and antioxidants.
Antioxidants Do Not Always Inhibit LDL
They may even accelerate it if/when:
1) The supplemented antioxidant acts as a pro-oxidant
by reducing transition metal ions (e.g. vitamin C [89,90])
Copyright © 2013 SciRes. OPEN ACCESS
D. Lichtenberg, I. Pinchuk / Advances in Bioscience and Biotechnology 4 (2013) 48-61
or via tocopherol-mediated peroxidation (TMP) [91].
2) The added antioxidant reduces the OS too much, to
levels below normal, thus causing toxic effects (e.g. due
to promotion of the adhesion of red blood cells to endo-
thelial cells [92]).
3) Modified lipoproteins are formed via free radi-
cal-independent mechanisms [46,87,93].
4) Free radicals initiate atherosclerosis but play only a
minor role in later stages of the complex processes that
yield AS, so that vitamin E does not alter the process
when added after initiation occurred [88].
5) The antioxidant interferes with the protective effect
of HDL against LDL peroxidation [60], as discussed
6) The antioxidant reduces the production of other an-
tioxidative factors, as indicated by the slight difference
between CVD patients and healthy controls. It is also
consistent with the interpretation of Ristow et al. [7] to
their disappointing results concerning the (negative) ef-
fects of vitamin E-supplementation on the health of
sportsmen. Furthermore, this possibility is supported by
the finding of high oxidizability of HDL in the serum of
vitamin E-treated patients [60]. This may also be true for
LDL and if it is not the case, we can still expect changes
in LDL oxidation due to HDL oxidation, as described
Given this long (yet partial) list of possible reasons for
the apparent contradictions between the observed and
predicted relationship of oxidative modification and
CVD, we can only conclude that this relationship is cer-
tainly uncertain.
Atherogenesis is a “chronic” disease that develops during
many years. It depends (at least partially) on alteration of
the antioxidant-pro-oxidant balance, which is responsible
for maintenance of the homeostatic level of the redox
state. In comparison to the homeostatic level, OS means
that the redox status in the relevant body compartment is
“oxidative”. Antioxidants may be sufficient to shift it to
normal levels but too much antioxidant may cause a shift
to a “reductive state”. Alternative terminology can be
based on the level of ROS in comparison to their homeo-
static level. Specifically, OS may be described as being
the result of “hyperOSemia”; an excess of antioxidants
can be attributed to “hypoOSemia”.
In fact, we have no data on the dependence of either
the functions or the toxic effects of ROS on their con-
centrations. In other words, the dependence of AS on OS
is not well understood. This does not undermine the va-
lidity of the hypothesis that peroxidation is responsible
for atherogenesis [20] but it certainly justifies reconsid-
eration of the hypothesis. To us, the present review indi-
cates that ROS is not just the “bad guys” and not the only
“bad guys” in the multi-factorial pathogenesis of coro-
nary artery disease and atherogenesis.
The major change in the field of free radical research,
particularly in biology and medicine, is the growing un-
certainties. Now that we realize the importance of the
tight control of homeostatic level of OS, the paradigm of
“bad free radicals versus good antioxidants” can not be
retained, not only with respect to cardiovascular issues.
Even Harman’s “free radical theory of aging” is ques-
tionable. This state of art (in Atherosclerosis) raises at
least three questions: 1) what is the sequence of proc-
esses that lead to CVD and AS? 2) what is the interrela-
tionship between LDL oxidation and CVD? and 3) who
is likely to benefit from antioxidant supplementation?
While the first two questions have been investigated
and partially answered [44,45], we have no clues to an-
swer the third [94]. Needless to say, high dose indis-
criminate supplementation of vitamin E can not be rec-
ommended to the general public (that still spends mil-
lions of dollars on vitamin E supplements). Yet, it is quite
clear that some specific groups may gain from vitamin E
supplementation. The challenge is to define a criterion to
predict who is likely to benefit from antioxidant supple-
In his commentary in 1989, Witztum raised the ques-
tion “To E or not to E?” [95] and proposed that antioxi-
dants can be expected to be beneficial for people under
oxidative stress. He then pointed out that the problem is
that we “lack measures to identify high-risk groups that
would theoretically benefit most from antioxidant inter-
ventions” as well as a reliable measure “to determine the
in vivo effectiveness of such interventions”. Unfortu-
nately, not only the existing data is still insufficient to
predict who is likely to benefit from vitamin E treat-
ment. We even don’t know which type of oxidative stress
has to be reduced. What we know is that based on the
results of the population-based Cache County Study,
Hayden et al. [52] advocate “further caution regarding
the use of vitamin E by those with existing cardiovascu-
lar disease”. This statement is inconsistent either with the
assumption that people under oxidative stress can be
expected to benefit from vitamin E or with the assump-
tion that CVD patients are under high oxidative stress.
We think that the apparent contradiction may be partially
due to unrealistic estimation of the role of oxidative
stress in CVD, in agreement with our conclusion regard-
ing the (modest) role of OS in CVD patients [49].
Furthermore, in his 2003 communication, Frei raised
the question of whether “to C or Not to C” and con-
cluded that “What we know with certainty, however, is
that a healthy diet and lifestyle lowers the risk of CHD,
and this is what we should advocate to CHD patients and
healthy people alike” [96]. Again, this kind of conclu-
sions does not help in our goal of understanding the
Copyright © 2013 SciRes. OPEN ACCESS
D. Lichtenberg, I. Pinchuk / Advances in Bioscience and Biotechnology 4 (2013) 48-61 57
questions defined above. In short, the ill-defined term
“oxidative stress” does not differentiate between the ex-
istence of “different types of oxidative stress” and the
possibility that different antioxidants may have different
antioxidative activity against different types of oxidative
Another disappointing result is that physical exercise,
which is likely to cause excessive oxidation (thus, OS),
does not increase the benefit from antioxidant supple-
mentation. In fact, antioxidants prevent health-promoting
effects of physical exercise [7].
In our future search for criteria to predict who is likely
to benefit from antioxidant supplementation, we can ei-
ther look for people who are under high (relevant type of)
OS or/and patients suffering from a disease that have
been associated with OS such as dialysis patients [97] or
diabetes mellitus type 2 (DM2) patients with haptaglobin
2-2 genotype [98].
As long as we do not have such criteria, we propose
basing the decision of whether or not to expect benefit
from chronic supplementation of antioxidants on whether
or not a relatively short period (e.g. one month) of sup-
plementation to a patient with low vitamin E level results
in a significant decrease of the blood level of lipid per-
oxidation markers (e.g. MDA).
This approach is supported by a previous study of the
effect of vitamin E on AD patients, in which the clinical
outcome of a group of “vitamin E respondents”, as de-
termined after a short period of supplementation, was
highly significant and excided by far the effect observed
in the group of AD patients that were classified as
“none-respondents”. In their words [99] “the decision of
whether or not to expect benefit from chronic vitamin E
supplementation should be based on whether or not one
month of vitamin E supplementation to patient with low
vitamin E level results in a significant decrease of the
blood level of lipid peroxidation markers”.
Regardless of the possible effects of food supplements,
fruits and vegetables are beneficial and those “many pa-
tients (that) view vitamins as a quick fix to compensate
for poor eating habits [Tara Parker-Pope, The Wall Street
Journal, March 20, 2006] should be advised of the dif-
ference between food ingredients and food supplements.
A glass of red wine, orange juice or a salad of vegetables
or fruits along with ROS-containing food is likely to
prevent absorption of unhealthy peroxides by quenching
ROS in the stomach [100]. Furthermore, it is tasty. Syn-
thetic supplements, commonly do not “meet” toxic food
ingredients in the gastrointestinal system. Maybe this is
why they are not as beneficial as expected.
We thank Dr Edit Schnitzer and Prof Menachem Fainaru of TAU and
Prof Yury Miller (UCSD) for their constructive remarks and the Lady
Davis Foundation for financial support.
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AAPH—2,2’-Azobis(2-amidinopropane) dihydrochlo-
ride; a water-soluble generator of free radicals;
MDA—malondialdehyde; a relatively stable peroxida-
tion product;
PAF-AH—platelet aggregation factor acetyl hydrolase;
HDL associated PLA—the enzyme that catalyzes the
hydrolysis of peroxidation products;
QALY—quality adjusted life years;
OS—oxidative stress; an ill-defined term commonly
used to describe intuitively the imbalance between pro-
oxidative and antioxidative species;
RO(N)S—reactive oxygen (and nitrogen) species; used
when we do not know which reactive oxygen (or nitro-
gen) species are involved;
FR—free radicals; used when we relate to established
free radical mechanism(s);
oxLDL—oxidatively-modified LDL; used when we
mean that the relevant active species is one or more LDL
oxidation product(s).
OS is tightly controlled. In CVD patients, it is only
slightly higher than normal.
The weak association of OS with CVD is insufficient
to prove causal relationship.
Oxidative modification of LDL is one of several
processes that govern atherosclerosis.
Antioxidants do not necessarily inhibit the multifac-
torial processes resulting in AS.
Our knowledge is insufficient to predict who is likely
to gain from antioxidants.