| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Biochemistry Group, The Heart Research Institute, Sydney, Australia
1Correspondence: Biochemistry Group, The Heart Research Institute, 145 Missenden Rd., Camperdown, Sydney, NSW 2050 Australia. E-mail: r.stocker{at}hri.org.au
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONROLE OF VITAMIN E...COANTIOXIDANTS FOR {alpha}-TOH...IS TMP RELEVANT TO...DOES INTIMAL LIPOPROTEIN...VITAMIN E SUPPLEMENTATION AND...COANTIOXIDANTS AS IN VIVO...CONCLUSIONSREFERENCES |
|---|
-tocopherol (-TOH, vitamin E), the
major lipid-soluble lipoprotein antioxidant, have been studied in
detail. Based on the known antioxidant action of -TOH and
epidemiological evidence, vitamin E is generally considered to be
beneficial in coronary artery disease. However, intervention studies
overall show a null effect of vitamin E on atherosclerosis. This
confounding outcome can be rationalized by the recently discovered
diverse role for -TOH in lipoprotein oxidation; that is, -TOH
displays neutral, anti-, or, indeed, pro-oxidant activity under various
conditions. This review describes the latter, novel action of -TOH,
termed tocopherol-mediated peroxidation, and discusses the benefits of
vitamin E supplementation alone or together with other antioxidants
that work in concert with -TOH in ameliorating lipoprotein lipid
peroxidation in the artery wall and, hence, atherosclerosis.—Upston,
J. M., Terentis, A. C., Stocker, R. Tocopherol-mediated
peroxidation of lipoproteins: implications for vitamin E as a potential
antiatherogenic supplement.
Key Words: atherosclerosis • lipid peroxidation • antioxidants • LDL
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONROLE OF VITAMIN E...COANTIOXIDANTS FOR {alpha}-TOH...IS TMP RELEVANT TO...DOES INTIMAL LIPOPROTEIN...VITAMIN E SUPPLEMENTATION AND...COANTIOXIDANTS AS IN VIVO...CONCLUSIONSREFERENCES |
|---|
. A
hallmark of early atherosclerosis is the accumulation within the
vascular wall of macrophages laden with LDL-derived lipid (i.e., foam
cells). Cholesterol is essential for cell growth and viability of
eukaryotic cells, yet unregulated cellular accumulation of cholesterol
predisposes to atherosclerosis. Peripheral cells obtain cholesterol
primarily via uptake of circulating LDL. Feedback inhibition by
cholesterol of the LDL receptor normally regulates the amount of
cholesterol entering the cell (2)
, and a loss of this regulation can
lead to foam cell formation and premature atherosclerosis. Cellular
cholesterol homeostasis therefore remains the subject of intense
investigation.
Excessive cellular lipid accumulation, such as that seen in
atherosclerotic lesions, can be induced in vitro
by LDL subjected to chemical modifications (3)
. For example, exposure
to various cells in the presence of transition metal ions (4)
oxidizes
and converts LDL into a high uptake form. Both lipid oxidation and
protein modification are critical to unabated cellular lipid
accumulation (5)
via scavenger receptors, the expression of which is
not feedback regulated. These early in vitro studies formed
the basis for proposing `oxidized LDL' as an inducer of foam cell
formation in vivo and hence contributing to and/or causing
human atherosclerosis (`oxidation hypothesis') (6
7
8
9
10
11)
. Subsequent
studies have shown that oxidized LDL has potentially proatherogenic
activities in vitro (reviewed in ref 10
), and oxidized
lipoproteins can be isolated from atherosclerotic lesions (8,
12
13
14
15)
.
Despite these associations, direct evidence in support of oxidized LDL
causing atherosclerosis is lacking.
In addition to cholesterol, LDL is a major vehicle for vitamin E
delivery to peripheral tissues. Vitamin E consists of two groups of
lipid-soluble compounds (tocopherols and tocotrienols) with four
structurally related forms in each group. In humans, -tocopherol
(-TOH) predominates and is generally considered the most active form
of vitamin E. -TOH is a powerful lipid-soluble antioxidant (16)
. It
is also quantitatively the major antioxidant in LDL lipid extracts and
is generally considered to protect LDL's lipid against oxidation (17)
and to be antiatherogenic. However, recent in vitro studies
have shown that -TOH can be pro-oxidative rather than protective for
lipids in isolated LDL (18
19
20
21
22
23)
. This pro-oxidant activity cannot be
explained by the classical mode of action of -TOH as a
chain-breaking antioxidant, and has been termed tocoperol-mediated
peroxidation (TMP) (19)
. In this review we summarize this activity of
-TOH, discuss whether TMP may occur in vivo and may be
relevant to atherogenesis, and address the implications of this to
vitamin E supplementation as an antiatherosclerotic strategy.
|
ROLE OF VITAMIN E IN LDL LIPID PEROXIDATION |
|---|
|
TOPABSTRACTINTRODUCTIONROLE OF VITAMIN E...COANTIOXIDANTS FOR {alpha}-TOH...IS TMP RELEVANT TO...DOES INTIMAL LIPOPROTEIN...VITAMIN E SUPPLEMENTATION AND...COANTIOXIDANTS AS IN VIVO...CONCLUSIONSREFERENCES |
|---|
and are more vulnerable to lipid peroxidation than
monounsaturated lipids or free cholesterol. This predisposes LDL to
oxidative lipid modification, which is thought to be the primary
oxidative event preceding and contributing to the modification of apo B
and formation of high-uptake LDL (5)
.
A detailed knowledge of how LDL's polyunsaturated lipids become
oxidized and how -TOH influences this process is therefore of
fundamental importance to an understanding of the overall process by
which LDL is converted to the high-uptake form and how this can be
prevented. As alluded to earlier, -TOH functions differently in LDL
lipid oxidation than in its classical role as a chain-breaking
antioxidant in homogeneous solution. We first describe the latter case
before summarizing the function of -TOH in lipoprotein lipid
peroxidation.
`Classical' antioxidant action of vitamin E in homogenous lipid
solution
The mechanism of peroxidation of lipid-containing bisallylic
hydrogens (LH) in homogeneous solution is well established. It proceeds
via the radical chain mechanism illustrated by reactions 1–3. The
process is initiated by the abstraction of a relatively weakly bound
bisallylic hydrogen on the lipid moiety to produce a carbon-centered
lipid radical (L•), which rapidly combines with
oxygen to produce a lipid peroxyl radical
(LOO•). Reaction of
LOO• with another LH gives rise to lipid
hydroperoxide (LOOH) while regenerating LOO•,
the chain-propagating species. In the absence of chain-breaking
antioxidants such as -TOH, lipid peroxidation eventually terminates
via bimolecular processes [reaction 4].
Initiation radical oxidant + LH —> inactive oxidant + L• [1]
Propagation L• + O2 —> LOO• [2]
Propagation LOO• + LH —> LOOH + L• [3]
Termination LOO• + LOO•/L• —> nonradical products (NRP) [4]
-TOH acts as a chain-breaking antioxidant by donating its phenolic
hydrogen to the otherwise chain-propagating
LOO• and replacing the latter with the less
reactive -tocopheroxyl radical (-TO•)
[reaction 5] (16)
. Alternatively, -TOH may react directly with the
initiating radical [reaction 6] to prevent
LOO• formation. LOO•
may also be eliminated via a radical–radical reaction with
-TO• [reaction 7]. There are no phase
barriers that restrict movement of -TO•,
hence its involvement in bimolecular reactions (Fig. 1
A).
|
Inhibition -TOH + LOO• —>
-TO• + LOOH [5]
Inhibition -TOH + radical oxidant —> inactive oxidant +
-TO• [6]
Termination -TO•+
LOO• —> nonradical products (NRP) [7]
Thus, each molecule of -TOH has the capacity to scavenge two
radicals (i.e., stoichiometric value of -TOH = 2), resulting
in 
1 mol of LOOH formed per mole of -TOH consumed.
The antioxidant activity of -TOH described above gives rise to a
well-defined period of strong inhibition of lipid peroxidation, termed
the `lag time' (16)
. After complete consumption of the vitamin, the
rate of lipid peroxidation increases rapidly. The key characteristic of
vitamin E action under these conditions is that increasing its content
results in enhanced inhibition of lipid peroxidation, reflected by a
prolonged lag time.
Tocopherol-mediated peroxidation: a model of in
vitro LDL lipid peroxidation that occurs in the presence of
vitamin E
Inhibition of lipid peroxidation by -TOH has also been observed
for isolated LDL oxidized with high concentrations of cupric ion
(Cu2+) (i.e., 
12–16
Cu2+ ions per LDL particle) (25)
. Using these
oxidizing conditions, Esterbauer and co-workers observed that lipid
peroxidation was minimal during the initial period in which -TOH and
other antioxidants were consumed rapidly. After -TOH depletion,
lipid peroxidation proceeded at much higher rates (25)
. The similarity
of these observations to those in homogeneous phase prompted the
authors to suggest that -TOH acts as a `conventional' antioxidant
in LDL. This view is supported by the finding that under the same
oxidizing conditions, supplementation of LDL with -TOH significantly
prolongs the lag time (see refs 17,
26
27
28
).
Close examination of the profile of LOOH accumulation in the
-TOH-containing stage of LDL oxidation reveals, however, that the
characteristics of lipid peroxidation cannot be explained by the
conventional antioxidant action described above (for a detailed review,
see ref 19
). Thus, with oxidizing conditions milder than those commonly
used by Esterbauer and co-workers, the rate of LOOH formation in the
-TOH-containing stage increases; supplementation of LDL
with -TOH increases the initial rate of lipid
peroxidation; the rate of lipid peroxidation is higher in the presence
of -TOH than immediately after its depletion and is retarded when
the lipoprotein is depleted of -TOH; and the maximal rate of lipid
peroxidation in the presence of the vitamin is independent of the rate
of initiation of lipid peroxidation (19,
22,
29
30
31)
. There also exists
a clear lack of correlation between endogenous vitamin E levels and lag
time in native LDL (17,
32)
, inconsistent with a conventional
antioxidant role of -TOH for LDL's lipids.
These discrepancies can be reconciled by proposing TMP as the mechanism
controlling lipid peroxidation in -TOH-containing lipoprotein
particles. This mechanism takes into account the discrete particle
nature of lipoprotein emulsions (Fig. 1B
) as well as
incorporating a radical phase and chain transfer role for -TOH (see
below) (19)
.
TMP has been proposed and verified experimentally using a variety of
oxidants including lipophilic or aqueous peroxyl radicals,
Cu2+ ions, hydroxyl radicals
(•OH), horseradish peroxidase in the presence
of hydrogen peroxide
(HRP/H2O2), myeloperoxidase
(MPO)/H2O2/chloride, SIN-1,
and 15-lipoxygenase (Table 1
) (33)
. However, for illustration, we assume the oxidant to be an
aqueous peroxyl radical (ROO•) (Fig. 2
). This oxidant can interact directly with either surface lipid (not
shown) or -TOH. Although the surface lipids are more abundant,
-TOH is approximately five orders of magnitude more reactive
toward ROO• and the redox-active
chromanol hydrogen that resides predominantly at, or close to, the
surface of the particle (34)
. Therefore, ROO•
tends to react preferentially with -TOH (reaction 1 in Fig. 2
). The
resulting -TO• is `trapped' within LDL and
therefore cannot undergo radical–radical termination unless a second
ROO• enters the oxidizing particle. This first
reaction alone may constitute a protective role by -TOH. However,
the fate of the resulting -TO• determines
whether the overall result of this initial reaction is pro- or
antioxidant.
|
|
The flux of aqueous radicals, i.e., the frequency with which LDL
encounters ROO•, fundamentally controls the net
effect of -TOH (19)
. Under high flux conditions, termination
reactions between -TO• and
ROO• are frequent (reaction 2 in Fig. 2
),
resulting in both the prevention of lipid peroxidation and the rapid
consumption of -TOH. A high frequency of radical–radical
termination readily accounts for why -TOH acts as an antioxidant in,
for example, the Cu2+:LDL = 12–16:1 system
mentioned above.
Under low flux conditions, however, termination reactions are
infrequent. The relatively (to -TOH) less polar (35)
-TO•moves within the particle where it can
gain access to both surface and core LH. Such transfer of radicals from
the outside to the inside of the particle is referred to as the phase
transfer activity of -TOH (reaction 1 in Fig. 2
). The relevance of
the phase transfer activity of vitamin E is most pronounced for mildly
reactive radicals (such as ROO•), whereas more
reactive radicals such as •OH can directly
attack LDL's LH. Thus, ROO• and
•OH react ~104- to
105- and 10-fold faster with -TOH than LH,
respectively (35,
36)
. Therefore, •OH will
react predominantly with LH because its concentration in LDL is ~100-
to 200-fold higher than that of -TOH; conversely,
ROO• will react predominantly with LDL's
-TOH. Independent of the nature of the radical oxidant, however,
under steady-state conditions -TO• is the
most stable and predominant radical that can be formed in LDL. Once
inside the particle, and in the absence of other radicals,
-TO• can react with LH and hence trigger
LOO• formation (reaction 3 in Fig. 2
). -TOH
rapidly scavenges LOO• to produce LOOH while
regenerating -TO• and continuing the cycle
(reaction 4 in Fig. 2
). Unless -TO• is
eliminated, a large proportion of LDL lipids can become oxidized
without substantial loss of the vitamin. The ability of
-TO• to act as the lipid peroxidation
chain-carrying molecule refers to the chain transfer activity of
-TOH (reactions 3 and 4 in Fig. 2
).
Given the lower reactivity of -TO• compared
with LOO•, the former is the predominant and
single radical within an oxidizing, vitamin E-containing particle under
steady-state conditions. The relative stability of
-TO• also means that TMP represents
`retarded' lipid peroxidation when compared with the uninhibited
peroxidation that can take place in -TOH-depleted particles.
Nevertheless the presence of -TOH increases the overall reactivity
of lipoproteins: the more -TOH LDL contains, the more likely the
particle reacts with ROO• and undergoes
oxidation (22)
.
For the remainder of this section we will expand on the concepts of
phase and radical transfer activities of -TOH, since these are
pivotal to TMP. We will also present experimental data that support
this mechanism under a variety of oxidizing conditions before
describing a strategy for preventing TMP.
Chain transfer activity of LDL's vitamin E
Enrichment of lipoproteins with -TOH increases the rate of
lipid peroxidation even under conditions of idential rates of lipid
peroxidation initiation (19)
. The impact of this chain transfer
activity of the vitamin is affected by the size of the lipoprotein
particle. Thus, under conditions of identical rates of lipid
peroxidation initiation, the lipid peroxidation chain length (defined
as the ratio of the rate of lipid hydroperoxide formation to the rate
of radical generation) decreases with decreasing particle size: very
low density lipoprotein > LDL > high density lipoprotein
(19,
37,
38)
. This order reflects an increase in both the surface area
to particle volume ratio and the relative residence time of
-TO• at the surface (vs. interior) of the
lipoprotein. The latter increases the likelihood of
-TO• participating in radical termination
reactions, which itself lowers chain transfer activity and hence the
extent of lipid peroxidation. The chain transfer activity of
-TO• is also reflected by the comparable
fractional peroxidation rates of surface and core lipids (19,
22,
39)
.
Phase transfer activity of LDL's vitamin E
Exchanging vitamin E's phenolic hydrogen with deuterium
attenuates the ability of -TOH to transfer radicals from the aqueous
compartment into LDL. Such exchange results in an overall twofold
decrease in both the rates of accumulation of CE hydroperoxides
(CE-OOH) and the consumption of -TOH in LDL exposed to
ROO• (30)
. By contrast, deuterium exchange
reduces the radical scavenging ability of -TOH (30,
40)
. Thus, if
lipid peroxidation were to proceed in LDL via the conventional
mechanism of vitamin E antioxidant activity, a reduction in radical
scavenging ability would be predicted to enhance rather than diminish
lipid peroxidation (41)
. However, the inverse deuterium kinetic isotope
effect observed (30)
is fully consistent with the reduced phase
transfer activity of -TOD v -TOH, thereby decreasing TMP of LDL
lipids.
Vitamin E can be required for effective initiation of LDL lipid
peroxidation
-TOH affects the oxidizability of LDL lipids induced by a
variety of radical oxidants (Table 1)
(33)
, consistent with -TOH
acting as the mediator of LDL lipid peroxidation. Most strikingly, the
initiation of lipid peroxidation in (hydroperoxide-free) LDL by soybean
lipoxygenase,
MPO/H2O2/Cl-/tyrosine,
or Cu2+ ions occurs only in the presence of
-TOH (22)
. In the latter case, this is true even when
Cu2+:LDL ratios of up to 20:1 are used (22)
. When
used at concentrations when the frequency of radical encounter by LDL
is low, these and other oxidants cause CE-OOH and hydroperoxides of
phosphatidyl choline to accumulate at lower rates in in
vitro -TOH-depleted than in the corresponding native LDL (22)
.
This applies even to highly reactive •OH,
although at the later stages of these oxidations CE-OOH accumulation is
greater in the vitamin-depleted than native LDL (22)
. This is
consistent with the idea that •OH can directly
react with LDL's LH and that a radical-containing, -TOH-free LDL
oxidizes more rapidly than the corresponding -TOH-containing
particle. Where studied, the oxidizability of lipids decreases
proportionally with LDL's -TOH content (22,
39)
. Replenishing
in vitro depleted LDL with -TOH, but not with the
redox-inactive -tocopheryl acetate, fully restores both its original
content of vitamin E and its lipid oxidizability (22)
. These
observations are consistent with -TOH actively promoting the
initiation of LDL lipid peroxidation.
In vivo -TOH-depleted plasma and lipoproteins obtained
from a patient with familial isolated vitamin E deficiency syndrome
(42)
behave similarly to the corresponding in vitro
-TOH-depleted samples (Fig. 3
). This demonstrates that the observed requirement for -TOH in the
initiation of lipoprotein lipid peroxidation is not the result of an
in vitro artifact.
|
Switch of LDL's vitamin E from pro- to antioxidant
With high fluxes of ROO• or
•OH, the activity of -TOH in LDL switches
from pro- to antioxidant. The `switching point' for
•OH is observed at a lower flux than that for
ROO• (22)
. For
Cu2+-induced initiation of LDL lipid
peroxidation, this switching point is achieved at Cu:LDL ratios ~3
(30,
43)
. Such a switch in -TOH's action is explained readily by
TMP (19,
35)
, so that complicated explanations involving several
different mechanisms for different Cu2+:LDL
ratios (43)
are not needed. As the radical flux increases, the
incidence of termination reactions increases, thereby reducing the
extent with which -TO• can engage in and
promote lipid peroxidation. This also allows vitamin E to appear to act
as a conventional antioxidant.
In summary, although developed initially for
ROO•, TMP represents a general model describing
LDL lipid peroxidation for radical (or radical-generating) oxidants
(Table 1)
. Even though differences exist between the various oxidizing
conditions (see, for example, ref 44
for Cu2+ vs.
Fe3+), the common features are that -TOH aids
the `entry' of radicals into LDL and that during the
-TOH-containing period, -TO• is the
primary lipid peroxidation chain-carrying radical. Therefore,
elimination of -TO• (rather than
LOO•) represents a unified general
strategy to inhibit radical-induced lipid peroxidation in LDL (see
below) and other lipoproteins. Where -TO•
formation is dependent on active enzymes (soybean lipoxygenase, HRP,
MPO, and likely other heme-containing proteins), their inhibition
represents an alternative and/or supplementary approach. Nevertheless,
oxidative modification of LDL may also be achieved by mechanisms
independent of TMP and lipid peroxidation, as for hypochlorite (45)
. In
this case, effective inhibition of oxidation is not achieved by -TOH
alone and/or prevention of TMP (46)
.
|
COANTIOXIDANTS FOR -TOH ARE INHIBITORS OF TMP AND PROTECT LDL
AGAINST LIPID PEROXIDATION
|
|---|
|
TOPABSTRACTINTRODUCTIONROLE OF VITAMIN E...COANTIOXIDANTS FOR {alpha}-TOH...IS TMP RELEVANT TO...DOES INTIMAL LIPOPROTEIN...VITAMIN E SUPPLEMENTATION AND...COANTIOXIDANTS AS IN VIVO...CONCLUSIONSREFERENCES |
|---|
-TO• within an oxidizing lipoprotein
particle that permits its reaction with LDL's LH although this
reaction is kinetically unfavorable (kTMP ~
0.01–0.1 M-1 s-1) (19,
47)
. It follows that prevention of TMP is dependent on the
destruction/elimination of -TO• from the
peroxidizing particle. This may occur via a bimolecular termination
reaction (reaction 2 in Fig. 2
) or by reduction of
-TO• by a suitable agent, termed
coantioxidant (XH; reaction 5 in Fig. 2
) (48,
49)
. The latter act in
concert with -TOH first by reducing -TO•
and, second, by transferring the radical character from LDL to the
aqueous environment (reaction 6 in Fig. 2
) (49)
. Therefore, the ability
of a compound to regenerate -TOH from
-TO• alone does not equate with
coantioxidation per se. Whether regeneration of -TOH
results in LDL antioxidation depends on the fate of the resulting
coantioxidant-derived radical (X•).
X• must be relatively unreactive toward LH and
be able to rapidly exit the particle or transfer its radical character
to another species that leaves the particle (19,
29,
50)
.
Because -TOH increases the likelihood that an LDL particle will
undergo oxidation, the more vitamin E the greater the lipoprotein's
need for coantioxidants to keep its lipids protected under all
conditions. In other words, TMP and the mode of its prevention predict
that the balance of -TOH and available coantioxidants, rather than
-TOH alone, determines whether LDL lipid peroxidation occurs in
biological systems.
A number of coantioxidants have been identified, including dietary
compounds such as ubiquinol-10
(CoQ10H2),
-tocopherylhydroquinone (51)
, ascorbate, bilirubin, and the
tryptophan metabolite 3-hydroxyanthranillic acid (Table 2
) (49,
52
53
54)
. Various synthetic coantioxidants are also described in
ref 53
(Table 2)
. The coantioxidant activity of two endogenous
compounds, ubiquinol-10 and ascorbate, are described below.
|
CoQ10H2
Relative to -TOH, freshly isolated LDL contains only small
amounts of CoQ10H2, the
reduced form of coenzyme Q10 (55,
56)
.
CoQ10H2 is the first
antioxidant consumed when an ascorbate-free solution of LDL is exposed
to ROO•, transition metal ions
(Cu2+, Fe3+), activated
human neutrophils, unstimulated monocytes/macrophages, authentic
hypochlorite, singlet oxygen, and nitric oxide ± superoxide anion
radical (55,
57
58
59)
. Although
CoQ10H2 itself is an
effective lipid-soluble antioxidant (60)
, in LDL it appears to act
predominantly as a coantioxidant for -TOH. Thus, the rate of
CoQ10H2 consumption is
independent of the rate of initiating radical generation (51)
.
CoQ10H2 may eliminate
-TO• indirectly (via the semiquinone
radical), resulting in the formation of superoxide anion radicals that
may diffuse from the particle or scavenge another
-TO• (29)
.
Dietary supplementation of human subjects with coenzyme
Q10 for 4–5 days increases LDL's
CoQ10H2 content from
~0.5–0.8 to ~2.0–2.4 molecules per particle (61
62
63)
. This
efficiently enhances LDL's resistance to lipid peroxidation initiated
by ROO• or the transition metal-containing
Ham's F-10 medium (63)
. Furthermore, and perhaps most important,
coenrichment of LDL with
CoQ10H2 and -TOH
demonstrates that the coantioxidant also efficiently attenuates the
pro-oxidant activity of vitamin E (63)
seen with LDL from subjects
enriched with -TOH alone. It is not known whether
CoQ10H2 in one particle can
reduce -TO• in another LDL particle.
As the majority of human LDL contains <1 molecule
CoQ10H2 per particle, an
intriguing question is how such small levels of this or other
coantioxidants can provide significant antioxidant protection to LDL
lipids. TMP of LDL lipids proceeds via a free radical chain reaction
(19,
64,
65)
whereby 20–40 LOOH may be formed per molecule of -TOH
consumed. Each molecule of
CoQ10H2 can potentially
terminate two radical chains, and therefore may readily cause the rate
of the peroxidation to decrease by 40- to 80-fold. In addition, the
degree of inhibition decreases only with the square root of
the concentration of the coantioxidant (see Eq. XXIV in ref 19
).
Together, this offers an explanation as to why small amounts of
coantioxidants offer substantial protection against LDL peroxidation,
at least under conditions of relatively low flux of initiating
radicals. An increase in the number of
CoQ10H2 molecules per LDL
particle from <1 to >1 will also increase the resistance of the
lipoprotein toward oxidation, because it provides all
particles with a coantioxidant molecule. This may be particularly
important if cellular systems exist that maintain this coantioxidant in
the quinol and hence antioxidant form (66
67
68)
.
For human plasma, CoQ10H2
consumption during peroxyl radical-induced oxidation shows a dependency
on the type of oxidant used. For example, treatment of freshly isolated
plasma with aqueous ROO• results in the
consumption of plasma ascorbate prior to that of
CoQ10H2 (54)
. By contrast,
oxidation of plasma using a lipophilic ROO•
results in CoQ10H2 being
consumed before ascorbate (52)
. This suggests that lipophilic
coantioxidants may be used in preference to aqueous counterparts if LDL
oxidation is initiated by a lipophilic oxidant and vice
versa.
Ascorbate
Among the aqueous biological coantioxidants, the
ubiquitous ascorbate (i.e., the reduced, antioxidant form of vitamin C)
most efficiently inhibits in vitro lipid peroxidation (69,
70)
. This activity is likely due to a combination of direct radical
interception (where aqueous radicals are involved) (22)
and interaction
with vitamin E as a coantioxidant. The regeneration of -TOH from
-TO• by ascorbate, with concomitant
generation of the ascorbyl radical, is well established (48,
53,
71
72
73
74)
. Addition of ascorbate to LDL undergoing oxidation induced by
aqueous ROO• results in immediate cessation of
-TOH consumption and lipid oxidation. Upon removal of ascorbate,
consumption of -TOH and oxidation of CE resumes, attaining the same
rates as those prior to the addition of ascorbate (19)
.
The induction of LDL lipid peroxidation by treatment with
HRP/H2O2 best illustrates
various aspects of TMP described above, including a coantioxidant
effect exerted by ascorbate (31)
(Fig. 4
). Thus, in LDL undergoing such oxidation, lipid peroxidation proceeds
in the presence of -TOH with concomitant detection of
-TO•(Fig. 4)
. Addition of catalase and
either ascorbate or urate to the oxidizing system results in immediate
cessation of -TOH consumption (Fig. 4B
). Catalase
degrades H2O2, and both
ascorbate and urate eliminate compound I (not shown), the one-electron
oxidant formed by HRP/H2O2
that is responsible for initiation of LDL oxidation. Addition of
ascorbate results in immediate elimination of
-TO• (Fig. 4A
), formation of the
relatively nonreactive ascorbyl radical (not shown), and immediate
cessation of lipid peroxidation (Fig. 4C
). By contrast,
urate is not able to eliminate -TO•, and
lipid peroxidation proceeds as long as this radical is detected (Fig. 4A, C
). Thus, ascorbate is a phase transfer agent that
facilitates the export of a radical from within the lipoproteins to the
aqueous compartment and thereby prevents lipid peroxidation.
|
|
IS TMP RELEVANT TO IN VIVO LDL OXIDATION? |
|---|
|
TOPABSTRACTINTRODUCTIONROLE OF VITAMIN E...COANTIOXIDANTS FOR {alpha}-TOH...IS TMP RELEVANT TO...DOES INTIMAL LIPOPROTEIN...VITAMIN E SUPPLEMENTATION AND...COANTIOXIDANTS AS IN VIVO...CONCLUSIONSREFERENCES |
|---|
-TOH in LDL oxidation
is complex and vitamin E does not simply act as an antioxidant. Whether
TMP of lipoprotein lipids occurs in vivo is not yet known.
However, an analysis of the putative in vivo oxidants
involved in atherosclerosis, the anticipated rate of free radical
generation in biological systems, and the properties of the
subendothelial space that induce LDL oxidation (discussed below)
suggest that TMP may indeed occur.
Retention of LDL within the arterial wall is proposed to stimulate
and/or increase the likelihood of lipoprotein oxidation. Intimal
proteoglycans that bind and may `trap' LDL (75)
in the extracellular
matrix could be proatherogenic. Thus, preexposure to proteoglycans
increases LDL's sensitivity to in vitro oxidation by
Cu2+ when compared with nonexposed, native LDL
(76,
77)
. Oxidation of such LDL proteoglycan complexes also proceeds
via TMP (K. Morris and R. Stocker, unpublished results).
Alternatively, oxidation may occur before LDL enters the arterial wall.
Autoantibodies to oxidized LDL are detected in the circulation and
sometimes (78)
correlate positively with atherosclerosis. Overall,
however, a relationship between immunologically detectable oxidized LDL
and oxidative events in circulation remains tenuous (79,
80)
. In
addition, plasma contains a myriad of antioxidant defenses (reviewed in
ref 55
) to protect its lipids from oxidative damage. Indeed, LDL itself
contains a substantial proportion of the lipid-soluble antioxidants of
plasma. Oxidized lipoproteins, should they exist in plasma, are cleared
rapidly by the liver (81)
. In support of this, only nanomolar
concentrations of lipid hydroperoxides are detected in the plasma of
healthy humans, and there is little evidence that lipid hydroperoxides
are specifically associated with LDL (37)
. Furthermore, LDL in patients
with severe cardiovascular disease at best show only small signs of
oxidative alterations and levels of -TOH remain fully intact (82,
83)
. Overall, it is therefore reasonable to expect that LDL lipid
peroxidation occurs in the intimal space rather than plasma.
How LDL's lipid and protein become oxidatively modified in the
arterial wall remains unknown, although several potential oxidants have
been proposed (10)
, including 15-lipoxygenase (84,
85)
, myeloperoxidase
(86,
87)
, transition metal ions (88)
, and reactive nitrogen species
(89)
. All of these putative in vivo oxidants oxidize
isolated LDL via TMP (Table 1)
, which suggests that TMP may be relevant
in vivo.
As described above, in the absence of coantioxidants, high rates of
radical encounter by LDL will result in rapid consumption of -TOH.
Under correspondingly lower radical fluxes, -TOH in the same
lipoprotein will be consumed more slowly and substantial lipid
peroxidation will occur, so that a (clear) lag phase is not observed
(19)
. The frequency of radical encounter is determined by the rate of
radical production, the efficacy with which aqueous antioxidants that
surround lipoproteins scavenge initiating radicals, and the
concentration of lipoproteins in the intima. Judged by the amounts of
apo B per protein, LDL is approximately half as concentrated in plaque
as in plasma (90)
, whereas the concentration of aqueous antioxidants in
extracellular fluids is only marginally lower than that in plasma (91)
.
The overall increased ratio of aqueous antioxidants to LDL can be
expected to maintain radical encounters of intimal LDL low.
Unfortunately, information on the rate of radical formation in the
arterial wall is not available. However, an upper limit may be
estimated from the maximal rate of superoxide anion generation of
maximally stimulated neutrophils at blood concentration and assuming
(according to the pKa-value) that 0.5% of this
becomes converted to hydroperoxyl radical, which can initiate LDL lipid
peroxidation (92)
. The obtained value is ~20-fold less than the rate
of radical generation in the commonly used
Cu2+/LDL `oxidizability' test (25)
, as judged
by -TOH consumption, i.e., 1 x 10-8 vs.
2 x 10-7 mol L-1
min-1. Obviously such calculations are largely
speculative and should be interpreted with caution. They indicate,
however, that it is not unreasonable to assume that the radical flux to
which arterial LDL is exposed are low, particularly as extracellular
antioxidants and other targets surrounding the lipoprotein will
scavenge the majority of these radicals (22)
before they react with
LDL's -TOH. Thus, it appears reasonable that TMP is physiologically
relevant.
Principally, TMP could explain formation of oxidized lipids in lesions
if ascorbate or other coantioxidants were absent or inaccessible at the
site of lipoprotein lipid peroxidation. However, advanced lesions
contain ascorbate (93)
. It is possible that aqueous antioxidants are
depleted only at localized sites of oxidation, so that an overall
decrease in antioxidants is not observed when the whole lesion is
assessed (e.g., in plaque homogenates). Alternatively, it may be that a
substantial proportion of lesion ascorbate resides within vascular
cells and hence may not be available for extracellular antioxidation.
In contrast to ascorbate, there is some evidence that
lipoprotein-associated coantioxidants (for which a putative
microenvironment is not relevant) are no longer present in lesions.
Indeed, only the oxidized, inactive quinone forms of the two relevant
lipid-soluble coantioxidants, ubiquinol-10 and
-tocopherylhydroquinone, have been detected in advanced plaques
(93)
. Therefore, a lack of available and suitable coantioxidants
remains a plausible explanation for the occurrence of intimal lipid
peroxidation in the presence of -TOH. Future studies are needed to
verify this and to test whether (and if so, to what extent) inhibition
of intimal lipid peroxidation by coantioxidants decreases the
progression of atherosclerosis.
|
DOES INTIMAL LIPOPROTEIN OXIDATION OCCUR IN THE ABSENCE OR PRESENCE OF VITAMIN E? |
|---|
|
TOPABSTRACTINTRODUCTIONROLE OF VITAMIN E...COANTIOXIDANTS FOR {alpha}-TOH...IS TMP RELEVANT TO...DOES INTIMAL LIPOPROTEIN...VITAMIN E SUPPLEMENTATION AND...COANTIOXIDANTS AS IN VIVO...CONCLUSIONSREFERENCES |
|---|
-TOH, are actually depleted in these early events of
atherogenesis is not clearly established. There are several ways to
address this issue. First, the total vitamin E content in lesions and
individual intimal lipoproteins can be analyzed. Second the extent of
vitamin E oxidation can be determined. Last, one can investigate
whether the major lipid oxidation products present in lesions are
characteristic for lipid peroxidation occurring in the presence or
absence of -TOH. By utilizing all of these approaches, we have
obtained evidence to suggest that vitamin E remains largely intact and
that the majority of intimal lipoprotein oxidation indeed occurs in the
presence of vitamin E.
Vitamin E levels in atherosclerotic lesions
How much -TOH remains in lesion LDL after isolation may reflect
the minimal concentration of this antioxidant in intimal LDL. If
-TOH in such LDL were essentially depleted and there were
significant oxidized lipids, one could reasonably assume that lipid
peroxidation has proceeded in the absence of vitamin E. The amount of
-TOH in macrophage foam cell-rich atherosclerotic lesions is
reported to be low when expressed relative to the unesterified
cholesterol (94)
. However, atherosclerotic lesions are characterized by
excessive accumulation of unesterified cholesterol, so that this
standardization may be misleading especially for comparisons of -TOH
in lesion lipoproteins vs. plasma lipoproteins. We have chosen to
express -TOH levels relative to CE, in particular, Ch18:2
(cholesteryl linoleate). Linoleate is ~27-fold more prone to
oxidation than cholesterol (95,
96)
and Ch18:2 is the major oxidizable
substrate in LDL. Thus, the ratio of -TOH to Ch18:2, should indicate
whether lesion lipoproteins are equipped with sufficient vitamin E to
protect their lipids against oxidative attack.
Using this approach, -TOH levels in homogenates of advanced human
lesions are either little altered or in fact elevated when compared
with plasma (93)
. Futhermore, the apo B-containing low density fraction
(LDF) derived from homogenates of advanced lesions also contains normal
levels of -TOH compared with plasma LDL (Table 3
). Despite this, lesions and lipoprotein fractions isolated from such
tissue also contain large amounts of oxidized lipids (93,
97,
98)
. The
coexistence of normal -TOH levels and substantial lipid peroxidation
is consistent with TMP, yet is difficult to explain if -TOH were to
act in the classical mode.
|
Oxidation products of vitamin E in atherosclerotic lesions
Oxidation products of -TOH arise from reactions between
-TO• and other radicals to primarily form
8a-substituted tocopherones (99)
, i.e., NRP in Fig. 1
. These may
subsequently hydrolyze to form the relatively stable oxidation product
-tocopheryl quinone (-TQ). Alternatively, -TQ may be formed
directly via two-electron oxidation of -TOH by oxidants such as
hypochlorite and peroxynitrite (100)
. Oxidation of -TOH by
ROO• also gives rise to epoxytocopherones and
epoxyquinones (99)
. All of these oxidation products can be detected in
advanced human plaques and, to a lower extent, in less severe lesions
(A. C. Terentis, J. A. Burr, D. C. Liebler, and R.
Stocker, unpublished results). The mole ratio of -TOH to total
tocopherol oxidation products ranges from 4 to 9 in advanced and
intermediate lesions, respectively. These findings are inconsistent
with the notion that intimal lipid oxidation progresses beyond -TOH
depletion. They also indicate that the extent to which intimal
-TO• engages in radical termination
reactions is limited. The results further suggest that -TOH
participates in the formation of oxidized lipids more so than it
prevents lipid peroxidation, because the ratio of oxidized lipid to
oxidized -TOH in advanced lesions is 5. If the vitamin were
to primarily engage in lipid antioxidation, we would expect this ratio
to be 1. However, such conclusions should be drawn with caution
as they assume that the -TOH oxidation products measured represent
the overall extent of vitamin E oxidation. Though there is no
information available with regard to this, there is evidence that the
products measured are indeed the major oxidation products (99,
101,
102)
.
Chemical evidence for intimal lipid peroxidation occurring in the
presence of vitamin E
There are several explanations for the coexistence of oxidized
lipids and the relatively normal level of -TOH in lesions (93)
. For
example, lipid peroxidation may occur when the vitamin was temporarily
depleted and subsequent replenishment of -TOH might follow, while
the oxidized lipids remain in the lesion. Unfortunately, this
possibility may be impossible to confirm when simply examining the
amounts of oxidized lipids and antioxidants in whole lesions or
lipoproteins derived from them. By contrast, analysis of the
distribution of specific isomers of the primary lipid oxidation
products in lesions can yield direct information as to whether
oxidation has occurred in the presence of -TOH.
Enzymatic oxidation of polyunsaturated fatty acid, e.g., by
lipoxygenases, yields highly characteristic regio- and stereospecific
products. In contrast, random, free radical-induced polyunsaturated
fatty acid oxidation generates a complex array of primary products, the
distribution of which is influenced by lipophilic hydrogen donors such
as -TOH (103,
104)
(but not ascorbate and urate). In the presence of
-TOH, oxidation of both free and esterified lipid (in either
homogenous solution or emulsion) yields primarily cis, trans
(Z,E) hydro(pero)xide isomers (Fig. 5
). In the absence of -TOH, the thermodynamically more stable,
trans, trans (E,E) isomers are primarily formed
(Fig. 5)
. This specificity also holds true for lipoprotein lipid
peroxidation (105,
106)
. Thus, a preferential formation of
cis, trans (Z,E) isomers indicates
lipoprotein lipid peroxidation occurring primarily in the presence of
an effective hydrogen atom donor, such as -TOH.
|
Hydroxy cholesteryl linoleate (Ch18:2-OH) is the predominant lipid
oxidation product in atherosclerotic lesions (93,
107)
. In the various
lipoprotein density fractions isolated from homogenates of advanced
carotid lesions, cis, trans (Z,E) isomers of
Ch18:2-OH are also more abundant than the corresponding trans,
trans (E,E) isomers (Fig. 6
). This suggests that most lipid peroxidation does occur in the presence
of -TOH rather than at putative sites of localized vitamin E
depletion. Further support for the occurrence of vascular lipoprotein
lipid oxidation in the presence of -TOH, and perhaps TMP, can be
obtained from a study reported by Killion et al. (108)
. Although not
reported, the author's data show a strong positive correlation between
tissue vitamin E and lipid oxidation products in human tissues with
various degrees of atherosclerosis (Fig. 7
). Thus, vessels containing the largest amount of vitamin E also
contained the largest amounts of lipid oxidation products.
|
|
In summary, lesion homogenates and lesion lipoproteins contain
concentrations of -TOH comparable to those in human plasma, with
only a small proportion of the vitamin being oxidized, along with
substantial amounts of oxidized lipid, the majority of which appears to
have been formed in the presence of vitamin E. In other words, only a
minor proportion of lesion LDL appears to be oxidized beyond -TOH
depletion. Further, the amount of oxidized lipid correlates positively
with the level of tissue vitamin E. Studying the -TOH-containing
stage of LDL oxidation is thus likely to be relevant for in
vivo lipoprotein lipid oxidation and atherogenesis, and the TMP
model may indeed be relevant for this.
|
VITAMIN E SUPPLEMENTATION AND ATHEROSCLEROSIS |
|---|
|
TOPABSTRACTINTRODUCTIONROLE OF VITAMIN E...COANTIOXIDANTS FOR {alpha}-TOH...IS TMP RELEVANT TO...DOES INTIMAL LIPOPROTEIN...VITAMIN E SUPPLEMENTATION AND...COANTIOXIDANTS AS IN VIVO...CONCLUSIONSREFERENCES |
|---|
Studies in humans
Observational studies link lowered incidence of diseases such as
coronary artery disease and cancer with increased intakes of dietary
vitamins, particularly vitamins C and E. Vitamin E supplementation is
proposed to be particularly beneficial in atherosclerosis, as
epidemiological studies show an inverse relationship between coronary
artery disease and plasma vitamin E levels (109)
. However, the
literature reports contradictory findings, even among the largest
studies. Thus, in the health professional studies, a reduced risk for
coronary artery disease with vitamin E supplements was observed for men
(39,910 participants) (110)
and women (87,245 participants) (111)
. By
contrast, the Iowa study found little evidence that supplemental
vitamin E is associated with a decreased risk of death from coronary
artery disease in 34,486 postmenopausal women (112)
. Short-term
treatment with vitamin E appears to have no effect on coronary artery
disease progression (113)
. Prospective randomized trials have also
failed to resolve whether vitamin E supplementation ameliorates
cardiovascular disease in humans. One study (29,133 subjects), with a
mean follow-up of 6 years, showed no effect on coronary artery disease
(114)
. A comparatively smaller study (2002 subjects) reported a
reduction only in nonfatal myocardial infarction with vitamin E
supplementation; other end points were unaffected (115)
.
Vitamin E supplementation in anim
, 
) and after (•, 
) vitamin E supplementation was oxidized
by aqueous ROO• under identical conditions and both
) was added. Levels of
, n=12), aorta (
,
n=26), and peripheral artery (