(Received for publication, August 29, 1994; and in revised form, December 6, 1994)
From the
Oxidation of low density lipoprotein (LDL) may be involved in
the development of atherosclerosis. It has recently been shown that
-tocopherol (
-TOH) can act either as an antioxidant or
prooxidant for isolated low density lipoprotein (LDL). In the absence
of an effective co-antioxidant,
-TOH is a prooxidant and this
activity is evidently due to reaction of the
-tocopheroxyl radical
(
-TO
) with the LDL's polyunsaturated lipids (Bowry, V.
B., and Stocker, R.(1993) J. Am. Chem. Soc. 115,
6029-6045). Herein we examined the effectiveness of selected
natural and synthetic radical scavengers as co-antioxidants for inhibiting peroxyl radical-induced peroxidation in LDL that is
devoid of ubiquinol-10 (an effective endogenous co-antioxidant) but
still contains most of its natural complement of
-TOH. Various
quinols, catechols, and aminophenols, as well as ascorbate, 6-palmityl
ascorbate, and bilirubin, were very effective co-antioxidants under our
test conditions, whereas ordinary phenolic antioxidants, including
short-tailed
-TOH homologues, were less effective. Reduced
glutathione, urate, and Probucol were ineffective. These findings
confirm that the prooxidant activity of
-TOH in LDL relies heavily
on the segregation of water-insoluble radicals (particularly
-TO
) into individual LDL particles, since it was those
compounds that are expected to either irreversibly reduce
-TO
or accelerate the diffusion of radicals between particles which most effectively inhibited the tocopherol-mediated
phase of peroxidation. Theoretical and practical implications of these
findings are discussed, as is their relevance to the ``LDL
oxidation'' hypothesis of atherogenesis.
The oxidation of human low-density lipoprotein (LDL) ()in the artery wall is thought to be an initiator of
atherosclerosis(1) . Evidence from in vitro, in
vivo, and epidemiological studies (2, 3, 4, 5, 6) suggests
that ``oxidatively modified'' LDL is recognized and scavenged
by macrophages, leading to transformation of the latter into lipid
laden cells, which in turn contribute to the formation of fatty streaks
and eventually plaque in the artery wall. If LDL oxidation helps to
initiate atherosclerosis, it follows that increasing the resistance of
LDL to oxidation will mitigate or even prevent atherosclerosis. This
idea is supported by studies showing that supplementation with
antioxidants can attenuate atherosclerosis in animal models (7, 8, 9) and possibly in
humans(10, 11) .
Free radical-mediated aerobic
oxidation (peroxidation) of LDL has thus become a widely studied model
for atherosclerosis research(12, 13, 14) . A
commonly used method for oxidatively modifying LDL is to incubate the
lipoprotein with a redox catalyst (e.g. copper) or with cells
in a transition metal-containing cell culture
medium(13, 14) . With relatively high concentrations
of a strong redox oxidant, such as copper ions, little lipid
hydroperoxide (LOOH) is formed until -tocopherol (
-TOH) and
the minor antioxidants have been depleted. Following this apparent
``inhibited'' phase lipid peroxidation may enter a rapid
``uninhibited'' phase, toward the end of which the epitope of
LDL's apolipoprotein B-100 may become ``modified,'' i.e. recognizable to the scavenger or oxidized LDL receptors
of monocyte/macrophages(1, 15, 16) . The
``susceptibility'' of LDL toward oxidative modification is
gauged by the duration of the inhibited phase of peroxidation, the rate
of peroxidation during the uninhibited phase, and the maximal amount of
peroxidized lipids accumulated(14) .
The relatively low rate
of LOOH formation in the inhibited phase of copper-initiated LDL
oxidation has led to the conclusion that -TOH (the major and most
reactive peroxyl radical scavenger in LDL) is an effective antioxidant
for LDL in vitro and that it protects LDL from oxidation in vivo(14) . In view of this, we were surprised to
find recently that the type of activity exhibited by
-TOH in LDL
actually depended on the time taken for the
-TOH to be consumed.
Thus, a rapid flux of peroxyl radicals from an azo initiator produced
relatively little LOOH in LDL in the brief period of
-TOH
consumption, whereas a comparatively mild flux of initiating peroxyl
radicals could led to LOOH formation that was faster during
than following the period of
-TOH consumption. Likewise, whereas
the rapid oxidation of LDL with high concentrations of copper ions
produces little LOOH in the brief (
0.4 h) period of
-TOH
consumption(14) , the low concentrations of copper and iron
ions in the mildly oxidizing F-10 cell culture medium afford a faster
maximal rate of LOOH formation during than following the period
(
30 h) of
-TOH consumption(17, 18) .
Remarkably, in the latter case up to 50% of the polyunsaturated fatty
acids in the LDL lipid were oxidized before
-TOH was
depleted(17) , clearly demonstrating that substantial LOOH
formation can occur in
-TOH-containing LDL.
These and other
unusual findings about LDL peroxidation in the presence of -TOH (17, 19, 20, 21) have led us to
conclude that
-TOH in LDL can have prooxidant activity arising
from a reaction between the
-tocopheroxyl radical (
-TO
)
and the active bisallylic methylene groups of polyunsaturated fatty
acids (LH) of the LDL.
We have deduced from kinetics measurements that this normally
slow reaction (k
0.1 M
s
) can lead to fairly
rapid peroxidation in LDL because in the absence of an effective
co-antioxidant the highly water-insoluble
-TO
radicals are
isolated from each other by the aqueous medium of the LDL suspension
(see below)(21) .
Herein we examined the effect of selected
biological and synthetic radical scavengers on the rate of LOOH
formation in the -TOH-containing phase of LDL peroxidation induced
by lipid- and/or water-soluble organic peroxyl radicals (ROO
).
The dramatic co-antioxidant effectiveness of ascorbate
(AscH
), quinols, and certain other radical scavengers
in this study is shown to arise neither by sparing of
-TOH nor by
competitive scavenging of peroxyl radicals, but by attenuation of the
prooxidant activity of
-TOH, most likely through elimination of
-TO
from LDL particles.
The
methods used for oxidizing LDL and analyzing the oxidized and
non-oxidized lipids have been
described(17, 19, 20, 24) . Most of
the non-water-soluble antioxidants were added in methanol (<1%, v/v)
to LDL and pre-incubated for 10 min before adding the azo-compound, a
procedure that caused no turbidity or precipitation of the protein at
the concentrations specified in the tables and figures. Incorporation
of these antioxidants into the LDL was almost instantaneous, as judged
by disappearance of turbidity In contrast, incorporation of the highly
lipophilic compounds -TOH, C13OH, and C15OH required preincubation
in plasma, with the extent of incorporation being determined by HPLC
following ultracentrifugal isolation of the LDL and gel filtration to
remove any non-incorporated material (see (17) for
-TOH).
We defined the ``effectiveness'' of a co-antioxidant by
the relative amount of Ch18:2OOH formed with versus without
the added inhibitor in the presence of -TOH but in the absence of
CoQH
, which is itself an effective co-antioxidant for
native LDL(19, 26) . The amount of inhibitor added was
[inhibitor]
= [
-TOH]
= 10 µM, and the amounts of Ch18:2OOH formed
was determined at a time point corresponding to 20% consumption of
-TOH in the ``without'' sample (
)(see
``Results''). Lipids from the core and surface regions of the
LDL particle oxidize in a uniform manner, with LH groups in the
phospholipids being consumed at about the same fractional rate
(-d[LH]/dt)/[LH]) as LH groups from
the lipid core(17) . The rate of conversion of Ch18:2 (which
contains
50% of LDL's total LH groups) into Ch18:2OOH thus
gauges the total rate of LDL lipid peroxidation (noting that
[LOOH]
2
[Ch18:2OOH]; (17) ). An LDL sample supplemented with butylated
hydroxytoluene (BHT) at a concentration equimolar to that of endogenous
-TOH was included as internal control sample in each set of
experiments to check for the effect of potential (inadvertent)
variations in the lipoprotein composition and oxidation conditions. The
variation in Ch18:2OOH formation was rate
BHT/rate
= 15
± 5% (mean value ± S.D. of 13 different experiments with
LDL from 13 different donors) for AAPH-induced oxidation and 4.8
± 2% (n = 9, with 9 different LDL preparations)
for AMVN-induced oxidation. This is comparable to the random analytical
uncertainties, rate
BHT/rate
= 16 ± 2% for a
triplicate analysis of one sample. At the time points indicated,
aliquots of the LDL samples were withdrawn, extracted, and analyzed for
antioxidants and Ch18:2OOH(18, 24) .
For AAPH, R =
(CN
H
)(CH
)
C
;
for AMVN, R
=
(Me
CHCH
)(CH
)(CN)C
. In the
absence of an added co-antioxidant, the ensuing LDL peroxidation
exhibits three quite distinct phases in which the rate of LOOH
formation: (i) is very slow until the endogenous CoQH
present in freshly prepared LDL is consumed, (ii) accelerates
markedly (20-40-fold) to a relatively high value immediately
after CoQH
consumption before decreasing to second minimum
just as the last few percent of the
-TOH are being depleted, and
(iii) accelerates again following consumption of all recognized
antioxidants in the LDL (see, e.g., the control in Fig. 1A). Under all but the mildest oxidation
conditions, the first phase is relatively brief because the amount of
CoQH
in LDL is small (i.e. CoQH
/
-TOH
0.05) even when special
precautions are taken to prevent its autoxidation during LDL
isolation.
Extensive LOOH formation occurs during the phase
of
-TOH consumption if a low concentration of AAPH or AMVN, or
F-10 medium, is used to induce LDL lipid
peroxidation(17, 18, 19, 20) . The
amount of LOOH formed and the maximal rate of phase ii lipid
peroxidation are greater in
-TOH-enriched than normal LDL. These
and other observations have led us to conclude that
-TOH can act
as a pro-oxidant for LDL, and that the mechanism of LDL peroxidation is
quite different in the presence of
-TOH than in its absence (see
below).
Figure 1:
Inhibition of AAPH-initiated
peroxidation in LDL. LDL containing 6.6 µM -TOH, 800
µM Ch18:2, < 0.1 µM CoQH2, 0.4 µM Ch18:2OOH, and 4 mM AAPH was supplemented with the
specified amount of antioxidant (e.g. 10 BHT = +10
µM BHT), and its peroxidation at 37 °C was monitored
by HPLC(20, 24) . Panel A shows
-TOH
consumption (squares) and Ch18:2OOH formation (circles) during the inhibited and uninhibited phases of LDL
oxidation in the absence (brokenlines) and presence
of 2 µM BHT (solidlines). PanelB represents an expanded view of the early Ch18:2OOH
formation rates during the inhibited period (see Table 1for
structures). The results shown are representative of 3-15
separate experiments.
The fact that strong
co-antioxidants like 3HAA or 2AP (Fig. 2) were often consumed
more rapidly than -TOH, and that the peroxidation rate accelerated
after their disappearance, meant that apparent stoichiometric factors (n
) for some of the compounds could be estimated
from the inhibition period, t
, before onset of
the more rapid tocopherol-mediated peroxidation (TMP). Defining n
= R
t
/[inhibitor], where R
is the radical initiation rate measured via the
rate of
-TOH consumption in native LDL (assuming n = 2.0 for
-TOH; (27) ), we calculated n
2.8, 2.2, 3.0, and 2.8 for 2AP, 3HAA,
BHT, and TBHQ, respectively. The relatively large n
values for 2AP and 3HAA are consistent with our earlier
observations(28) . n
values give a
practical index for LDL protection; they assess the relative capacities
of co-antioxidants to delay the onset of untrammeled phase ii
peroxidation in LDL, which may be quite different to their contribution
to the total radical trapping capacity (as defined in (29) ) of
the supplemented lipoprotein.
Figure 2:
Antioxidation of LDL by the aminophenols
2AP and 3HAA. Conditions were as per Fig. 1with LDL containing
16 µM -TOH, 1800 µM Ch18:2, < 0.1
µM CoQH
, 0.2 µM Ch18:2OOH, and 5 mM AAPH.
-TOH consumption
(squares) and Ch18:2-OOH formation (circles) were
monitored in the absence (brokenlines) and presence (solidlines) of 10 µM either 3HAA or
2AP. Effective inhibition times (t
) (see text),
estimated from the maximum slope tangents to the plots, are indicated
by arrows. The results shown are representative of three
separate experiments.
From the above and other studies, it
became evident that the rate-lowering effect of a co-antioxidant
([Ch18:
2OOH]/[Ch18:2OOH]
)
was independent of initiator concentration and incubation period,
provided that neither
-TOH nor the co-antioxidant was
substantially depleted. We therefore examined a wider selection of
co-antioxidants using carefully matched and internally controlled
conditions, described under ``Experimental Procedures.''
Under this protocol, the effectiveness of the antioxidants depended
very little on whether water-soluble AAPH or lipid-soluble AMVN was
used to initiate LDL oxidation (Table 1). Thus, by segregating
compounds according to their reactive functional groups, we were able
to describe a number of general and clearcut trends in the data, as
enumerated below.
The highly active phenolic antioxidant -TOH can interrupt
this radical chain by reacting with peroxyl radicals to afford LOOH and
the relatively unreactive
-TO
.
The latter eventually combines with a second radical to afford non-radical products (NRP),
thereby destroying two radical species and terminating two potential radical chains. This scheme (Reactions 3-6) adequately explains the products and kinetics of inhibited peroxidation for homogeneous solutions of lipids containing low concentrations of a phenolic or quinolic antioxidant(27) . The effectiveness of a radical scavenger for lowering the rate of LOOH formation via this mechanism is determined solely by its peroxyl radical reactivity.
Most compounds in Table 1are kinetically much weaker radical
scavengers than -TOH(27) . The fact that many of these
compounds strongly inhibited peroxidation in
-TOH-containing LDL
is thus contrary to expectations for a peroxyl radical-scavenging
mechanism (37) . That is, (a) the most reactive
hydrogen donor should be consumed first, and (b) the
total radical ``trapping rate'' of the mixture should simply be the sum of the component trapping rates. In contrast,
the addition of micromolar amounts of BHT to LDL markedly slowed the
rate of both
-TOH consumption and LOOH formation (Fig. 1),
even though BHT is 200-fold less reactive than
-TOH toward peroxyl
radicals and
-TOH is consumed before BHT in a homogeneous
system(38) . The fact that AscH
caused such a
large reduction in LOOH formation for AMVN-initiated LDL (Table 1) is even more at odds with conventional paradigms
because AscH
(in the aqueous phase of a lipid
dispersion) is a very weak inhibitor of LOOH formation initiated by
lipophilic peroxyl radicals in liposomes and
micelles(39, 40) .
The problems revealed above are
eliminated if it is assumed that the main LOOH forming mechanism in LDL
is not a peroxyl radical chain at all but, rather, the radical chain
sequence based on , which we have termed TMP (see Fig. 3). In essence, peroxidation is initiated in an LDL
``particle'' (17) when an initiating radical,
ROO, reacts with
-TOH to generate a single
-TO
,
which, being too hydrophobic to escape the LDL particle readily, must
``wait around'' for a second ``initiating'' radical
(ROO
) to strike the particle. This ``waiting period''
may be many min under our conditions of low rates of radical
generation(20, 21) , thus providing the
-TO
with ample time to generate several molecules of LOOH via and (Fig. 3)(17, 20, 21) .
Figure 3:
Tocopherol-mediated peroxidation (TMP) in
LDL. A TMP chain is initiated in an LDL particle by ROO (e.g. from ) and continues until a second
ROO
reacts with the particle (see text). Rate constants shown are
those reported for organic solutions (17, 21) . Note
however that lower values, k
8
10
M
s
and k
3
10
M
s
are reported
for liposomes(37) .
Effective inhibition of the early stages of LDL oxidation must
therefore depend on elimination of -TO
from the LDL particle
before this radical can react with LH. AscH
inhibits
LOOH formation in LDL because it reacts rapidly with
-TO
to
regenerate
-TOH(41) , thereby ``exporting'' the
radical center into the aqueous medium (21) (Fig. R7).
Figure R7: Reaction 7.
The charge and thermodynamic stability of the ascorbyl radical (Asc
ensure that it would terminate in the aqueous phase (e.g.k
3
10
M
s
) (42) rather than back-reacting with
-TOH to re-initiate
TMP. Since the antioxidation results from a simple competition between and Fig. R7, the kinetic chain-length (R
/R
) in LDL initiated with
lipophilic peroxyl radicals is given by ,
where [LH] is the concentration of LH
groups within the LDL particle (cf.(17) ). and our AMVN data therefore give a lower limit for k
under the test conditions; i.e.R
/R
(0.03
pM/s)/(0.15 pM/s)
0.2 implies k
> 5
10
M
s
(based on k
[LH]
0.1
s
; (21) ). In liposomes k
= 2
10
M
s
has been
measured (37) .
In contrast to AscH, data
from homogeneous phase studies indicate that most phenolic antioxidant
radicals (X
) (whether they are produced directly via
reaction with aqueous peroxyl radicals or indirectly via reaction of
-TO
with the antioxidant XH, Fig. R9), would react
rapidly with
-TOH to (re)generate
-TO
(Fig. R9).
XH would therefore have little effect on the LDL oxidation rate unless
X
can escape the particle (Fig. R10) before
back-reacting with the
-TOH ( Fig. R9and Fig. R10).
Figure R9: Reaction 9.
Figure R10: Reaction 10.
Even if radical export does take place, however, it will not lead to a reduction in the peroxidation rate unless the diffusing radical reacts with a peroxidizing particle (Fig. R12) rather than with a non-peroxidizing particle (Fig. R11).
Figure R12: Reaction 12.
Figure R11: Reaction 11.
This
``radical-shuttling'' mechanism neatly explains the trends
seen in the Table 1and Table 2since the antioxidant
effectiveness of XH depends on (i) its reactivity toward
-TO
, (ii) the rapidity with which X
escapes the
particle (ampiphilicity), and (iii) the relative rate at which an
``escaped'' radical, (X
)
, reacts
with non-oxidizing versus oxidizing particles (see
``Appendix'' for a more detailed analysis).
This study concerns the inhibition of free radical
peroxidation (LOOH formation) in LDL that is devoid of CoQH but which still contains normal amounts of
-TOH. Although
our list of co-antioxidants is by no means exhaustive, it is clear from
the selected examples that the antioxidation of LDL exposed to a slow
flux of peroxyl radicals follows definite trends, which can be readily
explained by mechanisms that inhibit TMP (Fig. 3) via the
elimination of
-TO
(Reactions 7-12). Our experimental
findings and kinetic analysis for
``
-TO
-inhibited'' LDL peroxidation suggest that
the reactivity, size, and water solubility of the antioxidant and its
radical are all relevant for suppressing TMP (see above and
``Appendix''). On this basis, we note the following.
(i)
Among the low molecular weight phenolic antioxidants, those with the
highest peroxyl radical reactivity tended to be the most
active co-antioxidants for LDL. This is because phenols with high
peroxyl radical reactivities usually also have high reactivities toward
phenoxy radicals, including -TO
(see (43) ), thereby
favoring Fig. R10. Accordingly, the activating paramethoxy group
in DBHA makes it both a more active radical scavenger and
2-3-fold more effective than BHT in preventing radical-induced
LOOH formation in LDL. Pyrroloquinoline quinone was inactive in our
test because it lacks a good hydrogen donor group (weak N-H or
O-H bond), whereas the reduced nitroxide TDPHI is extremely
active, probably because of the exceptional weakness of its NO-H
bond (i.e. from bond strength data,
H
(alkyl
NO-H +
-TO
)
-8 kcal/mol).
(ii) Compounds that react rapidly
with -TO
are not expected to inhibit TMP unless the radical
formed can escape the LDL particle (Fig. R10) before
back-reacting with
-TOH (Fig. R9). A rapid rate of radical
escape accelerates the ``shuttling'' of radicals between
particles and hastens radical termination (via Fig. R11).
Accordingly, low molecular weight ampiphilic compounds had greater
antioxidant activity for LDL than did bulkier, hydrophobic species of
similar type and radical reactivity. The stronger antioxidation
observed by the ``short-tailed'' compared to the
``long-tailed'' homologues of
-TOH (Table 2)
demonstrates that we are dealing with a diffusion effect and not with
the replacement of LH-reactive
-TO
with a less LH-reactive
antioxidant radical since the reactivity of a homologue radical would
not differ appreciably from that of
-TO
. The importance of
inter-particle diffusion is clearly manifested in the activities of the
-tocopherol homologues, with the effectiveness order HC1OH >
C1OH > C5OH > C11OH > C13OH > C15OH
-TOH
(``C16OH'') (Table 2) being the same order that one
would predict for their relative water solubilities, and with the least
water-soluble species (C15OH and
-TOH) actually having a net
pro-oxidant effect on AMVN-initiated LDL oxidation (cf. (17) ). The fact that HC1OH (R = CH
OH, Table 2) is significantly more effective than the lower molecular
weight C1OH (R = CH
) confirms that inter-particle
diffusion is the key factor for this chromanol series rather than
intra-particle mobility, as has been suggested by Niki and
co-workers(44) , especially since hydration of the extra
hydroxy group on HC1OH is likely to compound any (intra-particle)
``mobility'' difference between C1OH and HC1OH.
(iii) An
antioxidant radical that escapes an LDL particle may either react with
-TOH (to re-initiate TMP) or with
-TO
(to destroy two
radicals and thereby terminating two TMP chains). The marked
superiority of quinols and aminophenols over similarly constituted
monoprotic phenols (Table 1) might thus be due to a high selectivity by semiquinone-type radicals for the latter
terminating reaction (Fig. R10) since semiquinone radicals are
strongly reducing species that can undergo rapid termination reactions
of the type ``QH
+ Y
Q + YH''.
Any tendency for the semiquinone radical to deprotonate and/or to react
with O
would probably enhance this effect because the
product radicals (Q or O
) are likely to
be both more water-soluble and more reducing than the parent
(QH
). In order to distinguish deprotonation from a direct
termination selectivity, we examined DBH
(the
``dibenzyl dimer'' of C1OH), since its radical (DBH) very
rapidly reacts with
-TO
, (
)but is unlikely to
deprotonate or react with oxygen. The observed high effectiveness of
DBH
, relative to say C1OH, confirmed that a rapid cross
termination rate enhances anti-TMP activity.
Ascorbic acid, either
as water-soluble AscH or as the oil-soluble palmityl
ester (6PAH
), exhibited remarkable anti-TMP activity. This
is consistent with the observation that in human plasma the combination
of AscH
(10-50 µM) and
LDL-associated
-TOH fully protect the lipoprotein's lipids
from oxidation by lipid-and water-soluble peroxyl
radicals(45) . We deduce from our data and from bulk phase
studies (46) that AscH
prevented lipid
peroxidation in LDL by rapidly and irreversibly converting
-TO
into a more inert radical that either reacts with oxygen
(to produce O
) or undergoes
radical-radical termination in the aqueous medium ()(35, 39, 41) . (
)Although AscH
may scavenge some or even
most water-soluble ROO
radicals from AAPH in the aqueous phase
before they initiate LDL peroxidation(47) , the contribution of
this to its LDL antioxidant activity cannot be deduced from the data
because AscH
(or 6PAH
) eliminated
virtually all LOOH formation in AAPH- and AMVN-initiated LDL under the
standard test conditions. In any event, our previous results with
aqueous peroxyl radical scavenger urate have shown that the scavenging
of even a large proportion of initiating radicals in itself is not
necessarily sufficient to slow the rate of LOOH formation in LDL (see
the ``urate paradox'' in (17) ).
Strong inhibition
of LOOH formation in AMVN-initiated LDL by AscH implies that TMP was virtually the only peroxidation pathway
operating under our conditions. That is, the fact that AscH
eliminated the LOOH formation induced by a lipid-soluble
ROO
indicates the LOOHs must have been formed via direct reaction
of
-TO
(which can be eliminated by
AscH
/AscH
; (39) and (41) ) rather than via reactions of the lipid with ROO
or
LOO
(which are not eliminated by
AscH
/AscH
; (39) ).
Under our
test conditions, Probucol neither inhibited LOOH formation nor spared
-TOH in the ``TOH-inhibited'' phase of AAPH- or
AMVN-initiated peroxidation. This is consistent with recent data by
Gotoh et al.(48) , which clearly show that Probucol
had almost no effect on the rate of LOOH formation in AAPH or
AMVN-induced peroxidation as long as
-TOH was present. This is
hardly surprising according to the TMP model since Probucol is a
relatively weak antioxidant for homogeneous lipids (48) and,
perhaps more importantly, it is very insoluble in water (which makes
its radical less likely than low molecular weight phenoxyls to diffuse
from an LDL particle before back-reacting with
-TOH). Probucol
does, however, inhibit the post-
-TOH phase of oxidation, thereby
increasing LDL's resistance toward oxidative modification in a
copper oxidation test(48) . Whether this activity explains
Probucol's effectiveness as anti-atherosclerotic drug independent
of its cholesterol-lowering activity (10) remains unclear (see, e.g., (49) ). We speculate that the 4,4`-bisphenolic
oxidation product (50) would have greater anti-TMP activity
than the parent Probucol. As the bisphenol is present at relatively
high concentration in LDL of Probucol-treated rabbits, it may
contribute to the anti-atherogenic activity.
Bilirubin is less
effective than -TOH in preventing azo-initiated peroxidation in a
lipid solution or liposome dispersion under air(51) . It
therefore seems likely that a combination of
-TO
reactivity (31) and perhaps high termination selectivity (see iii on
previous page) makes this pigment an effective co-antioxidant for LDL
(see (31) for detailed discussion). Since BR is present
throughout most of the body and is constantly being replenished via the
breakdown of heme, BR could be an important LDL antioxidant in vivo(50, 52) . This is consistent with a recent study
suggesting low bilirubin levels to be associated with an increased risk
for development of atherosclerosis(53) .
With regard to the
LDL oxidation theory for atherosclerosis(1) , the inhibition of
LOOH formation in the early phase of LDL peroxidation may well be
critical to its overall antioxidation since circulating LDL is
virtually LOOH-free(19, 54) , and small amounts of
LOOH can cause rapid initiation and damaging oxidation of LDL in the
presence of redox catalysts(55) . Moreover, analysis of human
atherosclerotic lesion material for its antioxidant content has
revealed the presence of comparatively (to human plasma) high
concentrations of -TOH, whereas CoQH
could not be
detected. (
)Thus, the CoQH
-free LDL used for the
antioxidation studies here may be biologically relevant, particularly
if in vivo oxidation was indeed initiated by very mild flux of
free radicals or weak redox oxidant(s).
In conclusion, our TMP model
of LDL oxidation and antioxidation presents a novel concept of how
lipids in -TOHcontaining LDL can be converted into their
hydroperoxides and how small amounts of an effective co-antioxidant
would inhibit such oxidation. In light of the oxidation theory of
atherosclerosis, our model may therefore provide the basis for the
rational design of an efficient LDL antioxidant and, hence, potential
anti-atherosclerotic drug.
The kinetics of the LDL peroxidation inhibited via reactions
9-12 can be analyzed if it is assumed that: (a)
diffusion is relatively ``free,'' (b) critical
reinitiating and terminating reactions take place within the
LDL's lipid compartment, (c) all diffusing radicals are
formed by Fig. R10, and (d) the only significant
terminating reaction is between X and -TO
radicals (since
neither
-TO
nor LOO
diffuse readily between particles; (17) ). In this situation the concentration of X is defined by
the equilibrium of Fig. R9.
Since the flux of diffusing radicals R = k
[X] (Fig. R10), we find
The rate of termination will then depend on the proportion of
these diffusing X, which react with
-TO
(Fig. R12) rather than with
-TOH (Fig. R11), i.e.:
Steady-state radical concentrations require R = R
, so becomes
Combining with Rinh = k
[LH][
-TO
] (see ), we finally obtain the inhibited rate of lipid
peroxidation:
A co-antioxidant effectiveness factor C is
thus defined that depends on the forward rate of Fig. R9, the
size and water solubility of X
relative to the tendency for
X
to back-react with
-TOH (Fig. R9), and the rate of Fig. R12relative to Fig. R11(which might be estimated
from the corresponding homogeneous phase rate ratio). implies that the degree of inhibition will fall off as
the square root of the inhibitor concentration. The predicted
``R
([initiator]/[inhibitor])
''
relationship of / has been experimentally
observed for BHT (17) and HSA-BR (31) in
AAPH-initiated LDL and has now been verified for TBHQ, 3HAA, and 2AP
(data not shown).