(Received for publication, December 5, 1994)
From the
Cell-mediated oxidative modification of human low density
lipoprotein (LDL), most likely an important early step in
atherosclerosis, requires redox active metal ions such as copper or
iron. We have previously shown that iron-dependent, in contrast to
copperdependent, oxidative modification of LDL requires superoxide, a
physiological reductant. In the present study, we sought to explain
these discrepant results. LDL was incubated at 37 °C with
Cu (10 µM) and bathocuproine (BC, 360
µM), an indicator molecule which specifically complexes
Cu
, but not Cu
. In a time- and
concentration-dependent manner, LDL reduced Cu
to
Cu
. An LDL concentration as low as 10 µg of
protein/ml (about 20 nM) reduced about 7 µM Cu
within 1 h of incubation. Complexation of the
Cu
formed under these conditions with BC significantly
inhibited oxidative modification of LDL, as assessed by agarose gel
electrophoresis. Preincubation of LDL with N-ethylmaleimide
had no effect on the rate and extent of Cu
reduction
nor LDL oxidation, indicating that free sulfhydryl groups associated
with apolipoprotein B are not involved. Addition of either superoxide
dismutase or catalase or increasing the
-tocopherol content of LDL
from 11.8 ± 3.0 to 24.4 ± 2.8 nmol/mg of protein also had
no significant effect on the kinetics of Cu
reduction
by LDL. In contrast, incubation of LDL with
Fe
-citrate (10 µM) and the indicator
bathophenanthroline (BP, 360 µM) resulted in no
significant Fe
formation, even at LDL concentrations
as high as 200 µg of protein/ml. However, incubation of LDL with
Fe
-citrate and an enzymatic source of superoxide led
to rapid formation of Fe
and consequent oxidative
modification of LDL. Addition of BP inhibited iron-mediated LDL
oxidation under these conditions. Our results indicate that reduced
metal ions are important mediators of LDL oxidation, and that LDL
specifically reduces Cu
, but not
Fe
. These data, therefore, help explain why copper,
in addition to being chemically more reactive, is more potent than iron
at mediating LDL oxidation.
A primary event in early atherosclerosis is the unregulated
uptake of low density lipoprotein (LDL) ()by
monocyte-macrophages within the arterial wall to generate lipid-laden
foam cells characteristic of the fatty streak lesion(1) .
However, in vitro experiments have demonstrated that
incubation of monocyte-macrophages with native LDL does not result in
foam cell genesis(2, 3) , and that chemical
modification such as acetylation or conjugation with certain aldehydic
species is necessary to render the LDL particle
atherogenic(3, 4, 5) . Although the in
vivo nature of this chemical modification awaits unequivocal
identification, there is considerable evidence that oxidative
modification of LDL involving lipid peroxidation with subsequent lysine
residue modification in apolipoprotein B is
important(6, 7, 8) . Regardless of the
precise mechanisms, numerous in vitro studies have
demonstrated that copper and iron are essential for cell-mediated
atherogenic modification of
LDL(9, 10, 11, 12, 13, 14, 15, 16) .
Cell-free incubation of LDL with cupric ions (Cu
)
also results in generation of a lipoprotein particle with chemical and
physiological characteristics identical with those of cell-modified
LDL(17) .
In a recent study(18) , we reported that
copper-dependent oxidative modification of human LDL was independent of
a source of superoxide, hydrogen peroxide, or aqueous hydroxyl radicals
and occurred despite the absence of detectable amounts of pre-existing
lipid hydroperoxides in the lipoprotein particle. Oxidative
modification of LDL induced by ferric (Fe)-citrate
also was independent of hydrogen peroxide, aqueous hydroxyl radicals,
and pre-existing lipid hydroperoxides, but required a source of
superoxide(18) . Although it had previously been suggested that
either pre-existing lipid hydroperoxides in the LDL particle (16, 19, 20) or hydroxyl radicals generated
via a superoxide-driven Fenton reaction mechanism (21) serve as
initiators of metal ion-dependent lipid peroxidation, our in vitro studies showed that neither is required (18) for LDL
oxidation. However, the observation that superoxide, a known
Fe
reductant(22) , was needed for hydroxyl
radical-independent iron-mediated LDL oxidation suggested that
reduction of Fe
to ferrous ion (Fe
)
was essential for this process. Interestingly, Aust and
co-workers(22, 23, 24, 25, 26) have demonstrated that both Fe
and
Fe
must be present to initiate lipid peroxidation in
liposomes. We therefore investigated the hypothesis that copper- and
iron-mediated oxidative modification of human LDL depend upon
generation of the reduced metal ions. Furthermore, since
copper-dependent oxidative modification of LDL occurs in the absence of
an exogenous reductant, we also tested whether some redox activity
intrinsic to the lipoprotein particle itself could mediate cuprous ion
(Cu
) formation.
Figure 1:
Reduction of Cu by
LDL. Absorbance spectra of samples containing various combinations of
BC (360 µM), LDL (20 µg of protein/ml), and either
Cu
or Cu
(10 µM each)
in PBS are shown following incubation at 37 °C for 60 min.
Incubations contained BC (
), BC with Cu
(
), BC with Cu
(X), BC with LDL (
),
or BC with LDL and Cu
(
). Cu
was prepared by direct reduction of a Cu
solution (10 µM) with ascorbic acid (1 mM).
For each incubation, a single spectrum representative of four
independent experiments is shown.
Figure 2:
Lack of Fe reduction by
LDL. Absorbance spectra of samples containing various combinations of
BP (360 µM), LDL (20 µg of protein/ml), and either
Fe
-citrate or Fe
-citrate (10
µM each) in PBS are shown following incubation at 37
°C for 60 min. Incubations contained BP (
), BP with
Fe
-citrate (
), BP with
Fe
-citrate (X), BP with LDL (
), or BP with LDL
and Fe
-citrate (
). For each incubation, a
single spectrum representative of three independent experiments is
shown.
Fig. 1and Fig. 2show the results of
experiments in which Cu or Fe
(10
µM) were incubated with LDL (20 µg of protein/ml) at
37 °C for 60 min in the presence of indicator molecules (BC and BP,
respectively; 360 µM each) specific for the reduced forms
of these metal ions. Incubation of Cu
with LDL for 60
min resulted in reduction to Cu
as indicated by
formation of a peak with maximal absorbance at 480 nm characteristic of
the BC
Cu
complex (Fig. 1). In contrast,
following incubation of Fe
with LDL for 60 min, no
increase in the absorbance at 535 nm was observed, i.e. no
BP
Fe
complex was formed (Fig. 2).
Increasing the LDL concentration 10-fold to 200 µg of protein/ml
also did not result in significant Fe
reduction in
this experimental system (Fig. 3). Thus, LDL specifically
reduced Cu
to Cu
, but was unable to
reduce Fe
to Fe
. Furthermore,
complexation of the newly formed Cu
with BC in a redox
inactive form (31, 34, 35) during incubation
of LDL (200 µg of protein/ml) with Cu
(10
µM) significantly protected LDL from oxidative
modification, as assessed by electrophoretic mobility of LDL on an
agarose gel (Fig. 4).
Figure 3:
Reduction of Fe by the
MFO system. Fe
-citrate (10 µM) and LDL
(200 µg of protein/ml) were incubated in PBS at 37 °C with BP
(360 µM) in the absence (
) or presence (
) of
the hypoxanthine (1.5 mM)-xanthine oxidase (0.02 unit/ml) MFO
system. Superoxide dismutase (1000 units/ml, X) or catalase (1,000
units/ml,
) was added to incubations containing the MFO system.
Fe
formation was monitored as the increase in
absorbance at 535 nm. Each point represents the mean of at least two
independent experiments.
Figure 4:
Effect of BC on copper-dependent oxidative
modification of LDL. LDL (200 µg of protein/ml) was incubated in
PBS at 37 °C for 6 h without(-) or with (+)
Cu (10 µM) in the absence(-) or
presence (+) of BC (360 µM). Electrophoretic mobility
of LDL was measured on an agarose gel.
In contrast to the inability of LDL to
reduce Fe, when a standard mixed function oxidase
system (MFO, 1.5 mM hypoxanthine with 0.02 unit of xanthine
oxidase/ml) was added as a source of superoxide(18) , rapid
reduction of Fe
to Fe
was observed (Fig. 3). As shown in Fig. 5, LDL became oxidatively
modified under these conditions, in contrast to LDL incubated with
Fe
in the absence of MFO. Addition of BP to the LDL
incubation containing MFO and Fe
completely inhibited
LDL oxidation (Fig. 5). Reduction of Fe
by MFO
was dependent on superoxide, but not hydrogen peroxide, as addition of
superoxide dismutase, but not catalase, significantly inhibited
Fe
reduction (Fig. 3). In contrast, reduction
of Cu
by LDL was not inhibited by superoxide
dismutase, and addition of catalase also was without effect (Fig. 6).
Figure 5:
Effect of BP on iron-dependent oxidative
modification of LDL. LDL (200 µg of protein/ml) was incubated in
PBS at 37 °C for 6 h with Fe-citrate (10
µM) in the absence(-) or presence (+) of the
hypoxanthine (1.5 mM)-xanthine oxidase (0.02 unit/ml) MFO
system, and BP (360 µM). Electrophoretic mobility of LDL
was measured on an agarose gel.
Figure 6:
Effects of superoxide dismutase and
catalase on Cu reduction by LDL. Cu
(10 µM) and LDL (20 µg of protein/ml) were
incubated in PBS at 37 °C with BC (360 µM) in the
absence (
) or presence of either superoxide dismutase (1000
units/ml,
) or catalase (1000 units/ml, X). Cu
formation was monitored as the increase in absorbance at 480 nm.
Each point represents the mean of two independent
experiments.
To investigate the dependence of Cu reduction on LDL concentration, 10 µM Cu
was incubated for 60 min at 37 °C with BC (360
µM) and varying concentrations of LDL (0-30 µg
of protein/ml). Maximal levels of Cu
(approximately
7.3 µM) were observed at LDL concentrations as low as 10
µg of protein/ml (Fig. 7). Below this concentration, LDL
reduced Cu
in a dose-dependent manner (Fig. 7). To determine if the Cu
reducing
ability of LDL was saturable, LDL (10 µg of protein/ml) was
incubated for 60 min at 37 °C with BC (360 µM) and
varying concentrations of Cu
(0-80
µM). Independent of the initial Cu
concentration, incubation with LDL resulted in reduction of about
65% of the added Cu
(Fig. 8). Thus, a linear
relationship between initial Cu
concentration and
Cu
formed was observed, indicating that under the
experimental conditions used LDL's ability to reduce
Cu
was not saturated.
Figure 7:
Dependence of Cu
reduction on LDL concentration. LDL (0-30 µg of protein/ml)
was incubated in PBS at 37 °C for 60 min with Cu
(10 µM) and BC (360 µM). Cu
formation was monitored as the increase in absorbance at 480 nm.
Each point represents the mean of at least two independent
experiments.
Figure 8:
Effect of initial Cu
concentration on Cu
formation by LDL. LDL (10 µg
of protein/ml) was incubated in PBS at 37 °C for 60 min with BC
(360 µM) and Cu
(0-80
µM). Cu
formation was monitored as the
increase in absorbance at 480 nm.
The significance of free
sulfhydryl groups on apolipoprotein B as possible Cu reductants was investigated by preincubating LDL (470 µg of
protein/ml) with N-ethylmaleimide (6.3 mM) for 30 min
at 37 °C before exposure to Cu
. This treatment
had no effect on either the ability of LDL (20 µg of protein/ml) to
reduce Cu
(10 µM) (Table 1) or
Cu
-induced LDL oxidation (Fig. 9). Similarly,
increasing the
-tocopherol content of LDL from 11.8 ± 3.0
to 24.4 ± 2.8 nmol/mg of protein by preincubating plasma with
-tocopherol prior to LDL isolation (36) also had no
significant effect on the lipoprotein's ability to reduce
Cu
(Table 1).
Figure 9:
Effect of N-ethylmaleimide on
copper-dependent oxidative modification of LDL. LDL (470 µg of
protein/ml) was preincubated in PBS at 37 °C without(-) or
with (+) N-ethylmaleimide (6.3 mM) for 30 min.
These LDL preparations were diluted with PBS (final LDL concentration
= 200 µg of protein/ml), incubated at 37 °C with
Cu (10 µM), and aliquots were removed at
intervals (0-4 h). The reaction was terminated by addition of the
metal ion chelator diethylenetriaminepentaacetic acid (1 mM),
and the electrophoretic mobility of LDL was measured on an agarose gel. Numbers indicate incubation time
(h).
The results of this investigation demonstrate that
Cu, but not Fe
, is specifically
reduced by LDL in vitro ( Fig. 1and Fig. 2).
These findings have significance for our previous observations (18) that Cu
alone can oxidatively modify
LDL, while iron-dependent LDL oxidation requires a source of
superoxide, a known Fe
reductant (22) . Our
data suggest that reduced metal ions are an important factor mediating
oxidative modification of LDL. Consistent with this hypothesis,
chelation of nascent Cu
or Fe
by BC
or BP, respectively, significantly inhibited metal ion-dependent LDL
oxidation ( Fig. 4and Fig. 5). Although it has been
argued that pre-existing lipid hydroperoxides in isolated LDL are
required for initiation of lipid peroxidation by reduced metal
ions(16, 19, 20) , our previous (18) and present data do not support this hypothesis. The LDL
preparations used in the experiments reported here were routinely
analyzed for pre-existing lipid hydroperoxides by a sensitive and
specific HPLC procedure(27, 28) , and none were
detected. The low detection limit of this assay (1 lipid hydroperoxide
molecule in 200 LDL particles) makes it highly unlikely that
pre-existing lipid hydroperoxides were an important factor in the
initiation of lipid peroxidation in LDL or the observed reduction of
Cu
.
Heinecke et al.(37) and,
more recently, Sparrow and Olszewski (38) have shown that
extracellular cysteine or other sulfhydryl compounds are essential for
cell-mediated oxidation of LDL in media containing redox active metal
ions. Since cysteine is a physiological reductant capable of reducing
metal ions, we investigated the possibility that free sulfhydryl groups
in apolipoprotein B could mediate the observed reduction of
Cu by LDL. Preincubation of LDL with an excess of the
sulfhydryl group alkylating agent N-ethylmaleimide had no
effect upon subsequent reduction of Cu
(Table 1) nor copper-mediated oxidative modification of LDL (Fig. 9). Moreover, addition of superoxide dismutase did not
affect Cu
reduction by LDL (Fig. 6), whereas
cysteine-dependent oxidation of LDL by smooth muscle cells was
inhibited by superoxide dismutase(37) . These data indicate
that free sulfhydryl groups associated with apolipoprotein B are not an
important factor mediating copper-dependent LDL oxidation.
Recently,
some investigators have presented evidence that under certain
conditions -tocopherol may function as a pro-oxidant, rather than
an
antioxidant(39, 40, 41, 42, 43) .
Bowry et al.(39) have observed that the initial rate
of lipid peroxidation in LDL is directly related to its
-tocopherol content. Maiorino et al.(42) have
shown that
-tocopherol in a detergent dispersion is rapidly
oxidized by Cu
, and that phospholipids added to these
dispersions undergo lipid peroxidation at a rate determined by the
initial
-tocopherol concentration. It has been suggested that
lipid peroxidation observed in these systems results from a direct
interaction between Cu
and
-tocopherol with
generation of Cu
(42, 43) :
where -TocH denotes
-tocopherol, and
-Toc
the
-tocopheroxyl radical. In the presence of pre-existing lipid
hydroperoxides (LOOH), Cu
then can initiate lipid
peroxidation via formation of lipid alkoxyl radicals (LO
):
where LH denotes a lipid molecule with a polyunsaturated fatty
acid side chain containing a doubly allylic hydrogen, LOH a lipid
hydroxide, and L a carbon-centered polyunsaturated fatty acid
radical. The latter then can reinitiate the radical chain reaction of
lipid peroxidation(19, 43) . However, as discussed
above, we could not detect pre-existing lipid hydroperoxides in our LDL
preparations. It is, therefore, tempting to speculate that it is not
Cu
plus lipid hydroperoxides, but the
-tocopheroxyl radical formed in that initiates lipid
peroxidation in LDL, as has been suggested by Stocker and
colleagues(39, 40, 41) :
This is an attractive hypothesis, as it would not only explain
how Cu initiates lipid peroxidation in LDL devoid of
pre-existing lipid hydroperoxides, but also why LDL can reduce
Cu
in excess over its
-tocopherol content. For
example, we observed that LDL (20 µg of protein/ml) containing 11.8
nmol of
-tocopherol/mg of protein (i.e. 0.24 µM
-tocopherol) can reduce approximately 7.6 µM Cu
(Table 1), i.e. a 30-fold
molar excess of Cu
over the LDL-associated
-tocopherol (see also below). This phenomenon could be explained
by the regeneration of
-tocopherol in , so that the
reducing equivalents for Cu
reduction ()
would ultimately be provided by the polyunsaturated fatty acids in LDL,
the concentration of which is much greater than the concentration of
-tocopherol(19) .
However, there are at least two lines
of evidence that seem inconsistent with the above hypothesis. First,
measuring tocopherol consumption, Yoshida et al.(43) estimated that the apparent rate constant for the
interaction of endogenous
tocopherol in LDL with Cu
() is 2.9 M
s
. Based on this rate constant, the predicted
rate of Cu
reduction by
-tocopherol in LDL under
the conditions described in Table 1would be 0.42
nM/min; the observed rate, however, was almost 3 orders of
magnitude higher (0.27µM/min). Second, doubling of
LDL's
-tocopherol content had no significant effect on
either the rate or extent of Cu
reduction by LDL (Table 1). However, it is conceivable that under the conditions
employed the
-tocopherol content of LDL was not rate-limiting.
Thus, the potential significance of LDL-associated
-tocopherol as
the specific Cu
reductant is presently unresolved.
Regardless of the mechanism of Cu reduction by
LDL, it is obvious from Fig. 7that this process is highly
effective, as maximal reduction of 10 µM Cu
was observed during 60 min of incubation with LDL at a
concentration as low as 10 µg of protein/ml (
20 nM LDL). The maximum Cu
concentration observed under
these conditions was approximately 7.3 µM, corresponding
to reduction of about 360 Cu
ions by each LDL
particle. At LDL concentrations greater than 10 µg of protein/ml,
no further increase in Cu
reduction was observed,
indicating maximal activity of the Cu
reducing system
under these conditions (Fig. 7). Results from other experiments
in which the LDL concentration was held constant (10 µg of
protein/ml) and the initial Cu
concentration was
varied (Fig. 8) indicate that under the conditions employed the
Cu
reducing activity of LDL is nonsaturable with
respect to copper: a linear relationship between Cu
formation and initial Cu
concentration was
observed up to the maximum Cu
concentration tested
(80 µM, corresponding to approximately a 4,000-fold molar
excess of Cu
over LDL).
The observation of a
Cu specific reducing activity of LDL may be related
to the ability of LDL to bind
Cu
(44, 45, 46, 47, 48, 49) .
Thus, Cu
reduction may occur at specific binding
sites proximal to the reducing moieties within the lipoprotein
particle. Interestingly, Kuzuya et al.(44) have
reported that while both Cu
and Fe
may bind to LDL in 0.15 M NaCl, Fe
binding is significantly decreased in phosphate-containing
buffer. This may explain the Cu
specific reducing
activity of LDL observed in the experiments reported here, which were
performed in PBS. Although LDL did not reduce Fe
,
incubation with an enzymatic source of superoxide resulted in rapid
Fe
reduction (Fig. 3). This process was not
affected by catalase, indicating that hydrogen peroxide also generated
by the enzymatic MFO system used was without effect on Fe
formation. Addition of superoxide dismutase, however,
significantly inhibited Fe
reduction, demonstrating
the importance of superoxide in this reaction. Consistent with these
data, we reported previously that iron-dependent LDL oxidation requires
the presence of MFO, and that addition of superoxide dismutase, but not
catalase, inhibits LDL oxidation(18) . In the studies reported
here we have also shown that addition of BP to LDL incubations
containing MFO and Fe
completely inhibits LDL
oxidation (Fig. 5) indicating that iron-mediated LDL oxidation
requires nonchelated Fe
. Using an enzymatic system
similar to our MFO system, Miller et al.(22) reported
that lipid peroxidation in liposomes is initiated directly by iron,
probably via an Fe
Fe
complex.
It is conceivable that a similar complex is responsible for
iron-dependent initiation of lipid peroxidation in LDL, and, by
analogy, that copper-dependent oxidative modification of LDL requires
formation of a Cu
Cu
complex.
In summary, our results indicate that reduced metal ions
(Cu and Fe
) are important factors
mediating oxidative modification of human LDL. Under the experimental
conditions used, LDL specifically reduced Cu
to
Cu
, but could not reduce Fe
to
Fe
. This metal ion specificity may explain the
ability of Cu
to oxidize LDL and may be related to
binding of Cu
, but not Fe
, to LDL
under these conditions. Reduction of Cu
by LDL is
unlikely to be mediated by pre-existing lipid hydroperoxides in the
lipoprotein particle and is also independent of superoxide and free
sulfhydryl groups associated with apolipoprotein B. Although reduction
of Cu
by LDL occurred much faster than expected based
on the known reaction rate between Cu
and
-tocopherol, and
-tocopherol supplementation of LDL had no
effect on lipoprotein-mediated Cu
reduction, the
potential significance of
-tocopherol as the LDL-associated
Cu
reductant is presently unclear. In contrast to
copper, iron-dependent oxidative modification of LDL requires an
exogenous reductant such as superoxide to facilitate reduction of
Fe
to Fe
. Other physiological
reductants may also be important for iron-mediated oxidative
modification of LDL.