(Received for publication, December 1, 1995; and in revised form, February 14, 1996)
From the
Initiation of lipid peroxidation by Cu(II) requires reduction of
Cu(II) to Cu(I) as a first step. It is unclear, however, whether this
reaction occurs in the course of lipoprotein oxidation. It is also
unknown which reductant, if any, can drive the reduction of Cu(II) in
this case. We found that Cu(II) was rapidly reduced to Cu(I) by all
major human lipoproteins (high, low, and very low density lipoproteins
(HDL, LDL, and VLDL), and chylomicrons). Cu(II)-reducing activity was
associated with a lipid moiety of the lipoproteins. The rates of Cu(II)
reduction by different lipoproteins were similar when the lipoproteins
were adjusted to similar -tocopherol concentrations. Enriching
lipoproteins with
-tocopherol considerably increased the rate of
Cu(II) reduction. Cu(II) reduction by
-tocopherol-deficient LDL
isolated from a patient with familial inherited vitamin E deficiency
was found to occur much slower in comparison with LDL isolated from a
donor with a normal plasma level of
-tocopherol. Initial rate of
Cu(II) reduction by
-tocopherol-deficient LDL was found to be
zero. Enriching LDL with ubiquinol-10 to concentrations close to those
of
-tocopherol did not influence the reaction rate. When LDL was
treated with ebselen to eliminate preformed lipid hydroperoxides, the
reaction rate was also not changed significantly. Cu(II) reduction was
accompanied by a consumption of lipoprotein
-tocopherol and
accumulation of conjugated dienes in the samples. Increasing
-tocopherol content in lipoproteins slightly decreased the rate of
conjugated diene accumulation in LDL and HDL and considerably increased
it in VLDL. The results suggest that
-tocopherol plays a
triggering role in the lipoprotein oxidation by Cu(II), providing its
initial step as follows:
TocH + Cu(II)
Toc
+ Cu(I) + H
. This
reaction appears to diminish or totally eliminate the antioxidative
activity of
-tocopherol in the course of lipoprotein oxidation.
There is strong evidence that the oxidation of low density
lipoprotein (LDL) ()plays an important role in
atherogenesis(1, 2) . LDL oxidation appears to occur
in the arterial wall, giving rise to the formation of early
atherosclerotic lesions. The mechanisms by which LDL oxidation is
initiated in vivo, however, remain unclear.
In vitro, LDL can be oxidized by all major groups of arterial wall cells, namely macrophages, smooth muscle cells, endothelial cells, and lymphocytes(3, 4) . A number of agents, such as transition metal ions, lipoxygenases, peroxidases, and heme proteins, are capable in the absence of cells of oxidizing LDL(5, 6) . Of these oxidants, transition metal ions seem to be most likely involved in LDL oxidation in vivo because they are essential for oxidizing LDL in vitro in the presence of cells(3, 5, 6, 7) . In order to be oxidized by transition metal ions, LDL presumably must initially contain traces of lipid hydroperoxides(8, 9) . The decomposition of these hydroperoxides by metal ions provides free radicals that can directly oxidize LDL components. Current knowledge suggests that, in order to decompose lipid hydroperoxides to free radicals, transition metal ions must be reduced to the highly active low valency state(10, 11) . In the case of LDL oxidation, it means that the metal ions, which are tightly bound to the LDL surface(12, 13) , must be reduced by a reductant present in the LDL particle or in the aqueous environment. Such a reaction is expected to represent the initial step of LDL oxidation by transition metals. It has recently been shown that Cu(II) is reduced to Cu(I) by incubation with human LDL(14) . However, it is currently unknown which reductant drives this reaction in the course of LDL oxidation. The occurrence of this reaction in the course of the oxidation of other plasma lipoproteins has also not been demonstrated.
The LDL particle contains several reducing agents which may
potentially be involved in the reduction of transition metal ions. They
include antioxidants -tocopherol and
ubiquinol-10(10, 15) , surface SH groups of
apolipoprotein B(15, 16) , and preformed lipid
hydroperoxides (17) . It has been suggested by Esterbauer et al.(10) that
-tocopherol, the major, on a
molar base, antioxidant in LDL, can provide initial reduction of Cu(II)
in the course of LDL oxidation by this ion. Recently, it has been shown
that
-tocopherol can be oxidized by Cu(II) in homogeneous solution (11) as well as in phospholipid (18) and detergent (11) micelles. A similar reaction between
-tocopherol and
Fe(III) has been described earlier(19, 20) . These
data correspond well with the absence of any significant correlation
found between oxidizability by Cu(II) and
-tocopherol content of
native LDL unsupplemented with this
antioxidant(21, 22, 23, 24, 25) .
Prooxidative activity of
-tocopherol as a reductant for Cu(II)
might explain the weakening of its activity toward oxidation found in
these experiments. In agreement with these findings, we have recently
shown that
-tocopherol does not significantly contribute to LDL
oxidizability by Cu(II)(26, 27) . We have also
demonstrated that the antioxidant efficiency of LDL supplementation
with
-tocopherol is much weaker than, for example, with
ubiquinol-10(28) . In the present study we report on a
prominent Cu(II)-reducing activity of all major human lipoproteins,
specifically very low, low, and high density lipoproteins (VLDL, LDL,
and HDL, respectively) and chylomicrons, and show that
-tocopherol
plays a central role in the Cu(II) reduction.
In separate experiments, LDL was isolated
from a 27-year-old male patient with familial isolated vitamin E
deficiency (FIVE)(32, 33) . The patient was
homozygotic for a defect in the -tocopherol transfer protein gene (33) and required regular vitamin E supplements (1800 mg
daily). In experiment A, he was completely withdrawn from his vitamin E
supplementation 5 days before taking his first blood sample (day 0).
Then, he was resupplemented with increasing amounts of vitamin E (400,
1200, and 1800 mg on the 1st, 2nd, and 3rd days of the
resupplementation). Blood was taken from the patient on the first 3
days after resuming the supplementation (days 1, 2, and 3). In
experiment B, the patient was withdrawn from his regular vitamin E
supplementation for 5 days (days 1-5) and simultaneously
supplemented with ubiquinone-10. Amounts of antioxidants taken by the
patient were 1800 mg of vitamin E and 90 mg of ubiquinone-10 on day 0
and 180 mg of ubiquinone-10 on days 1-5. All patient's
blood samples were taken after an overnight fast into the
EDTA-containing tubes and centrifuged. The plasma obtained from the
patient was frozen at -80 °C under nitrogen immediately after
isolation and stored under these conditions until use. LDL was isolated
from the plasma as described above and used for experiments within 2 h.
Total cholesterol, free cholesterol, triglyceride and phospholipid content of lipoproteins were determined by commercially available enzymatic tests (Boehringer Mannheim, Mannheim, Germany). Lipoprotein cholesterol esters were calculated from total and free cholesterol values using data on average lipoprotein content of these lipids(10, 36) , on the assumption that fatty acid moiety of cholesterol esters contributes to 6.0%, 17.1%, and 6.3% of the total lipoprotein weight in VLDL, HDL, and LDL, respectively. Total protein was determined as described by Lowry et al.(37) using bovine serum albumin as a standard. To recalculate the values obtained into mol/mol lipoproteins, approximate HDL, LDL, and VLDL molecular masses of 0.25, 2.5, and 25 MDa, respectively, were used(10, 36, 38) .
The amount of Cu(II) reduced by lipoproteins was measured as the
amount of generated Cu(I) using bathocuproinedisulfonic acid (BCSA)
which is known to form a colored complex with Cu(I)(39) . To
measure Cu(I) accumulation, tandem cuvettes (Hellma,
Müllheim, Germany) were used(40) . This
enabled us to mix reagents in one compartment of the reaction cuvette
and separate them in two compartments of the reference cuvette.
200-µl EDTA-free stock lipoprotein suspensions were mixed with 50
µl of 5.0 mM BCSA solution and necessary volume of PBS.
The reaction was started by adding freshly prepared aqueous solution of
CuSO to a final concentration of 100 µM and
final volume of 1.0 ml, in order to achieve the same final dilution of
1:5 for all stock lipoprotein suspensions. Formation of the BCSA-Cu(I)
complex was followed at 480 nm(39) , starting 30 s after
addition of Cu(II). Presence of BCSA did not influence the course of
the reaction between Cu(II) and
-tocopherol, since similar time
courses were recorded in the presence and in the absence of the
chelator in experiments on Cu(II) reduction by
-tocopherol
incorporated into sodium dodecyl sulfate micelles. (
)
To
assess the contribution of lipoprotein SH-groups to Cu(II) reduction,
the SH-groups were blocked by addition of p-chloromercuriphenylsulfonic acid (PCMPS) to a final
concentration of 50 µM(16) . To characterize the
contribution of lipoprotein lipids and proteins to Cu(II) reduction,
they were separated using chloroform/methanol mixture. 400-µl
lipoprotein suspensions were mixed with 3 ml of chloroform/methanol
mixture (2:1 v/v). Chloroform and methanol phases were separated,
collected, and evaporated under nitrogen. The residues obtained were
resuspended in 0.9% NaCl (lipids) or 50 µM SDS (proteins)
and assayed for Cu(II) reduction at a Cu concentration of 50 µM. Sodium borohydride was added
to the lipoprotein/chloroform/methanol mixture before separation of the
fraction containing lipoprotein lipids to remove lipid hydroperoxides.
Such a treatment was found to efficiently remove high amounts of
hydroperoxides from linoleic acid dissolved in the chloroform/methanol
mixture.
In some experiments, ebselen was used to remove lipid hydroperoxides from isolated LDL(41) . 2 ml of LDL (0.79 mg of total cholesterol/ml) was mixed with 12.5 µl of 10 mM ebselen solution in ethanol. After adding 50 µl of 30 mM freshly prepared aqueous glutathione solution, the LDL was incubated at 37 °C for 30 min. Ebselen was removed from the LDL using gel filtration on Sephadex PD-10 columns (repeated twice). This procedure has been shown to efficiently reduce all lipid hydroperoxides present in the sample into corresponding hydroxides(41) .
It was found that all major human lipoproteins rapidly
reduced Cu(II) to Cu(I) (Fig. 1). The reaction occurred rapidly
and usually reached saturation after about 5 min of incubation. The
maximal amount of Cu(II) reduced was similar in different lipoproteins,
if they were preliminarily diluted to a similar final -tocopherol
concentration (Fig. 1).
Figure 1:
Cu(II)
reduction to Cu(I) by human VLDL (), LDL (
), and HDL
(
) from a healthy donor and by chylomicrons (+) from an
apoC-II-deficient patient. Cu(I) accumulation was monitored as the
increase in absorbance at 480 nm. Initial concentrations of the
reagents: Cu(II), 100 µM; BCSA, 250 µM; VLDL,
0.24 mg of triglyceride/ml (1.20 µM
-tocopherol);
LDL, 0.17 mg of total cholesterol/ml (1.80 µM
-tocopherol); HDL, 0.12 mg of total cholesterol/ml (1.54
µM
-tocopherol); chylomicrons, 0.34 mg of
triglyceride/dl (0.60 µM
-tocopherol). The results
shown are representative of three separate experiments performed with
three different plasma samples from the healthy donor and of three
separate experiments performed with one plasma sample from the
patient.
When fractions containing lipoprotein lipids and proteins were separated by extraction with a chloroform/methanol mixture, only the fraction containing lipids retained the ability to reduce Cu(II) (Fig. 2). The results shown here for VLDL are representative of similar experiments performed with LDL and HDL (data not shown). Micelles formed of the fraction containing lipoprotein lipids reduced Cu(II) even more rapidly than corresponding native lipoproteins, despite the fact that the lipoproteins were treated with sodium borohydride to remove preformed lipid hydroperoxides. Denaturated apoproteins showed no significant Cu(II)-reducing activity (1.16 ± 2.16 µM Cu(II) reduced after 5 min of incubation, averaged for 3 experiments with each VLDL, LDL, and HDL apoprotein). In accordance with this finding, blocking protein SH-groups with PCMPS did not suppress Cu(II) reduction by native lipoproteins (Fig. 2). The slight acceleration of the reaction actually observed could be due to the weak reductive activity of PCMPS itself. Such an availability of Cu(II) to exogeneous reductants was confirmed by a considerable acceleration of Cu(II) reduction by native LDL after addition of cysteine (4.85, 12.4, and 70.9 µM Cu(II) reduced in the presence of 0, 10, and 100 µM cysteine, respectively, after a 5-min incubation, means of 2 independent experiments).
Figure 2:
Cu(II) reduction to Cu(I) by different
fractions of human VLDL from a healthy donor. Fractions containing
lipids () and proteins (
) from VLDL (0.12 mg of
triglyceride/ml) were assayed for Cu(II) reduction after their
separation as described under ``Materials and Methods.''
Cu(II) reduction by native VLDL (0.12 mg of triglyceride/ml) in the
absence (
) and in the presence (
) of PCMPS (50
µM) is also shown. Cu(I) accumulation was monitored as the
increase in absorbance at 480 nm. Initial concentrations of the
reagents: Cu(II), 50 µM; BCSA, 125
µM.
When the plasma from a healthy donor
was incubated with increasing amounts of -tocopherol, the
lipoproteins were enriched with the antioxidant. Such an enrichment
considerably increased both the initial rate of Cu(II) reduction and
the maximal amount of Cu(II) reduced by the lipoproteins (Fig. 3). The initial rate of Cu(II) reduction by the
lipoproteins was related to their
-tocopherol content, increasing
with the latter (Fig. 3, insets). The maximal amount of
produced Cu(I) was similarly related to the amount of
-tocopherol
present, when Cu(II) was in excess to
-tocopherol. In this case,
the amount of Cu(I) produced was always slightly higher than the amount
of
-tocopherol present. When the initial concentration of
-tocopherol in the reaction mixture exceeded that of Cu(II) (in
the
-tocopherol-enriched VLDL), nearly all Cu(II) present was
reduced. When LDL from a healthy donor was treated with ebselen to
eliminate preformed lipid hydroperoxides, the reaction rate was not
changed significantly (23.1 and 24.2 mol of Cu(II) reduced/mol of LDL
for native and ebselen-treated LDL, respectively, after a 5-min
incubation, means of 2 independent experiments).
Figure 3:
Cu(II) reduction to Cu(I) by human VLDL,
LDL, and HDL enriched with -tocopherol. To perform the enrichment,
the plasma from a healthy donor was incubated for 3 h at 4 °C with
-tocopherol, added as a solution in Me
SO to the final
concentration of 0.5 (
) and 1.0 (
) mM. Control
incubation was performed with 1% Me
SO (
). Following
the incubation, HDL, LDL, and VLDL were isolated by density gradient
ultracentrifugation. Cu(I) accumulation was monitored as the increase
in absorbance at 480 nm. Initial concentrations of the reagents were as
described in the legend to Fig. 1. Numbers at the curves denote lipoprotein content of
-tocopherol (mol/mol
lipoprotein). The results shown are representative of three separate
experiments performed with three different plasma samples. Insets show dependences of the initial rate of Cu(II) reduction by
lipoproteins measured after a 30-s incubation on their initial
-tocopherol content for all samples
studied.
When the FIVE
patient was given vitamin E after his withdrawal from his regular
vitamin E supplementation (experiment A), the LDL isolated from the
patient's plasma was enriched with -tocopherol. Enriching
LDL with
-tocopherol considerably increased the rate of Cu(II)
reduction (Fig. 4A). Noteworthy was that the LDL
isolated on day 0 of the experiment, which contained a negligibly low
level of
-tocopherol (0.37 mol/mol of LDL), reduced Cu(II) at a
very low rate. No Cu(I) production was found in this LDL incubated with
Cu(II) for 2 min (Fig. 4A), implying that the initial
rate of Cu(II) reduction by
-tocopherol-deficient LDL equaled 0.
When the FIVE patient was supplemented with ubiquinone-10 instead of
vitamin E (experiment B), the LDL isolated from the patient's
plasma was enriched with ubiquinol-10 and depleted in
-tocopherol.
Such a modification considerably decreased the rate of Cu(II) reduction
by LDL (Fig. 4B), demonstrating that the enrichment
with ubiquinol-10 (from 0.05 to 0.83 mol/mol LDL) could not overcome
the effect of much more pronounced depletion in
-tocopherol (from
7.04 to 0.49 mol/mol) on Cu(II) reduction. Enriching LDL from a healthy
donor with higher amounts of ubiquinol-10 (up to 3.2 mol/mol LDL) by
incubating the plasma with this antioxidant also did not significantly
influence the reaction rate (16.9, 16.9, and 16.1 mol of Cu(II)
reduced/mol of LDL for 0.16, 2.6, and 3.2 mol of ubiquinol-10/mol of
LDL, respectively, after a 5-min incubation, means of 2 independent
experiments).
Figure 4:
Cu(II) reduction to Cu(I) by human LDL
from the FIVE patient supplemented with vitamin E (A) and
ubiquinone-10 (B). A and B correspond to
experiments A and B described under ``Materials and
Methods.'' Numbers at the curves denote LDL
content of -tocopherol and ubiquinol-10 (in parentheses)
expressed as mol/mol LDL. Cu(I) accumulation was monitored as the
increase in absorbance at 480 nm. Initial concentrations of the
reagents: Cu(II), 100 µM; BCSA, 250 µM; LDL,
0.17 mg of total cholesterol/ml. Each point represents the
mean of two independent experiments.
Cu(II) reduction was accompanied by a consumption of
lipoprotein -tocopherol in most samples obtained from a healthy
donor (Fig. 5). No systematic decrease in
-tocopherol
content was found in the experiments with
-tocopherol-enriched
VLDL (probably due to its highly increased
-tocopherol level,
complicating accurate determinations). The rate of
-tocopherol
consumption was generally lower than the rate of Cu(I) accumulation in
the same samples. This was in accordance with data previously reported
for human LDL(14) .
Figure 5:
-Tocopherol consumption in human
VLDL, LDL, and HDL enriched with
-tocopherol and incubated with
Cu(II). The enrichment was performed as described in the legend to Fig. 3. Lipoprotein content of
-tocopherol before and after
a 1-min incubation is shown with open and closed
bars, respectively.
-Tocopherol consumption in the control
lipoproteins and lipoproteins isolated from the plasma incubated with
0.5 and 1.0 mM
-tocopherol are shown from left to right. Initial concentrations of the reagents were as
described in the legend to Fig. 1. The results shown are
representative of three separate experiments performed with three
different plasma samples.
Cu(II) reduction was also accompanied by
an accumulation of conjugated dienes in the lipoproteins (Fig. 6). Increasing lipoprotein content of -tocopherol
slightly decreased the rate of conjugated diene accumulation in LDL and
HDL. Much more pronounced increases in the
-tocopherol content
found in VLDL resulted in considerable increases in the conjugated
diene accumulation in this lipoprotein.
Figure 6:
Conjugated diene accumulation in human
VLDL, LDL, and HDL enriched with -tocopherol and incubated with
Cu(II). The enrichment was performed as described in the legend to Fig. 3. Conjugated diene accumulation in the control
lipoproteins (
) and lipoproteins isolated from the plasma
incubated with 0.5 (
) and 1.0 (
) mM
-tocopherol was monitored as the increase in absorbance at
234 nm. Initial concentrations of the reagents were as described in the
legend to Fig. 1. Numbers at the curves denote
lipoprotein content of
-tocopherol (mol/mol lipoprotein). The
results shown are representative of three separate experiments
performed with three different plasma
samples.
All plasma lipoproteins
studied were found to bind Cu(II). The amount of bound Cu(II) increased
with increasing amounts of added Cu(II) and was found to be saturable
(data not shown). The relationship between bound and added Cu(II) was
linear, when expressed in reciprocal coordinates, permitting
calculation of binding parameters (Table 1). Both parameters
calculated (binding constant and maximal binding capacity) were lowest
for the lipoprotein with the lowest protein content studied
(chylomicrons) and highest for that with the highest protein (HDL),
when measured at the same concentration of total lipids in the reaction
mixture and expressed as µM. On the contrary, maximal
binding capacity decreased from chylomicrons to HDL, when expressed as
mol/mol of lipoprotein. The maximal amount of Cu(II) reduced by native
lipoproteins was much lower than their maximal Cu(II) binding capacity (cf. Fig. 3and Table 1). In contrast, the
maximal amount of Cu(II) reduced by -tocopherol-enriched
lipoproteins was lower than their maximal Cu(II) binding capacity only
in LDL and HDL.
-Tocopherol-enriched VLDL reduced more Cu(II) than
its native counterpart could bind, suggesting that such an enrichment
considerably changed the lipoprotein structure.
All major human lipoproteins (HDL, LDL, VLDL, and
chylomicrons) were found to possess Cu(II)-reducing activity.
Lipoprotein particles contain several reducing agents which may
potentially be involved in Cu(II) reduction. Our results indicate that
lipophilic antioxidant -tocopherol plays a central role in Cu(II)
reduction by lipoproteins. This was concluded from the following
findings: (i) Cu(II)-reducing activity was associated with the lipid
moiety of the lipoproteins; (ii) the rate of Cu(II) reduction by
different lipoproteins was similar when they were diluted to a similar
-tocopherol concentration; (iii) enriching lipoproteins with
-tocopherol considerably increased the rate of Cu(II) reduction;
(iv) Cu(II) reduction by
-tocopherol-deficient LDL isolated from
the FIVE patient was found to occur much more slowly in comparison with
LDL isolated from a donor with a normal plasma level of
-tocopherol, and (v) initial rate of Cu(II) reduction by
-tocopherol-deficient LDL was found to be 0. All other potential
candidates (ubiquinol-10, SH groups of apoproteins, and preformed lipid
hydroperoxides) did not contribute substantially to Cu(II) reduction.
Enriching LDL with ubiquinol-10 to the concentrations exceeding its
physiological range, blockage of SH groups of apoproteins by PCMPS, or
eliminating lipid hydroperoxides from LDL with ebselen had no influence
on the rate of Cu(II) reduction. Denaturated apoproteins also did not
reveal any significant Cu(II)-reducing activity. However,
-tocopherol may not represent the only factor determining the rate
of Cu(II) reduction by lipoproteins, as judged by quite different rates
of Cu(I) production found in different LDL with a similar
-tocopherol content (cf. Fig. 3and Fig. 4, A and B). This suggests that some other unknown
factors, such as lipoprotein Cu(II) binding capacity, might be
important for this reaction.
Cu(II) reduction was accompanied by
oxidation of -tocopherol, suggesting that a redox reaction between
these reagents occurred. The reaction between
-tocopherol and
Cu(II) in micellar solution presumably has a stoichiometry of 1:1 (11) and proceeds as follows: Cu(II) +
-TocH
Cu(I) +
-TocH
+ H
where
-TocH and
-Toc
denote
-tocopherol and
-tocopheroxyl radical, respectively. Our
results suggest that
-tocopherol reduces Cu(II) by the same
reaction in human lipoproteins. This scheme corresponds with the
hypothetical mechanism of interaction between Cu(II) and
-tocopherol in human LDL proposed in earlier
studies(10, 11, 14, 18) .
Cu(II)
reduction by -tocopherol is expected to occur on the lipoprotein
surface, where bound Cu(II) is located(12, 13) .
Increases in both the binding constant and maximal amount of bound
Cu(II) with increasing protein content in the lipoproteins suggest that
Cu(II) is bound predominantly to the protein moiety of the
lipoproteins. This protein-bound and surface-localized Cu(II) must be
available to
-tocopherol to provide the reaction between them.
This model is in good agreement with the location of
-tocopherol
in the surface lipoprotein monolayer(43) . The surface location
of
-tocopherol can also explain the slightly higher rate of Cu(II)
reduction by micelles formed of the fraction containing lipoprotein
lipids than by native lipoproteins, simply by a lower availability of
Cu(II) to lipids and, hence, to
-tocopherol in the lipoproteins
than in the micelles. The lack of Cu(II) reduction by ubiquinol-10 can
accordingly be explained by a deeper location of this antioxidant in
the lipoprotein particle (44) , hindering its direct
interaction with Cu(II).
Reduction of Cu(II) by -tocopherol
found in our study suggests that this classical antioxidant plays a
dual role in the lipoprotein oxidation by Cu(II). Our results indicate
that
-tocopherol triggers the oxidation, reducing Cu(II) to Cu(I).
Formation of Cu(I), a highly active prooxidant, by
-tocopherol
implies that this antioxidant can develop a prooxidant activity in the
course of lipoprotein oxidation. On the other hand, a part of
-tocopherol pool which is not directly oxidized by Cu(II) can
function antioxidatively, scavenging free radicals according to the
classical view(45) . We found that Cu(I) production was
accompanied by accumulation of lipid hydroperoxides in the
lipoproteins. Increasing lipoprotein content of
-tocopherol
decreased the rate of conjugated diene accumulation in LDL and HDL and
increased it in VLDL. This suggests that the antioxidant activity of
-tocopherol prevailed in LDL and HDL, while the prooxidant
activity prevailed in VLDL. There are two major mechanisms by which
-tocopherol can behave as a prooxidant in the course of
lipoprotein oxidation: reduction of transition metal ions to a highly
active low valency state (11, 14, 18) and
propagation of the oxidation via the
-tocopheroxyl
radical(15, 46) . The
-tocopheroxyl radical can
efficiently propagate lipid peroxidation exclusively under mild
oxidative conditions (in comparison with
-tocopherol
concentration) (15, 46) . The concentration of Cu(II)
used to initiate oxidation in our study was much higher than that of
-tocopherol in almost all samples. The
-tocopherol-enriched
VLDL represented the only exception due to its highly elevated
-tocopherol content. Decreased Cu(II)/
-tocopherol ratio
(implying milder oxidative conditions relative to
-tocopherol)
might result in increased propagation via
-tocopheroxyl radical
and a net prooxidative action of
-tocopherol. Classical
antioxidant activity known to prevail under strong oxidative conditions (15, 46) could be responsible for the inhibitory
action of
-tocopherol on HDL and LDL oxidation.
These results
indicate that a net effect of -tocopherol on lipid peroxidation is
determined by a subtle balance of its pro- and antioxidant activities.
The relative importance of different components of this balance seems
to depend on the kind of oxidative stress applied to
lipids(15, 47, 48) . There is evidence that
transition metal ions may be involved in the LDL oxidation in
vivo(6) . Nevertheless, assays using solely Cu(II) to
induce oxidation can overestimate the prooxidant activity of
-tocopherol, in comparison with its physiological role. This
assumption can explain the lack of any significant correlation found
between the oxidizability by Cu(II) and
-tocopherol content of
native LDL under relatively strong oxidative
conditions(21, 22, 23, 24, 25, 26, 27) .
Exaggerated production of Cu(I) and
-tocopheroxyl radical may
underlie the prooxidant activity of
-tocopherol in this case.
Cu(I) might be expected to accelerate lipid peroxidation, when
oxidizing lipids contain sufficient amounts of preformed lipid
hydroperoxides (such as in LDL isolated by time-consuming procedures).
-Tocopheroxyl radical can initiate peroxidation in virtually
``hydroperoxide-free'' LDL(9, 14) ,
interacting directly with lipoprotein polyunsaturated fatty
acids(15, 46) . Higher rates of Cu(I) production in
comparison to the rates of
-tocopherol consumption found in our
study imply that
-tocopherol is regenerated by unknown mechanisms
during the oxidation. Interaction of
-tocopheroxyl radical with
lipoprotein polyunsaturated fatty acids might represent such a
mechanism of regeneration.
It is unclear at present whether the
reaction between Cu(II) and -tocopherol is important under
physiological conditions. Cell-mediated oxidation of LDL in media
containing transition metal ions has been shown to require
extracellular cysteine or other sulfhydryl
compounds(49, 50) . In accordance with this finding,
considerable acceleration of Cu(II) reduction by LDL in the presence of
cysteine was found in our study. These data suggest that the initial
reduction of transition metal ions may represent an important step in
the lipoprotein oxidation in vivo.