© 1995 American Heart Association, Inc.
Articles |
Differing 
-Tocopherol Oxidative Lability and Ascorbic Acid Sparing Effects in Buoyant and Dense LDL
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, and published in abstract form in Circulation (1994;90[pt 2]:I-409).
From the Department of Molecular and Nuclear Medicine (D.L.T., P.M.T., R.M.K.), Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, Calif, and Children's Hospital Oakland (Calif) Research Institute (J.J.M.v.d.B.).
Correspondence to Dr Diane L. Tribble, Lawrence Berkeley National Laboratory, Donner Laboratory, Room 465, University of California, Berkeley, CA 94720.
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract The enhanced oxidizability of smaller, more dense LDL is explained in part by a lower content of antioxidants, including ubiquinol-10 and
-tocopherol. In the present
studies, we also observed greater rates of depletion of
-tocopherol (mole per mole LDL per minute) in dense
(d=1.040 to 1.054 g/mL) compared with buoyant
(d=1.026 to 1.032 g/mL) LDL in the presence of either
Cu2+ or the radical-generating agent
2,2'-azobis(2-amidinopropane)dihydrochloride. Differences were
particularly pronounced at the lowest Cu2+ concentration
tested (0.25 µmol/L), with a fivefold greater rate in dense LDL. At
higher concentrations (1.0 and 2.5 µmol/L Cu2+), there
was a greater dependence of depletion rate on initial amount of
-tocopherol, which was reduced in dense LDL, thus
resulting in smaller subfraction-dependent differences in depletion
rates. Inclusion of ascorbic acid (15 µmol/L), an aqueous antioxidant
capable of recycling -tocopherol by hydrogen
donation, was found to extend the course of Cu2+-induced
-tocopherol depletion in both buoyant and dense LDL,
but this effect was more pronounced in dense LDL (time to
half-maximal -tocopherol depletion was extended
15.6-fold and 21.2-fold in buoyant and dense LDL, respectively, at 2.5
µmol/L Cu2+; P<.05). Thus, dense LDL exhibits
more rapid -tocopherol depletion and conjugated
diene formation than buoyant LDL when oxidation is performed in the
absence of ascorbic acid, but these differences are reversed in the
presence of ascorbic acid. These results suggest that differences in
oxidative behavior among LDL density subfractions may involve
differences in antioxidant activity and thus that the efficacy of
antioxidant regimens designed to inhibit LDL oxidation in vivo may vary
in relation to interindividual variations in LDL particle distribution
profiles.
Key Words: lipoproteins, low-density • -tocopherol • antioxidants • ascorbic acid • atherosclerosis
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Oxidative susceptibility has been shown to differ across the LDL particle spectrum, with a greater susceptibility consistently observed for smaller, more dense LDL particles. De Graaf et al1 reported a greater susceptibility of the most dense of three LDL subfractions, and Dejager et al2 reported a greater susceptibility of the most dense of five LDL subfractions based on shorter lag times before accelerated formation of conjugated dienes induced by Cu2+. In studies of six LDL subfractions, we observed a progressive increase in susceptibility to Cu2+-induced oxidation from less dense to more dense LDL, as indicated by greater rates of formation of conjugated dienes, thiobarbituric acid–reactive substances, and fluorescent pigments, and more rapid changes in anionic electrophoretic mobility.3 4 In view of evidence that oxidation renders LDL more atherogenic, these data have been offered as a partial explanation for the increased risk associated with LDL distribution profiles relatively enriched in small LDL particles (LDL subclass pattern B) (1-4).
Examination of differences in oxidative susceptibility among LDL
density subfractions has proved particularly valuable for identifying
particle attributes governing oxidative behavior. Recently, using
site-specific oxidation-labile fluorescent probes, we
obtained evidence that subfraction-related differences in oxidative
susceptibility occur early in the oxidation process and involve
differences in susceptibility to initial peroxidation events within the
surface monolayer compartment.5 6 Surface susceptibility
appears to be influenced in large part by the content of the
antioxidants ubiquinol-10 and
-tocopherol,5 which intercept radical
species and interrupt radical-chain propagation. However, other
events preceding antioxidant intervention (eg, radical initiation) or
affecting antioxidant radical–scavenging efficacy also may be
important. Improved definition of these early events and their role in
determining the enhanced oxidizability of dense LDL is expected to
provide insight into the most effective strategies for inhibiting LDL
oxidation and the atherogenic consequences thereof, particularly in
individuals with the LDL subclass pattern B phenotype.
In the present studies, to further characterize early events
leading to divergence in oxidative behavior among LDL density
subfractions, we examined the course of oxidative depletion of
-tocopherol in buoyant and dense LDL subfractions.
Our results indicate that -tocopherol is more labile
in dense than in buoyant LDL particles. Thus, subfraction-related
differences in oxidative susceptibility appear to involve not only
differences in the content of -tocopherol but also
differences in the rate of utilization of this antioxidant. Differences
in -tocopherol depletion and conjugated diene
formation between buoyant and dense LDL are reversed in the presence of
ascorbic acid, an aqueous antioxidant capable of regenerating
-tocopherol from the -tocopheroxyl
radical, suggesting that intervention at this stage in the oxidation
sequence is particularly effective in dense LDL particles.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Materials
TROLOX (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was obtained from Aldrich Chemical Co. EDTA, butylated hydroxytoluene, CuSO4, pyrogallol, ascorbic acid, and
- and 
-tocopherol were from Sigma Chemical
Co. AAPH [2,2x-azobis(2-amidinopropane)dihydrochloride] was from
Wako Chemicals. All reagents, buffer components, and
high-performance liquid chromatographic
solvents were of the highest grade commercially available.
LDL Subfraction Isolation and Characterization
Blood was obtained from healthy normolipidemic adult male
volunteers not using vitamin supplements or taking hormones or drugs
known to alter plasma lipids or lipoproteins. Samples were collected by
venipuncture into Vacutainers containing 1 mg/mL EDTA and
10 µmol/L TROLOX (a water-soluble -tocopherol
analogue), and plasma was separated from cells by
centrifugation at 2000g under refrigeration
(4°C). LDL subfractions were isolated from separate plasma aliquots
by sequential ultracentrifugation at
d=1.026 and 1.032 g/mL (for buoyant LDL) or
d=1.040 and 1.054 g/mL (for dense LDL) as previously
described.5 6 7 These density intervals contain LDL
subclasses I and III, respectively,8 and yield
preparations containing particles of nonoverlapping size as assessed by
gradient gel electrophoresis. LDL was dialyzed immediately for
analysis of physicochemical properties and oxidation
experiments.
Particle diameters of predominant lipoprotein peaks were determined by nondenaturing 2% to 16% gradient gel electrophoresis according to published methods.9 LDL protein concentrations were determined by use of the Lowry method modified to include sodium dodecyl sulfate.10 Phospholipid phosphorus was analyzed according to the method of Bartlett,11 and values were expressed as phosphatidylcholine equivalents. Total cholesterol and triglyceride concentrations were measured by standard enzymatic methods on a System 3500 Gilford computer-directed analyzer. Free and esterified cholesterol levels were determined by gas-liquid chromatography on a Hewlett-Packard 5830A gas chromatograph.12
-Tocopherol was determined by
high-performance liquid chromatography with
UV detection as previously described.13 Aliquots from the
LDL incubation (500 µL) were prepared by addition of ethanol (2 mL)
containing ascorbic acid 57 mmol/L, pyrogallol 1.6 mmol/L, butylated
hydroxytoluene 10 µmol/L, and -tocopherol 1 nmol/L
as an internal standard. The mixture was then extracted three times
with 2 mL hexane. High-performance liquid
chromatographic separation was accomplished with the use of
a 5-µmol/L Supelguard LC-NH2 (20x4.6-mm) precolumn
linked to a 5-µm Supelcosil LC-NH2 (250x4.6-mm)
analytical column. The flow rate was set at 1.3 mL/min with isocratic
elution using a mobile phase of hexane/2-propanol (93:7, vol/vol). The
amount of -tocopherol was quantified by comparison
with standards of known amount and was expressed as moles
-tocopherol per moles LDL.
LDL Oxidation
LDL (100 µg protein per milliliter) was incubated in
phosphate-buffered saline, pH 7.4, at 37°C. Oxidation was
initiated either with CuCl2 (0.25, 0.50, 1.0, or 2.5
µmol/L) or AAPH (0.25 or 0.50 mmol/L). For studies of
-tocopherol depletion, incubations were performed
with 1 to 2 mL LDL in loosely capped 20-mL glass tubes, and aliquots
were removed for measurement of the amount of
-tocopherol at 5- and 10-minute intervals for up to
2 hours after oxidant addition. The coefficient of variation for rates
of Cu2+-induced -tocopherol depletion
was previously shown to be less than 2%.14 Ascorbic acid
15 µmol/L was added to some preparations immediately before addition
of the oxidant. In these experiments, -tocopherol
concentrations were monitored for up to 3 hours. Because
-tocopherol is unstable in isolated
LDL,5 depletion experiments and
high-performance liquid chromatographic
analyses were performed within 3 to 4 days after isolation and
corresponding buoyant and dense LDL subfractions were always
analyzed simultaneously.
Conjugated diene formation, used as an index of LDL lipid peroxidation, was monitored in separate incubations by following the change in absorbance at 234 nm in a Shimadzu model UV 2101 spectrophotometer equipped with a temperature-controlled six-position automatic sample changer. Initial absorbance was set at zero and was recorded every 2 minutes for up to 8 hours at 37°C. The oxidative susceptibility of LDL was assessed on the basis of lag time, which was defined as the time interval between initiation and the intercept of the slope of the absorbance curve.
Statistical Analyses
Statistical analyses were performed with the
STATVIEW II statistical program. The significance of
differences in physicochemical properties,
-tocopherol depletion rates, and conjugated diene
lag times between buoyant and dense LDL was assessed by paired
t test (buoyant versus dense LDL) analysis.
Correlations between LDL -tocopherol depletion rates
and physicochemical properties were determined by linear regression
analyses, and differences in these relationships between
buoyant and dense LDL were evaluated by ANCOVA. All significance levels
were based on two-tailed tests.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Physicochemical Properties and Oxidizability of Buoyant and Dense LDL
As shown in Table 1
, buoyant and dense LDL
exhibited characteristic differences in size and chemical composition.
In dense LDL, phospholipid, free cholesterol, and
triglyceride content were reduced and cholesteryl ester and
protein content were increased when expressed on the basis of percent
mass. Of particular relevance to the present studies, in dense LDL,
the amount of -tocopherol was approximately 25%
lower on a mole per mole basis and approximately 15% lower when
expressed relative to total lipid mass. Consistent with
previous reports, the oxidative susceptibility of dense LDL was
increased relative to buoyant LDL, as indicated by shorter conjugated
diene lag times at 2.5 µmol/L Cu2+.
|
Cu2+-Induced Oxidation of -Tocopherol in
Buoyant and Dense LDL
Subfraction-dependent differences in
-tocopherol oxidative lability were evaluated by
comparing the course of depletion of this antioxidant after addition of
Cu2+. On the basis of preliminary observations suggesting
that subfraction differences may be more apparent under milder
oxidizing conditions, we performed experiments at four Cu2+
concentrations ranging from 0.25 to 2.5 µmol/L. Fig 1
shows, for a representative subject, the extent of
depletion of -tocopherol during the initial 10
minutes after addition of each of the four Cu2+
concentrations. The extent of depletion was highly dependent on the
Cu2+ concentration, although this relation was not linear;
absolute values varied almost 100-fold over a 10-fold range of
Cu2+ concentrations. There was a particularly marked
difference in depletion between 0.25 and 0.5 µmol/L
Cu2+, suggesting the existence of a threshold
Cu2+ concentration below which LDL, particularly buoyant
particles, was very resistant to oxidation. Subfraction
differences in the course of -tocopherol depletion
were most apparent at the lowest Cu2+ concentration (0.25
µmol/L Cu2+); the extent of depletion during the first 10
minutes was approximately twofold greater in dense than in buoyant LDL
in this subject. In contrast, at the highest Cu2+
concentration (2.5 µmol/L), the response was nearly identical in
buoyant and dense LDL.
|
) and dense (
)
LDL at four Cu2+ concentrations. Shown is the extent of
loss (in moles
Mean rates of depletion for buoyant and dense LDL from nine subjects
are shown in Table 2, and rates for corresponding
buoyant and dense LDL preparations are shown in Fig 2.
There was considerable interindividual variation in
-tocopherol depletion rates, although for a given
pair of LDL subfractions rates of depletion were almost always greater
in dense LDL, particularly at the lower three concentrations of
Cu2+. Pairwise comparisons indicated significantly greater
rates of -tocopherol depletion in dense than in
buoyant LDL at 0.25, 0.5, and 1.0 µmol/L Cu2+ (see Table 2). Subfraction differences in -tocopherol depletion
rates were not apparent at 2.5 µmol/L Cu2+. This was
attributed in part to the rapid course of
-tocopherol depletion relative to our sampling
frequency (every 5 minutes). The importance of sampling
frequency was indicated in separate experiments involving three sets of
LDL subfractions in which -tocopherol concentrations
were monitored at 1-minute intervals up to 5 minutes after addition of
2.5 µmol/L Cu2+. Subfraction differences were apparent at
the earliest time points (eg, rates of loss during the first minute
were 1.84-fold greater in dense than in buoyant LDL,
P<.05), but diminished as time progressed and were
nonexistent at 5 minutes, by which time the extent of
-tocopherol loss was usually greater than 50%.
|
|
Another factor contributing to reduced subfraction differences in
-tocopherol depletion rates at the higher
Cu2+ concentrations may have been the dependence of such
rates on the initial amount of -tocopherol, which
was greater in buoyant LDL (see Table 1
). As shown in Fig 3, rates of depletion were highly correlated with the
initial amount of -tocopherol in both buoyant and
dense LDL at 1.0 and 2.5 µmol/L Cu2+. This may reflect
the fact that -tocopherol is rate limiting under
these conditions. Notably, at 1.0 µmol/L Cu2+ (Fig 3, left), the relation between initial content and rate
of depletion of -tocopherol differed in buoyant and
dense LDL, as indicated by ANCOVA (P<.05), providing
additional evidence of a greater oxidative lability of
-tocopherol in dense LDL particles. The initial
amount of -tocopherol was not predictive of the rate
of -tocopherol loss in either buoyant or dense LDL
at 0.25 and 0.5 µmol/L Cu2+, suggesting that the
amount of -tocopherol was not rate limiting under
these milder oxidizing conditions. Among the other lipoprotein
properties characterized in this study (see Table 1), particle diameter
was observed to be the best predictor of -tocopherol
depletion rates at 0.5 µmol/L both within buoyant LDL
(r=–.75, P<.05) and among all LDL samples
(r=–.56, P<.05), with an inverse relation
between particle diameter and depletion rate. Also observed was a
significant inverse relation between free cholesterol
content and -tocopherol depletion rate among all
subfractions (r=-.52, P<.05).
|
AAPH-Induced Oxidation of -Tocopherol in Buoyant and
Dense LDL
To determine whether the enhanced oxidative lability of
-tocopherol in dense LDL is specific for metal
ion–induced oxidation, we also performed experiments using the
radical-generating agent AAPH (0.25 and 0.5 mmol/L). Mean rates of
-tocopherol depletion for seven sets of LDL
subfractions are shown in Table 3. As with
Cu2+-induced oxidation, rates were significantly greater in
dense than in buoyant LDL. In contrast to results obtained with
Cu2+, subfraction differences were not dependent on
oxidant concentration. Experiments were performed over a narrow range
of AAPH concentrations, however, and this may not have coincided with
the range of concentrations over which a concentration dependence
occurs.
|
Ascorbic Acid Inhibition of -Tocopherol Depletion
and Conjugated Diene Formation in Buoyant and Dense LDL
Ascorbic acid has been shown to preserve
-tocopherol and extend the course of LDL oxidation,
presumably owing to its ability to regenerate
-tocopherol from the -tocopheroxyl radical.
To assess whether ascorbic acid reduces subfraction differences in
susceptibility to -tocopherol depletion, we compared
the effects of addition of this antioxidant on Cu2+-induced
-tocopherol depletion in buoyant versus dense LDL.
As shown in Fig 4, ascorbic acid 15 µmol/L markedly
extended the course of -tocopherol depletion, as
indicated by times to 50% -tocopherol depletion in
the absence and presence of this agent. This effect was considerably
greater in dense than in buoyant LDL (at 2.5 µmol/L
Cu2+, time to half-maximal
-tocopherol depletion was extended 15.6-fold and
21.2-fold in buoyant and dense LDL, respectively; P<.05).
Thus, under these conditions, -tocopherol was more
labile to oxidative depletion in buoyant LDL. Corresponding increases
in conjugated diene lag times also were observed in the presence of
ascorbic acid (Fig 5), and as with
-tocopherol depletion, the increase was greater in
dense than in buoyant LDL (P<.05). As Fig 6
illustrates for a representative subject, the
preferential protection of dense LDL was observed over a range of
physiological ascorbic acid concentrations (15 to
45 µmol/L).
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We previously reported that the amount of
-tocopherol is reduced in dense LDL and that this is
associated with the enhanced oxidizability of these
particles.5 In the present studies, we show both a
reduced content and a greater oxidative lability of
-tocopherol in dense compared with buoyant LDL in
the presence of both Cu2+ and the radical-generating
agent AAPH. Notably, while the enhanced response of dense LDL was
observed for all subjects, these differences tended to be obscured by
the larger interindividual variation in -tocopherol
depletion rates. Subfraction differences were especially enhanced at
the lowest Cu2+ concentration (0.25 µmol/L) and were not
apparent at the highest Cu2+ concentration (2.5 µmol/L)
unless samples were collected within minutes of oxidant introduction.
Since in vitro measures of LDL oxidative susceptibility usually involve
Cu2+ concentrations greater than 2.5 µmol/L, the observed
differences in -tocopherol oxidative lability are
likely to be of little significance to differences in LDL oxidative
susceptibility in vitro. Variations in -tocopherol
lability could be of considerable importance in vivo, however, where
oxidant stress conditions are expected to be mild.
Subfraction differences in -tocopherol depletion
rates could reflect differences in rates of oxidative events occurring
before the involvement of -tocopherol. As Fig 7 illustrates, initial events in
Cu2+-induced oxidation are suggested to include
Cu2+ binding and reductive activation to Cu+
(the proximate oxidant) (reaction I). Esterbauer and
colleagues17 18 proposed that Cu2+ binding
and/or reduction varies among LDL preparations, as determined by
variations in the presence of catalytically active
Cu2+-binding centers and intrinsic reducing power, and that
this may be a major factor contributing to differences in
susceptibility to Cu2+-induced oxidation. In addition to
these events, recent studies have shown that Cu2+-induced
LDL oxidation is dependent on the presence of performed hydroperoxides,
which serve as sites of radical initiation (reaction II, Fig 7).19 20 Consistent with these observations, Frei
and Gaziano21 have reported that the hydroperoxide content
varies in freshly isolated LDL and is predictive of variations in
conjugated diene lag times in Cu2+-exposed LDL. It appears
possible therefore that differences in rates of Cu2+
binding and reductive activation or rates of hydroperoxide scission may
lead to differences in -tocopherol depletion rates
in buoyant and dense LDL. This is unlikely to be the sole explanation,
however, because subfraction-dependent differences in
-tocopherol lability also were apparent in the
presence of AAPH, which does not require binding or activation, or the
presence of preexisting hydroperoxides but decomposes unimolecularly in
the aqueous phase to yield free radicals at a constant
rate.16
|
Alternatively or in addition to these factors, the enhanced lability of
-tocopherol in dense LDL could reflect a reduced
-tocopherol radical–scavenging efficacy, which
is determined by the balance between radical inhibition and propagation
reactions. Although typically considered to be of importance only after
depletion of chain-breaking antioxidants, radical propagation
reactions (eg, reactions IV, V, VII, VIII, and X; Fig 7) also may occur concurrent with inhibition reactions
(reactions III, VII, and IX; Fig 7). Under conditions
favoring propagation reactions, the hydroperoxide-radical pool
could increase rapidly and impose a much greater oxidant burden on the
existing -tocopherol pool. Differences in the
radical-scavenging or inhibitory efficacy of
-tocopherol have been observed in model lipid
systems22 and, more recently, among LDL
preparations.14 23 24 Results of the present studies
suggest that LDL subclass distribution profiles may contribute to
variations in -tocopherol inhibitory
efficacy among LDL preparations, but given the considerable
interindividual variation in -tocopherol depletion
rates among buoyant and among dense LDL preparations, other factors
clearly are involved.
One of the most important LDL attributes contributing to variations in
-tocopherol radical–scavenging activity may be
fatty acyl composition, specifically the degree of enrichment with
polyunsaturated fatty acyl components, which serve as substrates for
oxidation and compete with -tocopherol for
interaction with radical species. Since differences in fatty acyl
composition among LDL density subfractions appear to be small relative
to those observed among LDL from different
individuals,1 2 7 this factor is likely to be more
important for determining interindividual than subfraction-related
variations in -tocopherol depletion rates. Another
particle attribute of potential significance is the content of
ubiquinol-10, which is suggested to exert sparing and regenerating
effects on -tocopherol.25 Ubiquinol-10
content was not measured in the present studies but was previously
found to be substantially reduced in dense relative to buoyant LDL
preparations5 and thus potentially could be important to
subfraction differences in -tocopherol oxidative
lability.
Regardless of the mechanism or mechanisms involved, our finding of
greater -tocopherol depletion rates in dense LDL is
consistent with the hypothesis that subfraction differences in
oxidative behavior occur early in the oxidation
process.5 6 As an extension, we have proposed that
interventions targeting these early events are likely to be the most
effective strategies for inhibiting oxidation, particularly in dense
LDL particles. In the present studies, we examined the
inhibitory effects of ascorbic acid, an aqueous antioxidant
that has been shown to preserve -tocopherol and to
extend the oxidative resistance of LDL.26 27 28 This agent
does not appear to be efficient in directly intercepting lipid peroxyl
radicals within monolayer-bilayer systems but rather has been
suggested to protect lipids, including those in LDL, primarily through
regeneration of -tocopherol from the
-tocopheroxyl radical at the water–lipid
interface.29 30 31 Inclusion of ascorbic acid was found to
extend the course of -tocopherol depletion and
conjugated diene formation in both LDL subfractions, but this effect
was much more pronounced in dense LDL, thus resulting in a reversal in
the relative susceptibility of buoyant and dense LDL.
Prolongation of the course of -tocopherol depletion
is consistent with the premise that ascorbic acid protection
occurs through -tocopherol preservation, such as by
recycling of -tocopherol. Our observation that
ascorbic acid protection is greater in dense LDL therefore suggests
that the efficacy of -tocopherol recycling may be
greater in these particles relative to buoyant LDL. The results of
studies in model lipid systems indicate that a number of factors, both
compositional and physical, could be responsible for such differences.
In micellar and bilayer systems, for example, the reaction of ascorbic
acid with the -tocopheroxyl radical is decreased by addition of
negatively charged lipids and is increased by addition of positively
charged lipids.30 Surface charge properties are known to
differ between buoyant and dense LDL.32 However, the
greater negative surface charge in dense LDL would be expected to
reduce rather than potentiate interactions with ascorbic acid. The
efficacy of ascorbic acid recycling of -tocopherol
homologues in bilayer systems also has been shown to increase in
accordance with increased mobility of these homologues.29
A greater mobility of -tocopherol within the surface
monolayer and between surface and core in dense relative to buoyant LDL
thus could lead to more efficient -tocopherol
recycling in dense LDL. This effect could be further potentiated by a
greater surface disposition of -tocopherol in
smaller, more dense particles, which arises from the greater ratio of
surface volume to core volume in these particles.
Another factor possibly affecting the inhibitory capacity of ascorbic acid is its pro-oxidant activity. Ascorbic acid is known to serve as a source of reducing equivalents for the redox cycling of transition metal ions and as a result of this activity has been shown to potentiate lipid peroxidation in some oxidizing systems.33 34 35 The protective effect of ascorbic acid in Cu2+-exposed LDL observed in the present and previous studies26 27 28 29 indicates that any such potentiation, if it occurs, is overcome by the antioxidant effects of this agent. The balance between the pro-oxidant and antioxidant activities of ascorbic acid could differ in buoyant and dense LDL, leading to subfraction differences in its net inhibitory effect.
Protection by ascorbic acid may also occur independently of its
hydrogen-donating activities. Retsky et al.28 recently
provided evidence that inhibition of Cu2+-catalyzed LDL
oxidation may involve covalent modification by ascorbic acid oxidation
products, which in turn may inhibit LDL-Cu2+ binding. Such
modification could occur to a greater degree or displace
Cu2+ more effectively in dense LDL particles. This effect
would also be expected to preserve the -tocopherol pool, and this is
consistent with the present data. Further studies are needed to
determine the extent to which this and the aforementioned antioxidant
mechanisms are involved in the preferential protection of dense LDL by
ascorbic acid.
In summary, the results presented here indicate that
-tocopherol is expended more readily in dense than
in buoyant LDL under oxidizing conditions but that this effect as well
as the greater oxidative susceptibility of dense LDL may be overcome in
the presence of ascorbic acid. These findings raise the possibility
that the efficacy of antioxidant regimens in reducing LDL oxidation in
vivo may vary among individuals in relation to LDL particle size and
density distribution profiles. In particular,
-tocopherol may be less effective and ascorbic acid
may be more effective in individuals exhibiting the high-risk
lipoprotein pattern–B phenotype, which is characterized by a
predominance of smaller, more dense LDL particles. This and other
subject-specific variations in antioxidant effectiveness should be
considered when the antiatherogenic properties of antioxidants are
evaluated in human populations.
|
Acknowledgments |
|---|
We acknowledge Dr Nancy Cook-van den Berg for technical assistance with the analysis of
-tocopherol
and Robin Rawlings, RN, for subject recruitment and blood collection
and processing. This research was supported by National Institutes of
Health program project grant HL-18574 from the National Heart,
Lung, and Blood Institute, Bethesda, Md, and a grant from the National
Dairy Promotion and Research Board administered in cooperation with the
National Dairy Council, Rosemont, Ill, and was conducted at the
Lawrence Berkeley National Laboratory through the US Department of
Energy under contract DE-AC03-76SF00098. Received February 25, 1995; accepted September 9, 1995.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. de Graaf J, Hak-Lemmers HLM, Hectors MPC, Denmarcker PNM, Hendriks JCM, Stalenhoef AFH. Enhanced susceptibility to in vitro oxidation of the dense low density lipoprotein subfraction in healthy subjects. Arterioscler Thromb. 1991;11:298-306.
2. Dejager S, Bruckert E, Chapman MJ. Dense low density lipoprotein subspecies with diminished oxidative resistance predominate in combined hyperlipidemia. J Lipid Res. 1993;34:295-308. [Abstract]
3. Tribble DL, Holl LG, Wood PD, Krauss RM. Variations in oxidative susceptibility among six low density lipoprotein subfractions of differing density and particle size. Atherosclerosis. 1992;93:189-199. [Medline] [Order article via Infotrieve]
4. Chait A, Brazg RL, Tribble DL, Krauss RM. Susceptibility of small, dense, low-density lipoproteins to oxidative modification in subjects with the atherogenic lipoprotein phenotype pattern B. Am J Med. 1993;94:350-356. [Medline] [Order article via Infotrieve]
5.
Tribble DL, van den Berg J, Motchnik P, Ames B, Lewis
D, Chait A, Krauss RM. Oxidative susceptibility of low density
lipoprotein subfractions is related to their ubiquinol-10 and
-tocopherol content. Proc Natl Acad
Sci U S A. 1994;91:1183-1187.
6. Tribble DL, Krauss RM, Lansberg MG, Thiel PM, van den Berg JJM. Greater oxidative susceptibility of the surface monolayer in small, dense LDL may contribute to differences in copper-induced oxidation among LDL density subfractions. J Lipid Res. 1995;35:662-671.
7.
Reaven PD, Grasse BJ, Tribble DL. Effects of
linoleate-rich and oleate-rich diets in combination with
-tocopherol on the susceptibility of low-density
lipoproteins (LDL) and LDL subfractions to oxidative modification in
humans. Arterioscler Thromb. 1994;14:557-566.
8. Musliner TA, Krauss RM. Lipoprotein subspecies and risk of coronary disease. Clin Chem. 1988;33:B78-B83.
9. Krauss RM, Burke DJ. Identification of multiple subclasses of plasma low density lipoprotein in humans. J Lipid Res. 1982;23:97-104. [Abstract]
10. Markwell MAK, Haas SM, Bieber LL, Tolbert NE. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein systems. Anal Biochem. 1978;87:206-210. [Medline] [Order article via Infotrieve]
11.
Bartlett GR. Phosphorus assay in column
chromatography. J Biol
Chem. 1959;234:466-468.
12. Hindriks FR, Wolthers BG, Groen A. The determination of total cholesterol in serum by gas-liquid chromatography compared with two other methods. Clin Chim Acta. 1977;74:207-215. [Medline] [Order article via Infotrieve]
13. van den Berg JJM, Kuypers FA, Roelofsen B, Op den Kamp JAF. The cooperative action of vitamins E and C in the protection against peroxidation of parinaric acid in human erythrocyte membranes. Chem Phys Lipids. 1990;53:309-320. [Medline] [Order article via Infotrieve]
14. Cominacini L, Garbin U, Cenci B, Davoli A, Pasini C, Ratti E, Gaviraghi G, Lo Cascio V, Pastorino AM. Predisposition to LDL oxidation during copper-catalyzed oxidative modification and its relation to alpha-tocopherol content in humans. Clin Chim Acta. 1991;204:57-63. [Medline] [Order article via Infotrieve]
15. Bowry VW, Ingold KU, Stocker R. Vitamin E in human low-density lipoprotein: when and how this antioxidant becomes a pro-oxidant. Biochem J. 1992;288:341-344.
16.
Ingold KU, Bowry VW, Stocker R, Walling C.
Autoxidation of lipids and antioxidation by
-tocopherol and ubiquinol in homogeneous
solution and in aqueous dispersions of lipids: unrecognized
consequences of lipid particle size as exemplified by oxidation of
human low density lipoprotein. Proc Natl Acad Sci
U S A. 1993;90:45-49.
17. Esterbauer H, Jurgens G. Mechanistic and genetic aspects of susceptibility of LDL to oxidation. Curr Opin Lipidol. 1993:114-124.
18. Geiseg SP, Esterbauer H. Low density lipoprotein is saturable by pro-oxidant copper. FEBS Lett. 1994;343:188-194. [Medline] [Order article via Infotrieve]
19.
Thomas CE, Jackson RL. Lipid hydroperoxide
involvement in copper-dependent and independent oxidation of low
density lipoproteins. J Pharmacol Exp
Ther. 1991;256:1182-1188.
20. Thomas JP, Kalyanaraman B, Girotti AW. Involvement of preexisting lipid hydroperoxides in Cu2+-stimulated oxidation of low-density lipoprotein. Arch Biochem Biophys. 1994;315:244-254. [Medline] [Order article via Infotrieve]
21. Frei B, Gaziano JM. Content of antioxidants, preformed lipid hydroperoxides, and cholesterol as predictors of the susceptibility of human LDL to metal ion-dependent and -independent oxidation. J Lipid Res. 1993;34:2135-2145. [Abstract]
22. Niki E. Lipid antioxidants: how they act in biological systems. Br J Cancer. 1987;18:153-157.
23.
Esterbauer H, Dieber-Rothenender M, Striegl G, Waeg
G. Role of vitamin E in preventing the oxidation of
low-density lipoprotein. Am J Clin Nutr. 1991;53:314S-321S.
24. Esterbauer H, Gebicki J, Puhl H, Jurgens G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic Biol Med. 1992;13:341-390. [Medline] [Order article via Infotrieve]
25.
Stocker R, Bowry VW, Frei B. Ubiquinol-10
protects human low density lipoprotein more efficiently against lipid
peroxidation than does alpha-tocopherol.
Proc Natl Acad Sci U S A. 1991;88:1646-1650.
26. Sato K, Niki E, Shimasaki H. Free radical-mediated chain oxidation of low density lipoprotein and its synergistic inhibition by vitamin E and C. Arch Biochem Biophys. 1990;279:402-405. [Medline] [Order article via Infotrieve]
27. Jialal I, Grundy SM. Preservation of the endogenous antioxidants in low density lipoprotein by ascorbate but not probucol during oxidative modification. J Clin Invest. 1991;87:597-601.
28.
Retsky KL, Freeman MW, Frei B. Ascorbic acid
oxidation product(s) protect human low density lipoprotein against
atherogenic modification: anti- rather than prooxidant activity of
vitamin C in the presence of transition metal ions.
J Biol Chem. 1993;268:1304-1309.
29. Kagan VE, Serbinova EA, Packer L. Recycling and antioxidant activity of tocopherol homologs of differing hydrocarbon chain links in liver microsomes. Arch Biochem Biophys. 1990;282:221-225. [Medline] [Order article via Infotrieve]
30. Bisby RH, Parker AW. Reaction of ascorbate with the alpha-tocopheroxyl radical in micellar and bilayer membrane systems. Arch Biochem Biophys. 1995;317:170-178. [Medline] [Order article via Infotrieve]
31. Kagan VE, Serbinova EA, Forte T, Scita G, Packer L. Recycling of vitamin E in human low density lipoprotein. J Lipid Res. 1992;33:385-397. [Abstract]
32. Nigon F, Lesnik P, Rouis M, Chapman MJ. Discrete subspecies of human low density lipoproteins are heterogeneous in their interaction with the cellular LDL receptor. J Lipid Res. 1991;32:1741-1753. [Abstract]
33. Ohyashiki T, Adachi R, Matsui K. Changes in surface charge density of lecithin liposomes by lipid peroxidation: a fluorescence study with 8-anilino-1-naphthalenesulfonate. Chem Pharm Bull (Tokyo). 1991;39:3295-3298. [Medline] [Order article via Infotrieve]
34. Ghosh C, Dick RM, Ali SF. Iron/ascorbate-induced lipid peroxidation changes membrane fluidity and muscarinic cholinergic receptor binding in rat frontal cortex. Neurochem Int. 1993;23:479-484. [Medline] [Order article via Infotrieve]
35. Miura T, Muraoka S, Ogiso T. Effect of ascorbate on adriamycin-Fe(3+)-induced lipid peroxidation and DNA damage. Pharmacol Toxicol. 1994;74:89-94.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  W. L. Hall, Y. M. Jeanes, and J. K. Lodge Hyperlipidemic Subjects Have Reduced Uptake of Newly Absorbed Vitamin E into Their Plasma Lipoproteins, Erythrocytes, Platelets, and Lymphocytes, as Studied by Deuterium-Labeled {alpha}-Tocopherol Biokinetics J. Nutr., January 1, 2005; 135(1): 58 - 63. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Tribble, R. M. Krauss, B. M. Chu, E. L. Gong, B. R. Kullgren, J. O. Nagy, and M. La Belle Increased low density lipoprotein degradation in aorta of irradiated mice is inhibited by preenrichment of low density lipoprotein with {alpha}-tocopherol J. Lipid Res., October 1, 2000; 41(10): 1666 - 1672. [Abstract] [Full Text]
|
|
|
|
|
|
D. C. Schwenke, J. D. Wagner, and M. R. Adams In vitro lipid peroxidation of LDL from postmenopausal cynomolgus macaques treated with female hormones J. Lipid Res., February 1, 1999; 40(2): 235 - 244. [Abstract] [Full Text]
|
|
|
|
 |
|
 Y. B. de Rijke*, S. J.H. Bredie*, P. N.M. Demacker, J. M. Vogelaar, H. L.M. Hak-Lemmers, and A. F.H. Stalenhoef The Redox Status of Coenzyme Q10 in Total LDL as an Indicator of In Vivo Oxidative Modification: Studies on Subjects With Familial Combined Hyperlipidemia Arterioscler. Thromb. Vasc. Biol., January 1, 1997; 17(1): 127 - 133. [Abstract] [Full Text]
|
|
|
|
|
|
A. Sevanian, J. Hwang, H. Hodis, G. Cazzolato, P. Avogaro, and G. Bittolo-Bon Contribution of an In Vivo Oxidized LDL to LDL Oxidation and Its Association With Dense LDL Subpopulations Arterioscler. Thromb. Vasc. Biol., June 1, 1996; 16(6): 784 - 793. [Abstract] [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||