(Circulation. 1997;95:1378-1385.)
© 1997 American Heart Association, Inc.
Articles |
Antioxidant Protection of LDL by Physiological Concentrations of 17ß-Estradiol
Requirement for Estradiol Modification
From the Evans Memorial Department of Medicine and Whitaker Cardiovascular Institute, Boston (Mass) University Medical Center.
Correspondence to John F. Keaney, Jr, MD, Whitaker Cardiovascular Institute, Room W507, Boston University Medical Center, 80 E Concord St, Boston, MA 02118. E-mail jkeaney{at}acs.bu.edu.
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Abstract |
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Background Exposure to estrogens reduces the risk for coronary artery disease and associated clinical events; however, the mechanisms responsible for these observations are not clear. Supraphysiological levels of estrogens act as antioxidants in vitro, limiting oxidation of low-density lipoprotein (LDL), an event implicated in atherogenesis. We investigated the conditions under which physiological concentrations of 17ß-estradiol (E2) inhibit oxidative modification of LDL.
Methods and Results Plasma incubated with E2
(0.1 to 100 nmol/L) for 4 hours yielded LDL that demonstrated a
dose-related increase in resistance to oxidation by Cu2+ as
measured by conjugated diene formation. This effect was dependent on
plasma, because incubation of isolated LDL with E2 at these
concentrations in buffered saline produced no effect on
Cu2+-mediated oxidation. Incubation of plasma with
E2 had no effect on LDL 
-tocopherol content or
cholesteryl ester hydroperoxide formation during the 4-hour incubation.
Plasma incubation with [3H]E2 was associated
with dose-dependent association of 3H with LDL.
High-performance liquid chromatographic analysis of LDL derived from
plasma incubated with [3H]E2 indicated that
the majority of the associated species were not detectable as authentic
E2 but as nonpolar forms of E2 that were
susceptible to base hydrolysis consistent with fatty acid
esterification of E2. Plasma-mediated association of
E2 and subsequent antioxidant protection was inhibited by
5,5'-dithio-bis(2-nitrobenzoic acid), an inhibitor of plasma
acyltransferase activity.
Conclusions Exposure of LDL to physiological levels of E2 in a plasma milieu is associated with enhanced resistance to Cu2+-mediated oxidation and incorporation of E2 derivatives into LDL. This antioxidant capacity may be another means by which E2 limits coronary artery disease in women.
Key Words: hormones • lipoproteins • women • antioxidants
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Introduction |
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Coronary artery disease is more prevalent in men than in age-matched women1 and in postmenopausal than in premenopausal women.2 Furthermore, treatment of postmenopausal women with estrogen replacement reduces the incidence of coronary events in this population, even in those with established CAD.3 4 The precise mechanisms responsible for these observations are not clear, although estrogens favorably alter established risk factors for CAD.1 Oral estrogen therapy is associated with increased HDL cholesterol and decreased LDL cholesterol levels,5 although only 25% to 50% of the reduction in CAD with estrogen therapy is due to these alterations in plasma lipid profiles.6 7
The oxidative modification of LDL has been implicated in the initiation and progression of atherosclerosis.8 It has been proposed that LDL accumulates in the subendothelial space of lesion-prone arterial sites where vascular cells oxidatively modify this LDL into a form that is internalized by scavenger receptors of resident macrophages, resulting in the formation of "foam cells" that are characteristic of early atherosclerotic lesions.9 10 11 Epitopes from ox-LDL generated in vitro have been identified in atherosclerotic lesions,12 and LDL extracted from these lesions has physicochemical characteristics similar to ox-LDL.13 Moreover, antibodies to ox-LDL have been identified in sera of patients with atherosclerosis.14 Antioxidants that inhibit LDL oxidation have been shown to retard the development of atherosclerosis in animal models,15 and recent reports associate high intake of vitamin E with a reduced risk of CAD in both men and women.16 17
Emerging evidence suggests that estrogens may act as
antioxidants. Estrogens with a phenolic structure protect LDL from both
cellular and Cu2+-mediated oxidation in
vitro.18 19 20 21 One problem with these studies is the
requirement of supraphysiological estrogen concentrations (
1
µmol/L) for meaningful antioxidant protection of LDL. Recently, Sack
and colleagues22 demonstrated that E2
treatment of postmenopausal women is associated with increased LDL
resistance to ex vivo Cu2+-mediated oxidation. In
ovariectomized swine, we have found antioxidant protection of LDL with
physiological levels of E2 replacement.23 The
mechanism(s) for this antioxidant protection in vivo and the
discrepancy between effective doses in vivo and in vitro have yet to be
resolved. In the present study, we examined the conditions under which
physiological concentrations of E2, the most abundant form
of estrogen, provide antioxidant protection of LDL.
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Methods |
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LDL Isolation and Incubation With E2
Blood was collected from healthy, normolipidemic, male donors after an overnight fast into evacuated containers (Becton-Dickinson Co) containing 286 USP U sodium heparin per 15 mL of whole blood. Plasma was prepared by centrifugation (1200g) at 4°C for 15 minutes. Plasma was incubated with ethanolic additions of E2 or E2 17-stearate (both, Steraloids Inc) at final concentrations from 0.1 to 100 nmol/L (final ethanol concentration, 0.2% vol/vol) or vehicle alone for 4 hours at 37°C in the dark sealed under N2 gas. LDL was subsequently isolated by single vertical-spin discontinuous density gradient ultracentrifugation.24 Unbound estrogens and low-molecular-weight contaminants were removed by size-exclusion gel filtration with the use of a Sephadex G-25 column (Pharmacia Biotech, Inc) prepared as previously described.25 Contaminating metal ions were removed by brief treatment of the sample with Chelex-100 resin (Bio-Rad Laboratories) followed by centrifugation (10 000g) for 2 minutes. Protein was determined by a modified procedure of Lowry.26
Alternatively, LDL was isolated from fresh plasma and 
0.5 mg/mL LDL
(ie, the concentration approximating that in plasma) was incubated with
0.1 to 100 nmol/L E2 or E2 17-stearate in PBS
(154 mmol/L NaCl, 10 mmol/L NaH2PO4, pH 7.4) as
described above. After incubation, LDL was gel filtered and Chelex
treated as described above, followed by protein determination. LDL
isolated in this manner contained no detectable lipid hydroperoxides
(see below) and was used immediately for experiments.
Measurement of E2
Plasma and LDL E2 levels were quantified by RIA
after extraction with ethyl ether as previously
described.23 Antibody to E2 (supplied by Dr
G.D. Niswender, Colorado State University) showed <5%
cross-reactivity with other estrogens (data not shown). Limits of
detection for plasma and LDL E2 levels were 0.018 nmol/L
and 0.037 pmol/mg LDL protein, respectively. Standard curves were
performed with authentic E2 and were unaffected by the
presence of LDL or E2 17-stearate.
Oxidative Modification of LDL
For measurement of LDL susceptibility to oxidation, incubations
contained 100 µg LDL protein in 1 mL PBS and a final concentration of
3.3 µmol/L CuCl2 or 4.0 mmol/L AAPH (Eastman Kodak). LDL
oxidation was monitored by conjugated diene formation at 37°C in a
Varian Cary 3 spectrophotometer, and duration of the lag phase was
calculated as previously described.27 In selected
experiments, LDL oxidation was quantified as the formation of
TBARS28 or as an increase in LDL electrophoretic mobility
on 0.5% agarose gels29 with the use of a Paragon
electrophoresis system (Beckman Instruments) according to the
manufacturer's instructions.
LDL Antioxidant and Cholesteryl Ester Hydroperoxide Content
Isolated LDL (250 µg protein) was precipitated with an equal
volume of HPLC-grade methanol (Sigma Chemical Co), extracted with 10
volumes of hexane (Aldrich Chemical Co), and centrifuged at
500g for 10 minutes at 4°C. An aliquot of the hexane
extract was dried under N2 and resuspended in 0.1 mL
ethanol. Vitamin E, ß-carotene, and lycopene were determined by HPLC
with electrochemical detection as described previously.30
Aliquots of these samples were also analyzed for cholesteryl ester
hydroperoxides by HPLC with postcolumn chemiluminescence detection as
described previously.31
Incorporation of [3H]E2 Into LDL
Plasma or isolated LDL was incubated with 0.1 to 100 nmol/L
[3H]E2 (DuPont NEN; specific activity, 142
Ci/mmol) as described above for nonradiolabeled E2. After
4-hour incubations, samples were gel filtered through two successive
Sephadex G-25 columns to remove free [3H]E2,
and the amount of radioactivity associated with LDL was determined by
scintillation counting (LKB 1214 Betarack). Limits of detection were
0.7 fmol E2/mg LDL protein. Aliquots of LDL were
precipitated with heparin-MnCl232 to determine
the amount of [3H]E2 free in solution or
bound to albumin, which is reported to be a minor contaminant of the
LDL fraction isolated by the single vertical-spin
method.24 Electrophoresis of the heparin-MnCl2
precipitate yielded no detectable albumin by Coomassie staining, and
<8% of the radioisotope associated with the LDL fraction was present
in the albumin-containing supernatant.
HPLC Analysis of LDL and Associated
[3H]E2
LDL (200 µg) exposed to [3H]E2 in
plasma or PBS was extracted with methanol and hexane as described
above. The hexane layer was dried under N2 and
reconstituted in ethanol. LDL lipids were chromatographed by use of a
25-cm LC-18 column (Supelco, Inc) and a Hewlett-Packard series 1050
HPLC system with absolute methanol used as mobile phase at a flow rate
of 1.0 mL/min with UV detection at 210 nm. Elution of
[3H]E2 was determined by scintillation
counting of 0.3-mL fractions of eluate collected every 18 seconds. For
characterization of putative E2 fatty acid esters, aliquots
of LDL lipids from incubations with 100 nmol/L
[3H]E2 in plasma or PBS were hydrolyzed for
18 hours at 50°C in 90% aqueous methanol solution with 0.5%
NaHCO3 or 1% K2CO3 (both wt/vol),
as previously described,33 34 before extraction and
detection by HPLC as described above. Preliminary experiments with
E2 3-stearate (kindly provided by Dr R. Hochberg, Yale
University School of Medicine, New Haven, Conn) and E2
17-stearate confirmed earlier reports that C3 esters (phenolic esters)
are easily hydrolyzed by a weak base such as
NaHCO3,33 whereas C17 esters require
aggressive hydrolysis with
K2CO3.34
E2 Modification and LDL Oxidation
To inhibit E2 modification, plasma was incubated
with 10 nmol/L E2 or [3H]E2 as
described above in the presence of vehicle (DMSO, final volume 0.7%)
or 1.4 mmol/L DTNB (ICN Biomedicals, Inc), which inhibits LCAT activity
by 90%.35 After incubation, association of
[3H]E2 with isolated LDL was determined by
scintillation counting, and resistance to Cu2+-mediated
oxidation was measured as described above.
Data Analysis
All data are reported as mean±SE. The measures of LDL oxidative
resistance and LDL antioxidant content were compared among treatment
groups by ANOVA and a post hoc Bonferroni t test or Dunn's
multiple comparison test where appropriate. Statistical significance
was accepted if the null hypothesis was rejected with a value of
P<.05.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The coincubation of E2 with isolated LDL in PBS was associated with dose-dependent inhibition of LDL oxidation (Fig 1
). Consistent with previous reports,19 20
we did not observe meaningful inhibition of LDL oxidation with
E2 concentrations <1 µmol/L. At 1 µmol/L,
E2 equally protected LDL from Cu2+- or
AAPH-mediated oxidation, with corresponding increases in the lag phase
of 26% and 29%.
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), 10 (
), 100 (
), 1000 (
), or 5000 (
)
nmol/L E2. LDL oxidation was monitored at 5-minute
intervals as the increase in absorbance at 234 nm over time. For
clarity, symbols are shown for every fourth data point. Data are
representative of three independent experiments.
In vivo, LDL is exposed to E2 (0.15 to 2
nmol/L36 ) in a plasma milieu. Therefore, we incubated
plasma with 0.1 to 100 nmol/L E2 before LDL isolation and
determined the resistance of this isolated LDL to
Cu2+-mediated oxidation. Incubation of plasma with 0.1, 1,
10, and 100 nmol/L E2 prolonged LDL resistance to
Cu2+-mediated oxidation by -4±6%, 15±6%, 26±4%, and
44±3%, respectively (Fig 2A; P=.00014 for
trend by ANOVA). Assessment of LDL oxidation by TBARS or relative
electrophoretic mobility revealed a similar dose-dependent inhibition
of Cu2+-induced oxidation (Fig 2, B and C). In contrast, if
LDL was first isolated from plasma then incubated for 4 hours with 0.1
to 100 nmol/L E2 in PBS before E2 removal by
gel filtration, we did not observe protection against
Cu2+-mediated oxidation (data not shown). Thus, antioxidant
protection of LDL by physiological concentrations of E2
requires that LDL be exposed to E2 in a plasma milieu.
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To compare the effect of E2 with a more traditional
antioxidant, we incubated plasma with 60, 90, or 120 µmol/L
-tocopherol for 4 hours, isolated LDL, and examined LDL resistance
to Cu2+-mediated oxidation. In these experiments,
-tocopherol prolonged LDL resistance to oxidation by 16%, 29%, and
41% (mean of two independent experiments; data not shown), which is
comparable to previous reports.37 38 Thus, the antioxidant
protection afforded by physiological levels of E2 is
comparable to that obtained with physiological concentrations of
-tocopherol.
We investigated the time course for E2-mediated protection against LDL oxidation by incubating plasma with 10 nmol/L E2 for 1 to 4 hours before LDL isolation and exposure to Cu2+. Preincubation of plasma with 10 nmol/L E2 for 1, 2, or 4 hours increased LDL resistance to oxidation by 15±5%, 13±2%, and 25±10%, respectively (P=NS for 1 versus 2 or 4 hours, n=3; data not shown).
Persistent antioxidant protection by E2 after LDL isolation
from plasma suggests some alteration in the LDL particle that imparts
resistance to Cu2+-mediated oxidation. For example,
E2 is known to regenerate -tocopherol from its oxidized
form,39 and LDL enrichment with -tocopherol is
associated with increased LDL resistance to
oxidation.37 38 Similarly, "preformed" lipid
hydroperoxides are important determinants of LDL resistance to
oxidation.40 To investigate the effect of plasma
E2 incubation on these determinants, LDL was isolated from
fresh plasma or from plasma incubated with 0.1 to 100 nmol/L
E2 or vehicle for 4 hours, and LDL antioxidant and
cholesteryl ester hydroperoxide content were measured. As shown in
Table 1, the LDL content of antioxidants
(-tocopherol, lycopene, and ß-carotene) was not significantly
altered by incubation with E2 in plasma (P=NS
for before and after a 4-hour incubation). Similar results were
observed with all E2 concentrations. Additionally, there
was no detectable formation of cholesteryl ester hydroperoxides in LDL
incubated with E2 or vehicle for 4 hours.
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The incorporation of E2 into LDL as a means of protection
against Cu2+-mediated oxidation was investigated by
incubating plasma or isolated LDL (0.5 to 0.7 mg/mL in PBS, equivalent
to the LDL concentrations in donor plasma) with 0.1 to 100 nmol/L
[3H]E2 or unlabeled E2 for 4
hours. As shown in Table 2, incubation of plasma with
increasing concentrations of [3H]E2 was
associated with dose-dependent incorporation of the radioisotope into
LDL isolated from this plasma (P=.0013 for trend by ANOVA).
In contrast, incubation of LDL with [3H]E2 in
PBS resulted in 5-fold less association of the radioisotope (Table 2; P<.05 versus plasma incubation). Thus, plasma
facilitates the association of E2 with LDL.
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Parallel experiments performed with unlabeled E2 indicate
that this associated species is not recognized as authentic
E2 by RIA (Table 2
). With plasma incubations of 100 nmol/L
E2, the amount of LDL-associated E2 detected by
scintillation counting was 18 times that detected by RIA. In contrast,
LDL incubated with E2 in PBS demonstrated comparable
E2 associations with the use of either detection method.
Importantly, RIA detection of E2 was not impaired by the
presence of LDL at concentrations as high as 1 mg/mL (data not shown).
Thus, incubation of plasma with E2 results in enhanced
association of E2 with LDL, primarily in a form not
recognized as authentic E2.
To characterize the E2 that associates with LDL, we
incubated plasma or LDL (0.5 to 0.7 mg/mL in PBS) with 100 nmol/L
[3H]E2 for 4 hours, isolated and filtered the
LDL, and then subjected the LDL lipids to HPLC and collected fractions
for liquid scintillation counting. As shown in Fig 3,
incubation of LDL with [3H]E2 in PBS resulted
in the recovery of 3H principally in the fraction that
coelutes with authentic E2. In contrast, plasma incubations
yielded LDL carrying a small amount of authentic
[3H]E2 and three major radiolabeled species
that were more hydrophobic than authentic E2. None of the
fractions appeared to be conversions to either estriol or estrone
(retention times of 3.5 and 3.8 minutes, respectively). Thus,
incubation of E2 with LDL in a plasma milieu results in
conversion of E2 into nonpolar derivatives that associate
with the lipoprotein particle.
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One well-described set of hydrophobic E2 conjugates that
form in vivo are the fatty acid esters of
E2.33 The esterification of E2 may
occur at either of the hydroxy groups. To determine if the nonpolar
forms of E2 described herein were consistent with esters of
E2, we isolated LDL from plasma incubated with 100 nmol/L
[3H]E2 and hydrolyzed the LDL for 18 hours at
50°C. Hydrolyzed LDL was extracted with methanol/hexane and the LDL
extract subjected to HPLC as above. As shown in Fig 3C, the three major
nonpolar species of E2 were hydrolyzed by
K2CO3 but were resistant to hydrolysis by
NaHCO3. Additionally, the radiolabeled material from the
hydrolyzed species was quantitatively recovered in the more polar
fraction corresponding to authentic E2. Thus, incubation of
plasma with E2 results in the conversion of E2
to more nonpolar forms that have chemical characteristics of
E2 esterified with fatty acids at the C17 position.
The esterification of E2 with fatty acids in vivo is not
well characterized but is believed to be mediated by enzymatic
processes. One candidate for esterification of E2 is LCAT
associated with plasma HDL.41 LCAT has critical sulfhydryl
groups inhibited by thiol-blocking agents such as DTNB.35
We incubated LDL in PBS containing 10 nmol/L
[3H]E2 and 1.4 mmol/L DTNB and found a
12±7% inhibition of [3H]E2 association with
LDL (Table 3). In contrast, incubation of plasma with
[3H]E2 and DTNB resulted in a 69±19%
decrease in radiolabeled material associated with LDL
(P<.05 versus PBS incubation). In addition, these
incubation conditions prevented the antioxidant protection by
E2 of LDL as measured by the formation of conjugated dienes
in response to Cu2+ (Table 3). Thus, modification of
E2 appears to be necessary for antioxidant protection of
LDL at physiologically relevant concentrations.
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The oxidation of LDL by Cu2+ or AAPH proceeds through
fundamentally different mechanisms. AAPH-mediated LDL oxidation
involves direct peroxyl radical attack on LDL polyunsaturated fatty
acids.42 In contrast, Cu2+-mediated LDL
oxidation requires binding43 and subsequent reduction of
Cu2+ by LDL.44 To gain insight into the
mechanism(s) responsible for E2-mediated antioxidant
protection of LDL, plasma was incubated with 0.1 to 100 nmol/L
E2 for 4 hours, LDL was isolated, and it was subsequently
incubated with 3.3 µmol/L Cu2+ or 4 mmol/L AAPH. LDL
resistance to AAPH-mediated oxidation was not affected by plasma
incubation with E2 (Fig 4A), whereas
Cu2+-induced LDL oxidation was inhibited (Fig 4B).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The data presented herein demonstrate that incubation of plasma with physiological concentrations of E2 increases the resistance of LDL to Cu2+-mediated oxidation in vitro. However, antioxidant activity of E2 was not observed against AAPH-mediated modification, nor was oxidative resistance effectively increased when isolated LDL was exposed to E2 at concentrations
100 nmol/L in the absence of plasma. The increased
LDL oxidative resistance with E2 treatment in plasma was
not associated with LDL incorporation of E2 that could be
detected by RIA. However, incubations with radiolabeled E2
suggest that plasma facilitates the association of LDL with a
derivative of E2 that is consistent with E2
esterification at the C17 position. Moreover, this modification of
E2 appears to be required for antioxidant protection
because inhibiting E2 modification with DTNB prevents both
E2 association with LDL and antioxidant protection.
Our observations in vitro support those of Sack and
colleagues22 in which a single infusion of E2
in postmenopausal women resulted in a 36% increase in LDL resistance
to Cu2+-mediated oxidation. This infusion raised plasma
E2 from 55 pmol/L to 1.6 nmol/L, a level comparable to that
observed in premenopausal women.36 We found a similar 15%
to 26% increase in LDL resistance to Cu2+-mediated
oxidation by incubating plasma with 1 to 10 nmol/L E2. In
addition, this increase in LDL resistance to oxidation by
E2 is comparable to that observed with oral vitamin E
supplementation (400 to 800 mg/d)45 and with incubations
of plasma in vitro with 10x the physiological concentration of
-tocopherol.37 38 Sack and coworkers22
found that LDL oxidative resistance was not significantly associated
with plasma E2 concentration, although they did not assess
association of E2 with LDL. Thus, the study by Sack and
colleagues is in general agreement with our observations that exposure
of LDL in plasma to exogenous E2 alone is sufficient to
alter LDL oxidative susceptibility ex vivo. The greater increase in lag
time noted above in postmenopausal women compared with our incubations
(36% versus 26%) may be gender related or reflect a more efficient
mechanism for E2 association with LDL in vivo.
The increase in oxidative resistance of LDL exposed to
E2 in plasma did not result from changes in LDL
antioxidants during incubation (Table 1). Increases in LDL
-tocopherol, the most prevalent lipid-soluble antioxidant in LDL,
result in increased resistance to oxidation.37 38 As
previously described for ascorbate,46 estrogens regenerate
-tocopherol from the -tocopheroxyl radical.39
However, it is unlikely that this was the operative mechanism whereby
E2 altered LDL resistance to oxidation because no
significant changes in LDL -tocopherol resulted from a 4-hour
exposure to E2 in plasma.
We found no detectable cholesteryl ester hydroperoxides in LDL derived
from plasma incubated at 37°C for 4 hours (Table 1). The presence of
preformed lipid hydroperoxides in LDL is directly associated with
decreased resistance to Cu2+-mediated
oxidation.40 The absence of lipid hydroperoxides in our
preparations indicates that E2 did not inhibit
Cu2+-mediated LDL oxidation simply by limiting the
generation of preformed hydroperoxides in LDL.
Previous in vitro studies have demonstrated that E2 acts as
a classic chain-breaking antioxidant with free radical scavenging
activity against both aqueous and lipid peroxyl radicals47
and prevents LDL oxidation with a potency equivalent to
probucol.18 Our data with supraphysiological (micromolar)
levels of E2 show equivalent protection of LDL against
Cu2+- and AAPH-mediated oxidation (Fig 1), consistent with
free radical scavenging activity. In contrast, our data contain two
lines of evidence indicating that antioxidant protection of LDL at
physiological E2 concentrations is dependent on a different
mechanism. First, this protection requires incubation of E2
with LDL in plasma (Fig 2). Second, E2 protects LDL against
Cu2+-mediated but not aqueous peroxyl radical–mediated
oxidation (Fig 4). An important implication of the latter observation
is that antioxidant protection of LDL by physiological E2
concentrations may not be a consequence of general free radical
scavenging.
In the present study, we found that incubation of plasma with
[3H]E2 resulted in a dose-dependent
association of the radiolabeled material with LDL and subsequent
antioxidant protection, whereas this association and protection was
significantly decreased in PBS (Table 2). These observations parallel
those of Esterbauer and colleagues38 with -tocopherol.
Only LDL exposed to -tocopherol in plasma, not PBS, showed
significant enrichment of LDL with -tocopherol and subsequent
protection against Cu2+-mediated oxidation. Moreover, in
the present study, the enhanced association of E2 with LDL
in plasma is linked to the conversion of E2 into nonpolar
derivatives not recognized by RIA (Table 2).
One potential candidate for this E2 derivative that is
consistent with these data is a fatty acid ester of E2.
Fatty acid esters of E2 have been identified in plasma and
are concentrated in adipose stores of both premenopausal and
postmenopausal women.48 Consistent with the E2
derivatives observed in the present study, fatty acid esters of
E2 are not detectable by E2 RIA without base
hydrolysis.34 Lipid extracts of LDL incubated with
[3H]E2 in plasma contained
3H-compounds more hydrophobic than E2 (Fig 3).
Moreover, these compounds were resistant to NaHCO3 but
susceptible to K2CO3 hydrolysis, consistent
with conjugation at C17 and preservation of the antioxidant structure
of the phenolic E2 ring.33 34 Thus, these data
are consistent with the hypothesis that coincubation of E2
and LDL in plasma leads to the formation of E2 derivatives
with characteristics of E2 fatty acid esters, which are the
principal forms of E2 associated with LDL. These conjugates
most likely escape recognition by RIA, and thus plasma and perhaps more
importantly tissue estradiol concentrations in vivo are greater than
values reported by standard RIA.
Because incubation conditions that result in the enhanced association
of [3H]E2 with LDL are also those that lead
to increased LDL resistance to Cu2+-mediated oxidation, one
must consider that the modification of E2 is responsible
for the observed antioxidant protection. Indeed, incubation of plasma
with 10 nmol/L E2 resulted in a 26% increase in LDL
resistance to oxidation and significant association of
3H-material with LDL that was not detectable as authentic
E2 (Table 2; Fig 3B). Estradiol 17-stearate, a model
long-chain fatty acid derivative of E2, did not demonstrate
antioxidant protection when incubated with LDL in either plasma or PBS
(data not shown). Because E2 esters are extremely
hydrophobic, we cannot discount the possibility that E2
17-stearate is insoluble in the aqueous environment of our experiments
and therefore may not have associated with LDL. It is also possible
that E2 modification and associated antioxidant protection
take place on the surface of the LDL particle and thus, preformed
E2 17-stearate has no effect.
However, it is difficult to reconcile the quantity of E2
(or E2 derivative) that associates with LDL and the degree
of antioxidant protection we observed in the present study. Indeed,
incubation of LDL with 100 nmol/L E2 in PBS resulted in
nearly twice the association of E2 equivalents with LDL but
none of the antioxidant protection afforded by 10 nmol/L E2
in plasma (Table 2). Plasma incubation with 100 nmol/L
[3H]E2 resulted in stable association of
radiolabeled material with LDL in the amount of 3.4 pmol/mg LDL protein
(2% of [3H]E2 added; Table 2). This
corresponds to a ratio of 1 E2 equivalent to 500 LDL
particles. Although plasma incubations with DTNB inhibited the
association of E2 with LDL and prevented antioxidant
protection of LDL (Table 3), it is extremely unlikely that such a small
amount of E2 (or derivative) might directly inhibit
Cu2+-mediated LDL oxidation by direct interception of free
radical species, even though modification at C17 preserves the intact
phenolic hydroxyl group and thus antioxidant capacity. One could
speculate that incorporation of preformed E2 conjugates
into LDL or the enzymatic modification of E2 on the surface
of the LDL particle may catalyze a process that promotes a stable
modification of LDL and interferes with Cu2+ binding and/or
reduction by LDL. A similar process has been observed with antioxidant
protection of LDL by the ascorbic acid oxidation product,
dehydroascorbic acid. Incubation of LDL with dehydroascorbic acid
modifies LDL in a manner that inhibits Cu2+- but not
AAPH-mediated oxidation.25
To date, the mechanisms of LDL oxidation in vivo are unknown. It is hypothesized that vascular cells modify LDL into its atherogenic form,9 10 11 but redox active metals in the vascular wall may also act on subendothelial lipoproteins.49 The data presented herein indicate that short-term exposure to physiological concentrations of E2 has the potential to increase LDL resistance to Cu2+-mediated modification. This effect of E2 is dependent on a plasma milieu and results in the association of E2 with LDL in a form that is chemically similar to an E2 ester. These findings suggest a means by which physiological levels of E2 may limit the oxidation of LDL in vivo and perhaps help to explain the reduced incidence of CAD in premenopausal women compared with men.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from the Council for Tobacco Research-USA, Inc (No. 4073); the American Heart Association, Massachusetts Affiliate; and the National Institutes of Health (NIH; HL-52936). Joseph A. Vita, MD, is an Established Investigator of the American Heart Association and John F. Keaney, Jr, MD, is the recipient of a Clinical Investigator Development Award (HL-03195) from the NIH. We gratefully acknowledge Dr Richard Hochberg for providing 17ß-estradiol 3-stearate and Dr Gordon Niswender for providing antibody to 17ß-estradiol. We thank Balz Frei for assistance with the cholesteryl ester hydroperoxide assay and critical reading of this manuscript.
Received October 10, 1996; revision received November 11, 1996; accepted November 17, 1996.
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References |
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