© 2001 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Dietary Cosupplementation With Vitamin E and Coenzyme Q10 Inhibits Atherosclerosis in Apolipoprotein E Gene Knockout Mice
From the Biochemistry (S.R.T., S.B.L., R.S.) and Cell Biology (A.J.B.) Groups, The Heart Research Institute, Camperdown, NSW, Australia; Cardiovascular Pharmacology (K.P.), AstraZeneca R&D, Mölndal, Sweden; and Department of Medicine (K.D.C., T.A.M.), University of Western Australia, Royal Perth Hospital, Perth, Western Australia.
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Abstract |
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Abstract—Intimal oxidation of LDL is considered an important early event in atherogenesis, and certain antioxidants are antiatherogenic. Dietary coenrichment with vitamin E (VitE) plus ubiquinone-10 (CoQ10, which is reduced during intestinal uptake to the antioxidant ubiquinol-10, CoQ10H2) protects, whereas enrichment with VitE alone can increase oxidizability of LDL lipid against ex vivo oxidation. In the present study, we tested whether VitE plus CoQ10 cosupplementation is more antiatherogenic than either antioxidant alone, by use of apolipoprotein E–deficient (apoE-/-) mice fed a high-fat diet without (control) or with 0.2% (wt/wt) VitE, 0.5% CoQ10, or 0.2% VitE plus 0.5% CoQ10 (VitE+CoQ10) for 24 weeks. None of the supplements affected plasma cholesterol concentrations, whereas in the VitE and CoQ10 groups, plasma level of the respective supplement increased. Compared with control, plasma from CoQ10 or VitE+CoQ10 but not VitE-supplemented animals was more resistant to ex vivo lipid peroxidation induced by peroxyl radicals. VitE supplementation increased VitE levels in aorta, heart, brain, and skeletal muscle, whereas CoQ10 supplementation increased CoQ10 only in plasma and aorta and lowered tissue VitE. All treatments significantly lowered aortic cholesterol compared with control, but only VitE+CoQ10 supplementation significantly decreased tissue lipid hydroperoxides when expressed per parent lipid. In contrast, none of the treatments affected aortic ratios of 7-ketocholesterol to cholesterol. Compared with controls, VitE+CoQ10 supplementation decreased atherosclerosis at the aortic root and arch and descending thoracic aorta to an extent that increased with increasing distance from the aortic root. CoQ10 significantly inhibited atherosclerosis at aortic root and arch, whereas VitE decreased disease at aortic root only. Thus, in apoE-/- mice, VitE+CoQ10 supplements are more antiatherogenic than CoQ10 or VitE supplements alone and disease inhibition is associated with a decrease in aortic lipid hydroperoxides but not 7-ketocholesterol.
Key Words: antioxidant • atherogenesis • oxidation • 
-tocopherol • ubiquinol • ubiquinone
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The LDL oxidation theory of atherosclerosis proposes that oxidation of LDL lipid in the intima is an important early and proatherogenic event.1 2 In support of this theory, oxidized lipids3 4 and proteins5 6 7 8 are present in human lesions and lipoprotein-like particles isolated from them. For example, in advanced human atherosclerotic lesions,
5% of cholesteryl linoleate (C18:2), the major oxidizable
lipid in LDL, is oxidized and present primarily as hydroperoxides
and respective alcohols9 and
oxoderivatives.10 In
addition, atherosclerotic lesions also contain
oxysterols11 and
F2-isoprostanes,12 13
prostaglandin-like and nonenzymatic lipid oxidation
products of
arachidonate.14
However, these secondary lipid oxidation products are localized
predominantly in foam
cells12 15 and are
present at a lower concentration compared with primary oxidation
products of
C18:2.4 13 16
Although present in lesions, the extent to which
different oxidized lipids cause or promote atherogenesis remains
unknown. Lipid hydroperoxides (LOOH), the primary lipid peroxidation
products formed during the initial stage of lipoprotein
oxidation,17 may contribute
to oxidative modification of apolipoprotein B-100 of LDL in vitro by
means of secondary
reactions.18 Potential
atherogenic activities of 5-cholesten-3ß-OL-7-one
(7-ketocholesterol [7KC]) and
F2-isoprostanes also have been described in
vitro. For example, 7KC is cytotoxic to vascular cells and can impair
cholesterol efflux in
macrophages,11
whereas 8-epiprostaglandin F2
modulates platelet aggregation and is a smooth muscle cell
constrictor.19 20
Given the oxidation theory, inhibitors of lipoprotein lipid oxidation are considered to be potential antiatherogenic compounds. Indeed, several antioxidants inhibit atherosclerosis in various animal models of the disease.1 However, not all antioxidants that inhibit in vitro lipid oxidation attenuate atherogenesis,21 for reasons largely unknown. Also, in Watanabe hyperlipidemic rabbits, prevention of aortic lipid peroxidation itself is not sufficient for inhibition of atherosclerosis,22 which suggests that antioxidants that attenuate atherosclerosis may do so by means of actions in addition to or independent of inhibition of lipoprotein oxidation.23
Plasma lipoproteins contain several endogenous
antioxidants with -tocopherol (vitamin E [VitE]) and
ubiquinol-10 (CoQ10H2)
that represent important modulators of lipid
peroxidation.24 25
As the major antioxidant present in lipoprotein extracts, VitE is
commonly thought to be antiatherogenic. However, outcomes of VitE
intervention studies on atherosclerosis in experimental
animals and cardiovascular disease in humans overall
have been inconclusive, if not
disappointing.26 Despite
this, supplementation of apolipoprotein E-deficient mice (apoE-/-)
mice with 0.2% (wt/wt) VitE was recently reported to attenuate
atherosclerosis significantly in aortic root and to
decrease aortic content of
F2-isoprostanes.27
Compared with VitE, few studies have examined antiatherogenic potential of CoQ10H2. CoQ10 is used for dietary supplementation studies because it is stable and effectively converted into the antioxidant active CoQ10H2 on intestinal uptake.28 Recently, an antiatherogenic effect of CoQ10 was reported for rabbits fed an undefined preoxidized diet,29 and we observed that 1% wt/wt CoQ10 significantly reduced atherosclerosis in apoE-/- mice.30 CoQ10H2 is an effective coantioxidant,31 32 33 and coenrichment with VitE plus CoQ10H2 (VitE+CoQ10H2) effectively inhibits LDL lipid peroxidation, whereas VitE supplementation alone promotes this process under the mild in vitro oxidizing conditions used.33 We therefore proposed33 that cosupplementation with VitE plus CoQ10 (VitE+CoQ10) may be more antiatherogenic than either VitE or CoQ10 supplementation alone. In the present study, we show supporting evidence for this proposal and report differences with respect to the extent to which cosupplementation inhibits accumulation of different types of oxidized lipids within the vessel wall.
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Methods |
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Materials
C18:2 and cholesteryl arachidonate (together called cholesteryl esters [CE]), nonesterified cholesterol (NEC), 5-cholesten-3ß,19-diol (19-hydroxycholesterol), ascorbate, formalin, EDTA, glycerol, butylated hydroxytoluene and chloramphenicol were obtained from Sigma Chemical Co. 7KC was from Steraloids Inc (Wilton). VitE (RRR-
-tocopherol)
and CoQ10 were generous gifts from Henkel Corp
and Kaneka Corp, respectively. -Tocotrienol was purified as
described.34 Authentic
C18:2-OOH, used as a standard for LOOH, was prepared as
described.35 Authentic
CoQ10H2, prepared as
described,36 was used while
fresh as a standard for
CoQ9H2 and
CoQ10H2. Protease
inhibitor cocktail tablets were from Boehringer,
and gentamicin and chloramphenicol from Gibco BRL. Calcium- and
magnesium chloride–free Dulbecco’s PBS (Sigma) was prepared from
nanopure water and stored over chelating resin (Chelex
100; BioRad) at 4°C for 
24 hours to remove
contaminating transition metals. All buffers were filtered and
argon-flushed immediately before use. Organic solvents and all other
chemicals used were of the highest quality
available.
ApoE-/- Mice
Male C57BL/6J mice, homozygous for the disrupted apoE
gene (apoE-/-) were purchased from Jackson Laboratories (Bar
Harbor, Me), bred at The Heart Research Institute (Sydney, Australia),
and fed standard mouse chow (Laboratory-Feed) until the age of 8 to 10
weeks.37 Animals were then
fed for 24 weeks ad libitum a high-fat diet containing 21.2 wt/wt fat
and 0.15% wt/wt cholesterol without (controls; n=26) or
with VitE (0.2% wt/wt; n=25), CoQ10 (0.5%
wt/wt; n=24), or VitE+CoQ10
(CoQ10 0.5% wt/wt, VitE 0.2% wt/wt; n=24).
Diets were prepared by MJ Hoxey and Associates according to
specifications of the Harlan Teklad diet TD88137, packaged and sealed
under N2, and stored at 4°C until
use.
Plasma Analyses and Ex Vivo
Oxidation
Plasma was prepared from blood as
described.37 Aliquots (50
µL) of the resulting plasma were extracted immediately into hexane
and methanol35 and stored at
-80°C for lipid and antioxidant analyses or diluted in 5%
metaphosphoric acid (1:1 vol/vol) and stored at -80°C for ascorbate
analysis (see below). Plasma total cholesterol concentration
was measured with a total cholesterol assay kit (Sigma).
Residual plasma was pooled, argon-flushed, and stored at 4°C for 
12
hours before size-exclusion chromatography and ex vivo
oxidation with 2,2'-azo
bis(2-amidinopropane)
hydrochloride (5 mmol/L final concentration) as
oxidant.37 Such storage does
not significantly alter plasma lipid
oxidizability.33
Aortic Sampling for Biochemical and
Histologic Analyses
Procedures were performed largely as described
previously.37 38
Briefly, mice were perfused at near-physiological
pressure with buffer A (PBS with 1 mmol/L EDTA and 20 µmol/L
butylated hydroxytoluene). For biochemistry, aortas (control, n=12;
VitE, CoQ10, and
VitE+CoQ10, n=15 per group) were excised, and
hearts and ascending and descending aortas past the femoral bifurcation
were cleaned. Aortas then were randomly sorted into 3 separate groups
of 4 to 6 and placed immediately in cold buffer B (buffer A containing
1 protease inhibitor tablet per 150 mL, 0.008% gentamicin,
and 0.008 chloramphenicol). Aortas were then stored at -80°C until
processing for biochemical analyses, excluding determination of
F2-isoprostanes and arachidonic
acid. For histology, hearts and aortas of separate mice (control, n=14;
VitE, CoQ10, and
VitE+CoQ10, n=9) were excised. The upper portion
of each aorta (to the third pair of intracostal arteries) was then
placed in 4% vol/vol formaldehyde in saline overnight, before being
transferred into 0.1% vol/vol formaldehyde in saline solution. The
unfixed lower portion (from the fourth pair of intracostal arteries to
the femoral bifurcation) was placed in buffer B and kept frozen at
-80°C until analysis for
F2-isoprostanes and arachidonic
acid (see below). Fixed tissue was transported to AstraZeneca for
lesion assessment, performed in a blinded fashion by morphometry at the
aortic root and arch and descending thoracic aorta as described
previously in
detail.38
Aortic Biochemistry
Pooled aortas were pulverized in liquid
N2, resuspended in 1.5 mL of buffer A, and
homogenized as
described.37 A 50-µL
aliquot of homogenate was added to an equal volume of 5%
metaphosphoric acid for ascorbate analysis and a further 50
µL removed for protein determination with the bicinchoninic acid
assay kit (Sigma). Remaining homogenate was extracted in
500-µL aliquots added to 2 mL of methanol and 10 mL of hexane. The
sample was mixed vigorously for 1 minute and centrifuged at
4°C. The hexane fraction then was dried and lipids redissolved into
400 µL of isopropanol.35
This extract was analyzed for lipid-soluble antioxidants, NEC,
CE, and LOOH by high-performance liquid
chromatography (HPLC) as
described.35 39
LOOH were measured as a marker of primary lipoprotein lipid
peroxidation because it is the primary and major lipid oxidation
product formed in lipoproteins from apoE-/- mice undergoing
oxidation.40 A previous study
has shown that 70% of [3H]-C18:2-OOH
added to mouse aorta before pulverizing is recovered as the
hydroperoxide and 30% recovered as the corresponding alcohol and that
[3H]-C18:2 added to aortas before workup
is not converted to [3H]-C18:2-OOH or
[3H]-C18:2-OH.37
To confirm the presence of LOOH, postcolumn chemiluminescence detection
was used before and after borohydride treatment of
samples.41 All compounds
detected were quantified by peak area comparison with authentic
standards run under identical conditions.
For analysis of total cholesterol and 7KC, 10- and 100-µL aliquots, respectively, of the above isopropanol extracts were saponified after transfer to a screwcap tube and addition of diethyl ether (2.5 mL) and a methanolic solution of potassium hydroxide (20% wt/vol; 2.0 mL). 19-Hydroxycholesterol and cholesteryl propylether (both 100 µL of a 50-µg/mL solution in heptane/isopropanol 95:5 vol/vol) were added as internal standards for 7KC and total NEC, respectively. Tubes were flushed with argon and mixed overnight at 4°C before H2O (2.0 mL) and hexane (2.5 mL) were added. Extracts were then mixed vigorously (30 s) and centrifuged (1600g, 5 minutes, and 10°C). The ether/hexane phase was evaporated under vacuum and the extracts redissolved in heptane:isopropanol (95:5 vol/vol) or isopropanol:acetonitrile:water (54:44:2 vol/vol/vol) for 7KC or total cholesterol HPLC determination, respectively.16 42 For total cholesterol, a silica column (0.46x15 cm, 100 Å, 5 µm; Alltech) with guard column (3-µm particle size) was used with hexane:isopropanol:acetonitrile (94.8:4.6:0.6 vol/vol/vol) as the eluant at 1.0 mL/min monitored at 234 nm. Typical retention times of 7KC and 19-hydroxycholesterol were 14.8 and 28.3 minutes, respectively.
For analysis of
F2-isoprostanes, the pieces of thawed aortas
(20 mg wet wt) were blotted dry, weighed, and
homogenized in ice-cold chloroform:methanol (2:1 vol/vol).
[D4]-8-iso-prostaglandin
F2 (Cayman Chemicals) was added as an
internal standard. After removal of an aliquot for
arachidonate analysis (see below), the organic
phase was dried under nitrogen and hydrolyzed by addition of 15% KOH
and incubated for 1 hour at 45°C.
F2-isoprostanes were analyzed after
solid-phase extraction and HPLC purification by electron-capture
negative-ionization gas chromatography-mass
spectrometry as described in
detail.43 For
arachidonate analysis, phospholipids were separated
from total lipid extracts by thin-layer chromatography
on silica gel 60 F254-precoated aluminum sheets (Merck) with
hexane:diethyl ether:acetic acid:methanol (170:40:4:4 vol/vol) as the
solvent. Fatty acid methyl esters were prepared by treatment with 4%
H2SO4 in methanol at
90°C for 20 minutes and analyzed by gas-liquid
chromatography.43
Peaks were identified by comparison with known standards.
Arachidonate was quantified with heptadecanoic acid as the
internal standard.
Analysis of VitE and
CoQ10 Levels in Heart, Brain, and Skeletal
Muscle
Concentrations of VitE, CoQ10,
and ubiquinone-9 (CoQ9) were also determined in
heart, brain, and hind-limb skeletal muscle. Whole tissues were
homogenized in 2 mL of ice-cold buffer A, and a 50-µL
aliquot was removed for protein determination. The remaining
homogenate (200 µL) was extracted in 2 mL methanol and 10
mL hexane; the organic phase was resuspended in 200 µL of isopropanol
and analyzed for VitE, CoQ9, and
CoQ10 by
HPLC.39
CoQ10 and CoQ9 were
measured routinely due to the unstable nature of
CoQ10H2 and
CoQ9H2.44
Statistics
Data on lesion size are presented as
mean±SEM, and effects of supplements analyzed by 2-way ANOVA
(SAS software) by use of log-transformed data, with supplements and
aortic sites as factors. Because a significant interaction term existed
between supplement and aortic site, treatment effect at each site
measured was evaluated by Student’s
t test with log-transformed
data. Biochemical parameters were compared by use of 1-way
ANOVA or Student’s t test
(Systat 8.0 software; SPSS). Statistical significance was accepted at
P<0.05.
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Results |
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Plasma Lipids and Antioxidants
Effects of 24 weeks of supplementation of apoE-/- mice with VitE, CoQ10, or VitE+CoQ10 on plasma lipids and antioxidants are summarized in Table 1
. None of the supplements significantly altered
concentrations of total cholesterol, NEC, CE, and ascorbate.
Supplementation with VitE or VitE+CoQ10
significantly increased plasma concentration of VitE 3-fold
(P<0.001), whereas
supplementation with CoQ10 or
VitE+CoQ10 increased the concentration of total
CoQ 7-fold (P<0.001). This
increase was solely due to an increase in CoQ10
(Table 1).
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Plasma Lipoprotein Profile
Plasma from mice treated with
VitE+CoQ10 contained increased levels VLDL
compared with control mice, whereas supplementation with VitE or
CoQ10 had a comparatively minor effect
(Figure 1 and
Table 2). LDL levels were decreased in mice treated with
VitE, whereas HDL levels were similar in all groups
(Table 2). Lipid analysis of the individual
lipoprotein classes indicated that most of the increased plasma VitE
and CoQ10, which resulted from supplementation
with CoQ10 or VitE, respectively, was located in
VLDL
(Table 2).
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Plasma Oxidizability
Plasma lipids from mice supplemented with
CoQ10 or VitE+CoQ10 were
more resistant to peroxidation induced by peroxyl radicals
compared with plasma from control or VitE-supplemented mice
(Figure 2). As expected, 80% of supplemented
CoQ10 or endogenous
CoQ9 was present as
CoQ10H2 and
CoQ9H2, respectively (not
shown).30 Also, the time
during which lipid peroxidation was effectively suppressed corresponded
to the time required for the consumption of ascorbate and ubiquinols
(not shown).
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), VitE (
) and VitE+CoQ10 (
) mice (n=8 to 10 for each group) was exposed to 5 mmol/L AAPH and incubated in air at 37°C. At times indicated, aliquots of reaction mixture were removed and analyzed for LOOH. Table 1
Tissue Concentrations of VitE and
CoQ10
Supplementation with VitE or VitE+CoQ10 significantly
increased VitE in all tissues examined, including aortas
(P<0.05;
Figure 3 and
Table 3). In contrast, neither CoQ10
nor CoQ9 was increased significantly in heart,
brain, or skeletal muscle after supplementation with
CoQ10 or VitE+CoQ10 for
24 weeks
(Figure 3). However, aortic content of
CoQ10 increased >10-fold with
CoQ10 and VitE+CoQ10
supplements (P<0.001), which
resulted in a 2-fold increase in content of total CoQ
(P<0.02;
Figure 3 and
Table 3). Between 20% and 50% of aortic CoQ was
present as CoQ10H2 or
CoQ9H2 (not shown), which
indicates that the sample workup procedure used largely prevents
inadvertent oxidation (ubiquinols are more sensitive to
autoxidation than VitE). A consistent trend noted was that
CoQ10 supplements decreased VitE concentrations in all tissues. Thus,
extent of increase in VitE concentration in tissues was less in
VitE+CoQ10 supplemented mice than in mice
supplemented with VitE alone.
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). Results are mean±SEM. aP<0.05 vs control; bP<0.05 vs CoQ10; cP<0.05 vs VitE.
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Aortic Levels of Neutral and Oxidized
Lipids
Table 3
summarizes aortic concentration of lipids after 24
weeks of intervention. Compared with controls, aortas from all treated
groups exhibited a significant decrease in levels of NEC and total
cholesterol, with the order of efficacy
VitE+CoQ10>VitE>CoQ10
(Table 3). Such a decrease in aortic cholesterol
content in the absence of a hypolipidemic effect
(Table 1) is consistent with an antiatherogenic
activity of the treatments. Aortic concentrations of C18:2 and
cholesteryl arachidonate, the 2 major oxidizable CE, were
not decreased significantly by any treatment.
To assess the effect of VitE and
CoQ10 supplementation on aortic lipid oxidation,
aortic concentrations of LOOH and 7KC were measured. Both types of
oxidized lipids were detected in the aortas
(Table 4). Identity of LOOH was verified by treating the
organic extract with
NaBH4.41
This resulted in disappearance of chemiluminescence-positive peaks
coeluting with standards of LOOH and appearance of
chemiluminescence-positive peaks coeluting with standards of ubiquinols
(not shown). Treatment with VitE+CoQ10
significantly decreased both absolute concentration and the ratio of
LOOH:CE (P<0.05;
Table 4). Treatment with CoQ10 alone
also decreased lipid-standardized levels of LOOH by 40%, although
this did not reach statistical significance. In contrast, mean values
of lipid-standardized LOOH were increased nonsignificantly by 25% in
aortas from VitE-treated animals.
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All treatments decreased absolute concentration of aortic
7KC, with a significant decrease apparent in mice treated with VitE or
VitE+CoQ10 (P<0.05). However,
these effects were no longer seen when aortic 7KC concentrations were
standardized for total NEC content
(Table 4). However, concentration of
F2-isoprostanes determined varied greatly
between different aortas within each treatment group and was
100-fold and 10-fold lower than LOOH and 7KC, respectively, when
expressed per parent molecule (data not shown). Compared with controls,
none of the treatments significantly altered content of
F2-isoprostanes when expressed in absolute
amount or per arachidonate (not
shown).
Morphometry
After 24 weeks of high-fat diet, lesions of grossly
comparable morphology were found at all aortic sites examined
(Figure 4), with necrotic cores containing
cholesterol crystals observed frequently (not shown). ANOVA
indicated that treatments significantly affected lesion size in a
site-dependent manner, as indicated by a significant interaction term
(P0.001). Supplementation
with VitE+CoQ10 significantly decreased lesion
size at all sites examined
(P0.05)
(Figure 4). Extent of disease inhibition increased from the
aortic root to the aortic arch and descending thoracic aorta, with
30%, 50%, and 80% inhibition, respectively, compared with
respective sites in control animals. Supplementation with
CoQ10 alone significantly decreased the size of
lesions in the aortic root and arch
(P0.05) but not in descending
thoracic aorta. VitE supplementation significantly decreased lesion
size only in the aortic root
(P0.05). No significant
differences were observed in size of lesions among any of the treatment
groups except that animals supplemented with
VitE+CoQ10 exhibited significantly smaller
lesions in the aortic arch compared with VitE-supplemented mice
(P0.05).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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A previous study27 showed that 0.2% VitE supplements decreased lesions in the aortic arch and thoracic aorta of apoE-/- mice by 60%. The aim of the present study was to test whether cosupplementation with CoQ10 further enhanced this antiatherogenic effect of VitE. Because 1% CoQ10 alone significantly decreases lesions in this animal model,30 we compared the effect of 0.2% VitE with that of 0.5% CoQ10 and 0.2% VitE+0.5% CoQ10. We show that overall the antiatherogenic activity of VitE+CoQ10 was greater than that of VitE or CoQ10 supplementation alone. Inhibition of lesion formation was associated with a decrease in the aortic content of LOOH, a marker of primary lipoprotein lipid peroxidation. A further key finding was that VitE+CoQ10 increasingly inhibited disease along the aortic tree, similar to that reported recently for probucol in apoE-/- mice,38 except that the intervention used in the present study reduced lesions also in the aortic root, whereas probucol enhances atherogenesis at that site.38 45
How VitE+CoQ10 supplements inhibited atherosclerosis is unknown, although it significantly increased plasma and aortic concentrations of VitE and CoQ10 without lowering circulating concentration of cholesterol. This phenomenon indicates that a hypocholesterolemic effect cannot explain the observed effect, in contrast to several previous animal interventions with antioxidants, including VitE, in which a confounding hypolipidemic effect was reported.24 In consideration of the LDL oxidation theory,1 antioxidants commonly are thought to attenuate atherosclerosis by inhibiting lipoprotein oxidation. Several lines of evidence suggest that such activity may have contributed to the antiatherosclerotic effect of VitE+CoQ10 supplements. Thus, VitE+CoQ10 treatment increased VitE and CoQ10H2 in plasma and the oxidation resistance of plasma lipids toward ex vivo peroxidation by aqueous peroxyl radicals, consistent with our previous study with human LDL.33 This increased resistance of plasma lipids to peroxidation was associated with a significant decrease in content of aortic LOOH, independent of whether this oxidation parameter was expressed in absolute terms or lipid standardized. Together, these results support but do not prove the LDL oxidation theory.
Similar to VitE+CoQ10, supplementation with CoQ10 alone also inhibited ex vivo plasma lipid peroxidation and decreased aortic LOOH, although the latter did not reach significance. We observed recently that 1% wt/wt CoQ10 significantly decreased parent lipid-standardized LOOH in the vessel wall.30 In the present study, the lower dose of CoQ10 used resulted in somewhat lower aortic concentrations of CoQ10, which may explain the lower efficacy found in the present study. In both studies, supplementation with CoQ10 was associated with inhibition of atherosclerosis of similar magnitude, except for aortic arch in the present study. Together, the findings support an antiatherogenic effect of CoQ10 in apoE-/- mice, albeit less pronounced than that seen with VitE+CoQ10.
Treatment with VitE alone failed to lower aortic LOOH, which indicates that VitE supplements alone may not inhibit primary lipid peroxidation despite the observed 3-fold increase in tissue content of the vitamin. In contrast, cosupplementation with VitE+CoQ10 significantly decreased aortic LOOH even though aortic VitE was increased only 2-fold. These results are consistent with, although not conclusive proof of, lipoprotein lipid peroxidation in the vessel wall proceeding through tocopherol-mediated peroxidation and indicate that coantioxidants inhibit lipoprotein oxidation in vivo.24 The results also are consistent with the proposal33 that cosupplementation of VitE with a lipid-soluble coantioxidant such as CoQ10H2 is a more effective antioxidant strategy than supplementation with VitE alone.
We also measured 7KC as an index of in vivo lipid oxidation.
In contrast to LOOH, this parameter of secondary lipid
oxidation, when expressed in a parent lipid-standardized manner, was
not affected significantly by any of the treatments. In vitro
experiments with LDL suggest that substantial accumulation of 7KC does
not occur until after depletion of
VitE.46 However, our
present
(Table 3) and previous
study37 clearly show that
VitE does not become depleted in the aortas of apoE-/- mice even
after 6 months of high-fat diet, when substantial lesions have formed.
Thus, VitE supplements may not be expected to decrease aortic 7KC. This
assumes that the measurement of 7KC reflects lipoprotein
oxidation, although its accumulation in
cells15 suggests that it may
represent cellular rather than lipoprotein lipid oxidation.
What is clear from the present study is that compared with LOOH,
7KC is a minor product of lipid oxidation in the vessel wall of
apoE-/- mice, given that only 0.02% of total cholesterol was
present as 7KC, whereas 0.2% of CE was detected as LOOH. This is
consistent with the fact that compared with NEC, CE are
chemically more susceptible to oxidation. We are not aware of a
previous study that examined the effect of VitE (or
CoQ10) supplement on oxysterols in apoE-/-
mice. However, a recent study reported that feeding apoE-/- mice
the isoflavan glabridin reduced both atherogenesis and the aortic
levels of oxysterols including 7KC when standardized for wet weight of
tissue.47 Similarly, in the
present study, all supplements decreased aortic content of 7KC when
expressed per aortic protein. However, attenuation of the extent of
atherosclerosis by definition means attenuation of
aortic cholesterol content as observed in the present
study. Therefore, to distinguish an effect on disease burden (or lipid
load) versus lipid oxidation within the vessel wall, it is necessary to
standardize aortic content of oxysterols to cholesterol.
When standardized, none of the supplements inhibited aortic content of
7KC.
Direct evidence for a causative link between inhibition of
lipoprotein oxidation and atherosclerosis is scarce.
Perhaps the most direct support for such a correlation is the
observation that VitE reduced aortic content of
isoprostane-F2-VI and lesion size in
apoE-/- mice.27 However,
how precisely aortic isoprostane-F2-VI
relates to in vivo lipoprotein oxidation and/or atherogenesis is not
known. For example, Pratico et
al27 determined isoprostanes
in hydrolyzed total lipid extracts of aortas, so that this measure is
not specific for lipoproteins. Also, where examined,
F2-isoprostanes in atherosclerotic lesions were
reported to be associated primarily with foam
cells.12 In contrast, the
LOOH measured in the present study are found in lesion
lipoproteins9 and are derived
from the major oxidizable lipids associated with lipoproteins (ie, CE
and triglycerides), so that this measure may reflect in
vivo lipoprotein oxidation. Measuring accumulation of these LOOH in the
vessel wall of LDL receptor-deficient rabbits, we recently observed
that complete prevention of lipid peroxidation was not associated with
inhibition of
atherosclerosis.22
This finding suggests that lipoprotein lipid oxidation in the vessel
wall can be dissociated from atherogenesis. Antioxidants such as
CoQ10H2 and VitE may
inhibit atherosclerosis by means other than inhibition
of lipoprotein oxidation.23
For example, at the pharmacological dosage used in the present
study, VitE can inhibit smooth muscle cell
proliferation,48 platelet
aggregation49 , and
interleukin-1ß release from monocytes in
vitro.50 Also,
VitE51 and
CoQ1052
may improve endothelial dysfunction in
vivo.
In the present study, supplementation with 0.2% VitE
alone had a moderate antiatherogenic effect in the aortic root only,
whereas Pratico et al27
observed a 60% decrease in aortic lesion area. Several differences
between the 2 studies may explain the apparent discrepancy. We used a
high-fat diet, which resulted in total plasma cholesterol
of 845 mg/dL (22 mmol/L), whereas Pratico et al used a
normal chow and reported plasma total cholesterol of 500
mg/dL.27 Previous studies by
others in hamsters suggest that the antiatherosclerotic activity of
VitE is lost at plasma cholesterol concentrations >270
mg/dL.53 Thus, the
comparatively higher cholesterol levels observed in the
present study may have masked an antiatherogenic effect of VitE,
although this requires further investigation. Shaish and
coworkers54 recently reported
that a combination of 0.05% VitE and 0.05% ß-carotene was
ineffective in preventing atherosclerosis in
apoE-/- mice, consistent with both the moderate
antiatherogenic activity of VitE observed in the present study and
the overall disappointing results obtained with VitE supplements in
animals24 26 and
humans.55
Extent of antiatherogenic activity observed in the present study with 0.5% CoQ10 is comparable to that observed recently with 1% CoQ10.30 This suggests that with the dosage used in the present study, we achieved an antiatherogenic effect near the maximum that can be achieved with this CoQ10 alone. This effect was obtained with a slightly smaller increase in aortic content of CoQ10 compared with that observed with a 1% supplementation.30 Therefore, 0.5% CoQ10 appears to be a suitable dose to test a beneficial effect of the coenzyme on the antiatherosclerotic activity of 0.2% VitE reported by Pratico et al.12 By showing that VitE+CoQ10 cosupplementation is more antiatherogenic than VitE or CoQ10 supplement alone, the present study shows a benefit of the combination over the single antioxidant supplement.
We chose CoQ10 because it enriches lipoproteins with CoQ10H2 that provides coantioxidation localized to where oxidation takes place. Interestingly, many antioxidants that inhibit atherosclerosis in animals, such as butylated hydroxytoluene,56 N,N’-diphenyl-phenylenediamine,57 and BO-653,58 are also lipid-soluble coantioxidants.59 Just as normal chow for laboratory animals is supplemented with VitE,60 addition of a coantioxidant alone may be seen as a form of cosupplementation with VitE plus coantioxidant. However, supplementation with a coantioxidant does not always attenuate atherosclerosis,22 although it prevents aortic lipid peroxidation.
Antiatherogenic efficacy of VitE+CoQ10 increased with increasing distance from the heart for presently unknown reasons, in a manner similar to what we described recently for probucol.38 However, even if this regional variability is considered, we cannot establish a clear link to any of the measures of oxidation used, and in the case of probucol, inhibition of atherosclerosis was observed without inhibition of aortic lipoprotein lipid (per)oxidation.38 What is clear is that combining antioxidants with different properties increased overall antiatherogenic efficacy to an extent comparable to that of probucol in the aorta but also reduced lesion size in the aortic root.38
A discrepancy seems to exist between efficacy by which the
supplements decrease lesions versus aortic cholesterol.
However, the difference in antiatherosclerotic effect between the
combined treatment versus either antioxidant alone is most pronounced
in the descending thoracic aorta
(Figure 4). At that site, mean cross-sectional areas are only
10% of that in the arch. Thus, in mass terms, the contribution of
the descending thoracic aorta to lipid accumulation in the entire aorta
(which we used for biochemistry) is limited.
In summary, the present study shows that supplementation of apoE-/- mice with VitE+CoQ10 is a more effective antiatherogenic treatment than supplementation with CoQ10 or VitE alone. This antiatherogenic activity is associated with a decrease in the aortic concentration of LOOH but not 7KC. Further studies are required to establish whether the antiatherogenic activity of VitE+CoQ10 reflects the ability of the antioxidants to inhibit lipoprotein oxidation in the vessel wall.
|
Acknowledgments |
|---|
This work was supported by the Australian National Health and Medical Research grant 970998 to R.S. We thank Kaneka Corp and Henkel Corp for the generous gift of CoQ10 and VitE, respectively. We also thank J. Letters and P. Gabrielsson for excellent technical assistance and the animal house staff at the Heart Research Institute for the maintenance of apoE-/- mice.
|
Footnotes |
|---|
Reprint requests to Dr Roland Stocker, The Heart Research Institute, 145 Missenden Rd, Camperdown NSW 2050, Australia.
Received November 8, 2000; accepted December 18, 2000.
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References |
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A. C. Terentis, S. R. Thomas, J. A. Burr, D. C. Liebler, and R. Stocker Vitamin E Oxidation in Human Atherosclerotic Lesions Circ. Res., February 22, 2002; 90(3): 333 - 339. [Abstract] [Full Text] [PDF]
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A. Braun, B. L. Trigatti, M. J. Post, K. Sato, M. Simons, J. M. Edelberg, R. D. Rosenberg, M. Schrenzel, and M. Krieger Loss of SR-BI Expression Leads to the Early Onset of Occlusive Atherosclerotic Coronary Artery Disease, Spontaneous Myocardial Infarctions, Severe Cardiac Dysfunction, and Premature Death in Apolipoprotein E-Deficient Mice Circ. Res., February 22, 2002; 90(3): 270 - 276. [Abstract] [Full Text] [PDF]
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