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Regular Articles |
From the Departments of Biochemistry*
andCell Biology,
Heart Research Institute,Camperdown, Sydney, Australia; and the Department ofPathology,
Free Radical Research Group,Christchurch School of Medicine, Christchurch, New Zealand
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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-tocopherol was also present and maintained at a
comparable level over the disease process. Of the oxidized protein
moieties measured only o,o-dityrosine increased
with disease, although chlorotyrosines were present at
relatively high levels in all lesions compared to healthy vessels. Our
data show that accumulation of nonoxidized lipid precedes that of
oxidized lipid in human aortic lesions.
The definitive mechanisms leading to the initiation and development of atherosclerosis are unknown. However, retention of LDL in the vessel wall because of interaction and complex formation with extracellular matrix components is well-documented3 and is thought to be important. Further, a bulk of evidence suggests that oxidative modifications to LDL contribute to its retention.4-6 Both arterial wall retention and oxidative modification of LDL are thought to contribute to foam cell formation.
In vitro studies with plasma LDL have shown that oxidative
modifications proceed in different stages and involve changes to LDL’s
antioxidants, lipids, and protein components. The nature and extent of
these alterations vary depending on the type of oxidant involved, the
oxidant to lipoprotein ratio used, and the extent to which oxidation is
allowed to proceed. Originally4
the pathway was described
by the sequential depletion of endogenous antioxidants including
-tocopherol (-TOH), followed by the formation of lipid
hydroperoxides and their fragmentation to aldehydes and other reactive
secondary products of lipid oxidation. The latter can modify
apolipoprotein B-100 so that the altered particle becomes a ligand for
the scavenger receptor,7
and in vitro exposure
of macrophages to such oxidized LDL can lead to foam cell
formation.8
More recently, it has become apparent that not
all oxidants convert the lipoprotein along the above pathway. Thus,
hypochlorite (a two-electron oxidant produced by myeloperoxidase)
generates a high-uptake LDL by primarily oxidizing the protein
component, although some consumption of -TOH and formation of lipid
hydroperoxides is always observed.9
The fact that oxidized
high-uptake LDL can be formed via different pathways raises questions
of their relative importance and, hence, the extent of lipid
versus protein modification during the oxidative
modification of LDL in vivo.
Whether in vivo LDL lipid peroxidation proceeds beyond the
depletion of -TOH, LDL’s major antioxidant, is unclear. This is an
important issue, as secondary lipid oxidation products including
aldehydes are not formed to any substantial extent in the presence of
the vitamin.10
Low -TOH to cholesterol content has been
reported in advanced lesions,11
although our earlier
studies with advanced human lesions showed normal levels of -TOH per
cholesteryl ester (CE).12,13
Expressing -TOH per CE is
more appropriate as the vitamin scavenges peroxyl radicals produced
during the peroxidation of fatty acids containing bisallylic
hydrogen;14
CE represent the major source of such fatty
acids in lipoproteins and lesions, whereas cholesterol does not contain
bisallylic hydrogen.
The mechanisms responsible for oxidation of LDL within the vessel wall
remain undefined. Several oxidants have been proposed to be involved,
including macrophages, 15-lipoxygenase, myeloperoxidase and oxidants
derived from its action, reactive nitrogen metabolites, NAD(P)H
oxidases, and transition metals.15
Unlike the situation
with atherosclerosis in animals in which a gene knock-out approach can
be used, much of the evidence supporting the involvement of the
different oxidants in human atherosclerosis is derived from the
detection of generic or specific oxidation products in different types
of lesion materials. A problem with this approach has been that to
date, the disease stage-dependent accumulation of several different
lipid and protein oxidation products has not been studied
systematically in the same tissue samples and together with the
accumulation of nonoxidized lipids. We therefore examined various
parameters that reflect the oxidation status of both lipid (CE,
cholesterol) and protein residues (tyrosine (Tyr), phenylalanine (Phe),
and the content of lipophilic antioxidants (-TOH, ubiquinol-10) in
the intimal lipoprotein-containing fraction of human aortic lesions
spanning atherosclerotic disease development.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Cholesteryl linoleate (C18:2), cholesteryl arachidonate (together
referred to as CE), and coenzyme Q9
(ubiquinone-9) were purchased from Sigma Chemical Co.(St. Louis, MO).
D,L--TOH was purchased from Eastman-Kodak
(Rochester, NY). [3H]C18:2
[cholesteryl-1,2,6,7-3H(N)], 91.5 Ci/mmol, was
from Dupont-NEN (Boston, MA).
(25R)-Cholest-5-en-3ß-26-diol (27HC) was from Research
Plus Inc. (Bayonne, NJ) and cholest-5-en-3ß-ol-7-one (7KC) was from
Steraloids Inc. (Wilton, NH). Organic solvents of HPLC quality were
obtained from Mallinckrodt (Clayton, Australia) and Merck (EM Science,
Gibbstown, NJ). Before use, all aqueous solutions were stored over
Chelex-100 (Bio-Rad Laboratories, Richmond, CA) to remove contaminating
transition metals. All other chemicals were of the highest available
purity and nanopure water (Millipore Systems, Australia) was used
throughout.
Specimens
Postmortem materials were obtained from 20- to 66-year-old patients from the NSW Institute of Forensic Medicine, Sydney, Australia, with the approval of the local Human Ethics Review Committee. Aorta were resected within 24 hours of death and washed in ice-cold 50 mmol/L phosphate buffer containing 3-aminotriazole (10 mmol/L), diethylenetriamine pentaacetic acid (0.1 mmol/L), and butylated hydroxytoluene (20 µmol/L), pH 7.4 (buffer A). Aortas were stored in buffer A at -80°C.
Lesion Classification
Lesion stages were initially classified macroscopically according
to Stary and colleague’s16
nomenclature and definition.
The descending thoracic and abdominal aorta from a total of 35 patients
was resected and designated into four distinct groups; initial lesions
(Stary classes I-II), fatty streak lesions (II-III), fibro-fatty
lesions (IV-V), and complex/ulcerated lesions (V-VI), representing the
spectrum of atherosclerosis. A tissue section from each specimen was
removed and fixed in 10% formalin in phosphate-buffered saline and
transferred to 70% (v/v) ethanol for histology, using hematoxylin and
eosin staining (Table 1)
. Remaining
aortas were then stored in buffer A at -80°C until used.
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Sample preparation was essentially as described.17
Briefly separated intima was weighed and tissue samples from the same
group pooled to yield 
10 g. Each 10-g pool represented a single
sample (n = 1) for analysis. Intimas were frozen
in liquid N2, pulverized, and reconstituted in 50
mL of buffer B [10 mmol/L phosphate/0.15 mol/L NaCl, pH 7.4,
containing 0.3 mmol/L ethylenediaminetetraacetic acid, 0.1 mmol/L
diethylenetriamine pentaacetic acid, 10 mmol/L 3-aminotriazole, 20
µmol/L butylated hydroxytoluene, 1 mmol/L phenylmethyl sulfonyl
fluoride, 0.002% (w/v) elastatinal, 2 mmol/L benzamidine, 1 µmol/L
D-phenylalanyl-L-prolyl-L-arginine
chloromethyl ketone, 0.008% (w/v) gentamicin, 0.008% chloramphenicol,
and 10 µmol/L reduced glutathione]. Ubiquinol-9 (5 µmol/L)
prepared from ubiquinone-9,18
and -tocotrienol (1
µmol/L12
) were also added as internal standards for
estimation of recoveries during the processing steps. The samples were
gently agitated end-over-end overnight in the dark at 4°C and then
centrifuged at 2750 x g for 30 minutes at 4°C in a
swing-bucket centrifuge. The resulting tissue supernate (SN-1) was
further ultracentrifuged at 100,000 x g for 30 minutes
at 4°C, the pellet and the top lipemic layer discarded, and a tissue
supernate (SN-2) collected for subsequent analyses. SN-2 contains all
lesion lipoproteins as it represents the starting material for
sequential floatation ultracentrifugation.17
Aortic lesion SN-2 samples (<500 µL) were added to hexane:methanol
(5:1, v/v) for extraction of CE hydro(pero)xides (CE-O(O)H),
cholesterol, CE, and -TOH and the hexane phase reconstituted in
propan-2-ol and subjected to HPLC as described
previously.19,20
For oxysterol analyses aliquots (500
µL of SN-2) were extracted with chloroform/methanol (2:1, v/v) with
added 19-hydroxycholesterol as an internal standard (5 µg/tube). The
chloroform phase was evaporated and transferred with diethyl ether (2.5
mL) to a screw-cap tube before cold alkaline saponification. Total
cholesterol was also determined in separate aliquots (50 µL) after
saponification initiated by a methanolic KOH solution (20% w/v, 2.0
mL). Cholesteryl propyl ether was used as an internal standard (5
µg/tube). The tubes were flushed with argon and the contents mixed
overnight at 4°C before water (2.0 mL) and hexane (2.5 ml) were
added. The tubes were then mixed for 30 seconds and centrifuged at
1600 x g for 5 minutes at 10°C. Oxysterol and
cholesterol in the ether/hexane phase were then determined.
To account for inadvertent oxidation occurring during aortic lesion
work-up, [3H]C18:2 (50 µCi) was added to four
separate aortic (1 g wet weight) lesions. The lesions were subjected
to the tissue and analyte extraction processes described above in
buffer B (5 mL). Radiolabeled C18:2 and/or C18:2-O(O)H fractions were
identified by RP HPLC coupled with on-line radiometric detection.
The hydroxylation product of Tyr (3,4-dihydroxyphenylalanine; DOPA) and those of Phe (o-Tyr, m-Tyr) and o,o-dityrosine (diTyr) in aortic tissue were determined as described previously.21 Briefly SN-2 aliquots (900 µL) were mixed with sodium deoxycholate (100 µL, 0.3% w/v) and trichloroacetic acid (100 µL, 50% w/v). The delipidated protein samples were then freeze-dried and subjected to gas phase, acid-catalyzed hydrolysis using 6 N HCl containing mercaptoacetic acid (5% v/v) and phenol (1% v/v) under anaerobic conditions. Freeze-dried hydrolysates were reconstituted in water (100 µL) and filtered (0.22 µm) before HPLC.21
HPLC Analyses
CE-O(O)H, cholesterol, CE, and -TOH in lesion supernates were
quantified using RP HPLC coupled with UV, electrochemical, and
postcolumn chemiluminescence detection as described.19,20
For detection of radiolabeled compounds the above HPLC conditions were
combined with on-line radiometric detection and a scintillant-to-flow
ratio of 1.
Oxysterols were separated by normal phase HPLC using an Alltima Silica column (0.46 x 15 cm; 100 Å, 5 µm) with a 3-cm guard column (Ultremex, 3-µm particle size; Phenomenex, Thornleigh, Australia) and eluted with hexane/propan-2-ol/acetonitrile (94.8/4.6/0.6, v/v/v) at 1 mL/min. The mobile phase was sparged with helium during analysis and lipids detected at 210 and 234 nm. Total cholesterol was determined by RP HPLC using propan-2-ol:acetonitrile: water (54:44:2, v/v/v). All chromatography assignment and quantification of individual peaks was based on co-elution and area comparison with authentic standards.
DOPA, m-Tyr, o-Tyr, and diTyr in hydrolysates were isolated on a Zorbax ODS column at 1 mL/min elution and a gradient of solvent A (100 mmol/L sodium perchlorate in 10 mmol/L sodium phosphate buffer, pH 2.5) and solvent B (80% methanol in water) as described.22 The eluant was monitored by in-series UV (280 nm) and fluorescence detection (280 nm excitation for all components and emission wavelength of 320 nm for DOPA, m-Tyr and o-Tyr and 410 nm for diTyr). The identity and quantity of representative samples were checked by electrochemical detection23 and mass spectroscopy.21,22 Artifactual formation of m-Tyr, o-Tyr, DOPA, and diTyr during protein hydrolysis of atherosclerotic tissue using the methodology described herein has been investigated in detail.21,23,24 The maximum artifactual formation of the analytes determined by this method consistently ranges from 20 µmol/mol of m-Tyr, o-Tyr, and diTyr to 200 µmol/mol of DOPA per parent amino acid.
Gas Chromatography/Mass Spectroscopy (GC/MS) Determination of Chlorinated Tyrosine
3-Chlorotyrosine (ClTyr) and 3,5-dichlorotyrosine (diClTyr) were determined by stable isotopic dilution GC/MS as described.25 Briefly, samples were dried and proteins hydrolyzed using methanesulfonic acid (4 mol/L) containing 1% (v/v) phenol (100 µL). The hydrolysate was diluted in 0.1% trifluoroacetic anhydride (200 µL) and purified using solid phase extraction. Tyr, ClTyr, and diClTyr were eluted using 80% methanol containing 0.1% trifluoroacetic anhydride (1.1 mL) and dried. Amino acids were derivatized with n-propanol/HBr (3.5 mol/L) (300 µL) and then with trifluoroacetic anhydride/ethyl acetate (1:4, v/v) (100 µL). Derivatized samples were finally diluted in ethyl acetate (300 µL) before GC/MS analysis. The initial column temperature of 65°C was maintained for 2 minutes and then increased to 150°C (30°C/minute), 170°C (4°C/minute), 200°C (5°C/minute), and to 260°C (30°C/minute). The injector, auxiliary channel, and ion source temperatures were set to 250, 280, and 150°C, respectively. Pulsed splitless injection was used with selective ion monitoring. The background levels of diClTyr and diTyr in bovine serum albumin determined by this method range from 15 to 40 µmol/mol.25
Statistical Analysis
Kruskill-Wallis one-way analysis of variance on ranks and Mann-Whitney t-tests were used to evaluate significant differences at a P value 0.05 (two-tailed).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-TOH is not extensively
depleted despite the co-existence of oxidized CE.12,13
Herein we report on the characterization of antioxidants, lipid,
oxidized lipid, and proteins from the intimal lipoprotein fraction of
human aorta representing the earlier spectrum of the disease process.
Aortic intimal tissue was designated into four groups based on disease
status according to Stary and colleagues:16
I-II, initial
lesions; II-III, fatty streaks; IV-V, fibro-fatty lesions; and V-VI,
ulcerated/complex lesions (Table 1)
. A total of 35 aortas were used,
and all materials were obtained from patients 
20 years old. Thereby
the material in group I-II contained intima with diffuse thickening and
initial lesions and is distinct from fatty streak lesions and normal
artery tissue.16
Consequently a large number
(n = 24, Table 1
) of individual tissue samples
representing stage I-II were required to generate four separate pools
of tissue for analysis. Macroscopic assessment enabled processing of
the specimens before histology thereby averting a freeze/thaw cycle and
potential generation of artifacts. Although visual assessment does not
reflect intimal thickness we obtained >80% agreement (Table 1)
,
demonstrating that visual classification was useful and appropriate.
After low-temperature pulverization of the intima, the reconstituted tissue (SN-1) was subjected to centrifugation and removal of a lipemic layer to yield a tissue supernate designated SN-2. This supernate contains all tissue lipoproteins and thereby all analytes reported below reflect lipoprotein-associated parameters. Next we investigated the potential autoxidation of lipid (C18:2) and recovery of the internal standard, ubiquinol-9 to assess artifactual oxidation during sample work-up. Overall, tests with [3H]C18:2 (see Materials and Methods) indicated that the radiolabel eluted as unmodified C18:2 (n = 4), whereas on average, 60% (mean, n = 4) of added ubiquinol-9 was recovered as total CoQ9 in SN-2 (data not shown). The contents of lipid, both unmodified and oxidized, lipid-soluble antioxidants, and protein oxidation markers in SN-2 from the various lesions were then determined to compare relative changes in lipid, protein, and oxidation parameters in the disease process more systematically and comprehensively than reported previously.
An increase in cholesterol is a prominent early disease
marker.1
In agreement, the content of cholesterol per
protein increased with increasing lesion severity, with significant
changes noted in the early disease stages (Table 2)
. Lipoprotein-associated CE (the sum of
cholesteryl arachidonate and C18:2) also accumulated (Table 2)
,
although significant changes were not obtained until fibro-fatty
lesions were present. The level of CE per cholesterol in lesion
lipoproteins decreased in the most advanced lesions (V-VI).
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. However, they were detected
in fibro-fatty and advanced lesions, albeit at much lower amounts
(<2.5%) than the sum of CE hydroperoxides and hydroxides (CE-O(O)H)
(Table 2)
indicating that CE hydroxides (ie, CE-OH) were the major
product. Similar to hydroperoxides, CE-O(O)H were significantly
increased only in fibro-fatty and more complex lesions (stage IV)
(Table 2)
. We also measured 7KC as a marker of one-electron-mediated
cholesterol oxidation and 27HC, an enzymatic product of the
mitochondrial cytochrome P450 enzyme, sterol 27-hydroxylase. Of these,
27HC was present at higher levels than 7KC (Table 2)
. Whereas 7KC
content increased significantly only in the most complex lesions (group
V-VI), disease stage-dependent increases in 27HC were significant in
fibro-fatty lesions (Table 2)
.
To assess the dependency of the changes in nonoxidized and oxidized
lipids on lesion development, we expressed the content of each type of
lipid relative to the corresponding parent molecule in the intima
showing the least disease, ie, stage I-II (Figure 1)
. Thus, cholesterol increased up to
eightfold with lesion development (Figure 1A)
. Also, the relative
changes in CE were similar to cholesterol except in the most complex
lesions where CE markedly decreased. Similar to unmodified lipid,
lipoprotein-associated CE-O(O)H, 27HC, and 7KC increased significantly
with lesion severity (Figure 1B)
.
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. The amount of CE-O(O)H per CE
increased significantly in advanced lesion stages (V-VI) and comprised
2.4% of the parent molecule (Figure 2)
. Compared to CE-O(O)H however,
the percentage of 27HC per parent molecule comprised 0.9% even at the
most advances stages of the disease. Furthermore, the intimal content
of 7K per total cholesterol did not increase significantly (Figure 2)
.
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-TOH and ubiquinol-10 (measured as
CoQ10) remained detectable at each disease stage
(Table 3)
. In fact, the intimal content
of -TOH expressed per protein was similar for most lesions except
for a significantly elevated level in stages IV-V (Table 3)
. In
general, the -TOH/CE ratio remained constant independent of lesion
severity (Table 3)
. CoQ10, the reduced form of
which is an effective endogenous antioxidant for LDL
lipid,28
was present at 2 to 5 times lower concentrations
than -TOH and the levels of this antioxidant did not seem to alter
with disease progression (Table 3)
.
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or to parent molecule
(Figure 3)
. A notable exception was diTyr
that increased significantly in the most advanced lesions (Table 4
and
Figure 3
).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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According to the LDL oxidation theory of atherosclerosis,4 oxidation of LDL occurs early in the disease process and precedes foam cell accumulation and fatty streak formation. Despite the popularity of the theory, the accumulation of native and oxidized lipid and protein in relation to disease stage is rarely quantified and compared. Rather, single oxidation parameters are commonly determined at only a single disease stage or, alternatively, lesion parameters are compared to the concentration of total plasma or LDL cholesterol. We therefore compared the relative changes in protein and selected lipids that represent the major oxidizable components of plasma lipoproteins, and oxidation parameters of both in the intima of human aortic tissue displaying lesions of increasing severity. Importantly, tissue was resected within 24 hours of death. Within this time frame there are no significant changes to the biochemical composition of arterial tissue and the contents of free and esterified cholesterol, and other lipids and protein remain stable.29 In addition, the levels of CE-O(O)H per parent detected in the most severe aortic lesions (2.4%) were less than that observed for carotid lesions (3.5%) processed freshly within 2 hours of resection.13 Further, we compared analytes from various grades of lesion derived from the same aortic tissue sample and verified that artifactual oxidation of CE did not occur during sample processing. As CEs are more susceptible to oxidation than cholesterol, Tyr and Phe, the results obtained therefore likely represent the situation in vivo.
LDL lipid accumulation is a prominent and early marker for
atherosclerosis. Consistent with this, the content of cholesterol
significantly and linearly increased with disease severity (Table 2)
.
As expected, the CE content also increased with increasing lesion
severity, although the CE load per cholesterol in lesion lipoproteins
tended to decrease in the most advanced lesions. This is consistent
with an earlier report showing a decrease in C18:2 in fibrous
plaques,30
as CE measured herein represents the sum of
C18:2 and cholesteryl arachidonate. This suggests increased hydrolysis
of lipoprotein CE in advanced lesions and/or preferential loss of CE
rather than cholesterol from lipoproteins.
We selected CE-O(O)H and 7KC as markers of nonenzymatic, free
radical-mediated lipid (per)oxidation. The former are the major lipid
oxidation products formed when lipoproteins undergo in vitro
oxidation and contribute to the in vitro formation of
high-uptake LDL.7
In addition, CE-O(O)H are the major
lipid oxidation products in human lesions1,12,26,31
and
hydroxylated fatty acids are associated with plaque
instability.31
Notably, CE-O(O)H and oxysterols are not
found in normal iliac arteries12,32
or in circulating
plasma lipoproteins.13
Our data show a disease
stage-dependent increase in intimal CE-O(O)H (Table 2)
suggesting that
overall CE-O(O)H is a suitable parameter to monitor the extent of
lipoprotein lipid oxidation in the vessel wall.
As lesion severity increased, the percentage of lipoprotein-associated
CE present as CE-O(O)H increased to a maximum of 2.3%. Although
significantly greater than any other oxidation parameter determined
here, this value is at the low end of the range of values reported
previously for carotid plaque samples.12,13
The
configurational isomer distribution of C18:2-OH (JM Upston, AC
Terentis, R Stocker, unpublished results) indicate that particularly in
the early stages of atherosclerosis, the majority of CE-O(O)H detected
were formed in the presence of -TOH. Indeed, we show herein that
-TOH did not decrease with advancing atherosclerosis, consistent
with our previous data12,13
and a separate detailed
GC/MS-based study of -TOH oxidation products in human lesion
homogenates.33
The notion that oxidation of lipoprotein
lipid in the vessel wall proceeds via tocopherol-mediated
peroxidation34,35
suggests a likely mechanism for the
formation of most CE-O(O)H in the presence of -TOH in lesions.
Our observation that even advanced lesions contain normal -TOH and
at most 2.3% of its CE oxidized to CE-O(O)H has potentially important
implications for several reasons. First, the overall extent of lipid
oxidation is substantially less than that needed for the in
vitro conversion of LDL to a high-uptake form by
Cu(II).10,36
This conclusion is consistent with the
finding that the extent of oxidative change to aortic LDL, as assessed
by electrophoretic mobility or other physical parameters, is less than
that required for scavenger receptor-mediated uptake.37
Second, our observation has potential implications for the formation of
both minimally modified LDL and isoprostanes. Oxovaleroyl, glutaroyl,
and epoxyisoprostane (the three active moieties identified in minimally
modified LDL generated in vitro38
) and
isoprostanes are secondary lipid oxidation products. The formation of
isoprostanes is essentially prevented as long as -TOH is
present.39
Consistent with this, isoprostane levels in
atherosclerotic lesions are 20-fold to 300-fold lower per parent
molecule than lipid hydroxides.31
Like isoprostanes,
oxovaleroyl and glutaroyl are generated from arachidonic
acid-containing phospholipids, most likely via fragmentation of
arachidonic acid hydroperoxides, a process that is inhibited strongly
in the presence of -TOH.40
Indeed, the generation of
fragments of polyunsaturated fatty acids is almost completely prevented
during LDL oxidation as long as -TOH remains present.10
This indicates that the bioactive phospholipids generated during
in vitro oxidation of LDL and present in minimally modified
LDL may not be formed in substantial amounts in lesion lipoproteins the
-TOH content of which remains essentially intact. Arachidonic acid
is also present in cellular phospholipids and detection of its
secondary oxidation products in homogenates of lesion material does
not, by itself, establish their presence in lipoproteins. In fact,
immunohistochemical studies suggest that lesion isoprostanes are
predominantly associated with foam cells.41
Similarly,
using EO6, a monoclonal antibody specific for oxovaleroyl-containing
phospholipid,42
Palinski and
colleagues43
showed predominant co-localization of this
epitope with foam cells in lesions of cholesterol fed New Zealand White
and LDL receptor-deficient rabbits. Additional studies are needed to
fully establish the precise extent of oxidation of lesion LDL, whether
the oxidized arachidonate moieties detected41,43
represent
cellular membrane lipids (eg, from apoptotic or necrotic cells) or
lipids from lipoproteins taken up, and how these oxidation parameters
relate to lesion CE-O(O)H.
In contrast to CE-O(O)H, the oxidation of cholesterol was primarily
enzymatic. Of the analytes measured, 27HC showed the greatest and
earliest relative changes with disease progression (Figure 1)
, and
accounted for 0.9% of total cholesterol (Figure 2)
. The
disease-associated increase in the cholesterol-standardized 27HC
content is consistent with a previous report44
and infers
a disease-associated increase in the activity of sterol 27-hydroxylase
that co-localizes with macrophages in lesions.45
Interestingly, the 27-hydroxylase pathway has been suggested to be
important in the removal of extrahepatic cholesterol.46
As
the accumulation of cholesterol preceded that of 27HC, the formation of
the latter may be interpreted as a physiological (potentially
anti-atherogenic) response to cholesterol load.
Similar to 27HC, the presence of increased protein-standardized CE-O(O)H in fibro-fatty lesions may reflect the increased lipid load of that lesion stage and suggests that LDL lipid oxidation follows lipid accumulation in the temporal sequence of atherogenic events. However, it is possible that in the early disease stages lipid oxidation products are effectively metabolized or removed from the artery wall, but that their formation may still contribute to promotion of atherogenesis. Indeed, one study of human fetal fatty streak lesions reported the presence of malondialdehyde- and 4-hydroxynonenal-modified lysine (measured indirectly by immunological techniques) in the absence of monocytes/macrophages.47 These authors suggested that intimal LDL accumulation and oxidation is an early event that contributes to monocyte recruitment into the vessel wall. To more precisely implicate a causal role of lipid (per)oxidation in atherogenesis the metabolism and/or removal of oxidation products is worthy of further study.
Consistent with our findings, the content of the free radical-derived, cholesterol-standardized 7KC, was similar in human fatty streak and advanced lesions,48 yet homogenate of endarterectomy plaque contains more 7KC than that of normal arteries.12,32 A possible explanation for this difference is that we analyzed the lipoprotein fraction rather than the entire intima. Indeed, in pilot studies with the first tissue supernate (SN-1, containing SN-2 plus the lipemic layer; see Materials and Methods) we observed oxysterols and cholesterol-standardized 7KC to increase with increasing disease severity (data not shown). A lower 7KC and 27HC content in the SN-2 fraction compared to SN-1 is consistent with oxysterols being concentrated in the core of lesions, rather than in lipoproteins or the fibrous cap, as has been reported for 27HC (referred to as "26-OH-CHOL" by Garcia-Cruset and colleagues49 ).
Several groups have demonstrated the presence of oxidized protein
moieties in atherosclerosis including ClTyr,50
diTyr,21,51
DOPA, m-Tyr, and
o-Tyr.21
However, of the protein oxidation
products measured herein only diTyr increased significantly from early
to late disease stage (Table 4
, Figure 3
). These data are consistent
with increased diTyr levels found in aortic lesion-derived LDL compared
to plasma LDL.51
Chlorinated Tyr was determined as both mono- and dichloro forms. ClTyr
was present in higher amounts than diClTyr (Table 4)
, with levels
similar to those previously determined for lesion LDL (300
µmol/mol Tyr).50
Interestingly, the diClTyr to ClTyr
ratio in lesion lipoproteins was high (Table 4)
compared to that found
for reactions of HOCl with free and peptide-bound Tyr.24
In contrast, analysis of SN-2 after removal of all lipoproteins showed
comparatively fewer products and lower ratios of diClTyr to ClTyr (data
not shown). A high ratio may be obtained under conditions of high local
oxidant production (AJ Kettle, unpublished results), and our data
supports the previous suggestion by Heinecke52
that Tyr
chlorination via myeloperoxidase activity is relatively specific for
lesion lipoproteins. This is also consistent with the observation that
myeloperoxidase binds to LDL.53
Excepting diTyr, no other Tyr modification showed a clear disease stage
dependency and elevated levels of diTyr were detected only in the most
advanced lesion stages. diTyr may be produced via myeloperoxidase
activity and its increase in complex lesions is consistent with a
recent study showing increased numbers of myeloperoxidase-expressing
macrophages in advanced, but not early, atherogenesis.54
However an increase in the content of Tyr modification, particularly
diTyr, appeared to be present in even early lesions compared to those
values derived for normal iliac tissue,21
normal
aorta,51
and normal LDL55
(Table 5)
. Thus, our results do not exclude the
possibility that specific Tyr modifications may be early disease events
and that such oxidation could contribute to the initiation of
atherogenesis.
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-TOH. These data show that vitamin E does not
generally become depleted and suggest, although do not prove, that
appreciable lipoprotein lipid (per)oxidation represents a response to
rather than a cause for lesion development.
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Acknowledgements |
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Footnotes |
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Supported by the National Heart Foundation, the National Health and Medical Research Council of Australia, and Blackmores Ltd.
Accepted for publication November 14, 2001.
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References |
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-tocopherol and ascorbate. Arterioscler Thromb Vasc Biol 1995, 15:1616-1624-tocopherol in all lipoprotein fractions isolated from advanced human atherosclerotic plaques. Arterioscl Thromb Vasc Biol 1999, 19:1708-1718-tocopherol. Proc Natl Acad Sci USA 1991, 88:1646-1650This article has been cited by other articles:
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S. Sugiyama, K. Kugiyama, M. Aikawa, S. Nakamura, H. Ogawa, and P. Libby Hypochlorous Acid, a Macrophage Product, Induces Endothelial Apoptosis and Tissue Factor Expression: Involvement of Myeloperoxidase-Mediated Oxidant in Plaque Erosion and Thrombogenesis Arterioscler. Thromb. Vasc. Biol., July 1, 2004; 24(7): 1309 - 1314. [Abstract] [Full Text] [PDF]
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R. Stocker and R. A O'Halloran Dealcoholized red wine decreases atherosclerosis in apolipoprotein E gene-deficient mice independently of inhibition of lipid peroxidation in the artery wall Am. J. Clinical Nutrition, January 1, 2004; 79(1): 123 - 130. [Abstract] [Full Text] [PDF]
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 G. Pankhurst, X. L. Wang, D. E. Wilcken, G. Baernthaler, U. Panzenbock, M. Raftery, and R. Stocker Characterization of specifically oxidized apolipoproteins in mildly oxidized high density lipoprotein J. Lipid Res., February 1, 2003; 44(2): 349 - 355. [Abstract] [Full Text] [PDF]
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) and CE (
)
CE-O(
)H (•), 27HC
(
), and 7KC
(µ) were expressed as a
fraction of those observed in tissue containing no/initial lesions
(group I-II). The data
represent mean values ± SEM with asterisks indicating
P < 0.05 compared with group I-II.
