Electrospray MS/MS reveals extensive and nonspecific oxidation of cholesterol esters in human peripheral vascular lesions

Article abstract of DOI:10.1194/jlr.M019174

Although LDL is rendered proatherogenic by various experimental treatments (e.g., acetylation), the exact structural changes that drive LDL transformation in vivo remain enigmatic. Among the many hypothesized targets of oxidative modification are cholesterol esters (CE). This family of neutral lipids, which carries a highly unsaturated pool of fatty acyl groups, is the main component of both LDL particles and atherosclerotic plaques. Tandem mass spectrometry (MS/MS) was employed to reveal abundant and diverse oxidized CEs (oxCE), including novel oxidation products, within human peripheral vascular lesions. These oxCE species composed up to 40% of the total CE pool, with cholesteryl linoleate being oxidized to the greatest extent. Imaging mass spectrometry studies showed that oxCE was entirely confined within the plaque, along with unmodi- fied CE and triacylglyceride (TAG). Interestingly, we found no evidence for TAG oxidation, although polyunsaturated species were abundant. Enzymatic oxidation of cholesteryl linoleate by 15-lipoxygenase (15-LO), an enzyme often invoked in CE oxidation, initially results in a regio- and stereospecific product. Analysis of intact cholesteryl hydroxyoctadecadienoate isomers in human atheromata revealed no regio- or stereospecificity, indicating 15-LO was either not a major source of oxCE or nonenzymatic processes had eroded any product specificity. Copyright

Full text of DOI:10.1194/jlr.M019174

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patient-oriented and epidemiological research
Electrospray MS/MS reveals extensive and nonspecific
oxidation of cholesterol esters in human peripheral
vascular lesions
Patrick M. Hutchins,* Ernest E. Moore,† and Robert C. Murphy1,*
Departments of Pharmacology* and Surgery,† University of Colorado at Denver, Aurora, CO 80045
Abstract Although LDL is rendered proatherogenic by
various experimental treatments (e.g., acetylation), the ex-
act structural changes that drive LDL transformation in vivo
remain enigmatic. Among the many hypothesized targets of
oxidative modification are cholesterol esters (CE). This
family of neutral lipids, which carries a highly unsaturated
pool of fatty acyl groups, is the main component of both
LDL particles and atherosclerotic plaques. Tandem mass
spectrometry (MS/MS) was employed to reveal abundant
and diverse oxidized CEs (oxCE), including novel oxidation
products, within human peripheral vascular lesions. These
oxCE species composed up to 40% of the total CE pool,
with cholesteryl linoleate being oxidized to the greatest ex-
tent. Imaging mass spectrometry studies showed that oxCE
was entirely confined within the plaque, along with unmodi-
fied CE and triacylglyceride (TAG). Interestingly, we found
no evidence for TAG oxidation, although polyunsaturated
species were abundant. Enzymatic oxidation of cholesteryl
linoleate by 15-lipoxygenase (15-LO), an enzyme often
invoked in CE oxidation, initially results in a regio- and
stereospecific product. Analysis of intact cholesteryl
hydroxyoctadecadienoate isomers in human atheromata
revealed no regio- or stereospecificity, indicating 15-LO was
either not a major source of oxCE or nonenzymatic pro-
cesses had eroded any product specificity.—Hutchins, P. M.,
E. E. Moore, and R. C. Murphy. Electrospray MS/MS
reveals extensive and nonspecific oxidation of cholesterol
esters in human peripheral vascular lesions. J. Lipid Res.
2011. 52: 2070–2083.
reported (1, 2). Cholesteryl esters (CE) constitute the bulk
of the accumulated neutral lipid, and a number of studies
have identified several families of oxidized cholesteryl
esters (oxCE). These oxCE species include hydroxy-octadec-
adienoate-CE (HODE-CE), hydroperoxy-octadecadieoate-CE
(HpODE-CE), and oxo-octadecadienoate-CE (oxoODE-CE),
as well as chain-shortened ␻-aldehyde-CE, often referred
to as “core aldehydes” (3–6).
Oxidation of CE is frequently cited in the context of the
oxidative modification theory of atherogenesis (7, 8). This
well-supported theory proposes that LDL is oxidized and
subsequently taken up by macrophages through surface
receptors other than the LDL-receptor. This unregulated
process leads to foam cell formation and atherogenesis.
There is evidence that oxidation of CE, the chief compo-
nent of LDL, may be involved in this process (9). First,
abundant oxCE has been detected and shown to accumu-
late in human atheromata (4, 10). Oxidized CE has also
been detected in human plasma (11). Second, CE is read-
ily oxidized when LDL is incubated with macrophages
(12). Finally, LDL particles oxidatively modified by mac-
rophages are taken up at much greater rates than are
unmodified LDL particles (13). For these reasons, a
proatherogenic role of CE oxidation is widely assumed,
although recent studies using foam cells as well as in vivo
models suggested that oxidation of CE may increase re-
verse cholesterol transport; however, this hypothesis is
controversial (3, 14, 15).
Supplementary key words atherosclerosis • 15-lipoxygenase • imag-
ing mass spectrometry • MALDI • ammonium adduct • sodium ad-
duct • triacylglyceride
The accumulation of neutral lipid in the arterial wall is
a defining feature of atherosclerosis, and since the 1950s,
oxidized lipid species within arterial plaques have been
Abbreviations: BSTFA, N,O-bis(trimethylsilyl)-trifluoroacetamide;
CE, cholesteryl ester; oxCE, oxidized cholesteryl ester; CID, collision-
ally induced decomposition; DCM, dichloromethane; EpOME, epoxy-
octadecenoate; HETE, hydroxy-eicosatetraenoate; HODE, hydroxy-
octadecadienoate; HpETE, hydroperoxy-eicosatetraenoate; HpODE,
hydroperoxy-octadecadieoate; IMS, imaging mass spectrometry;
15-LO, 15-lipoxygenase; MRM, multiple reaction monitoring; MTBE,
methyl tert-butyl ether; NH4OAc, ammonium acetate; NP, normal
phase; oxoODE, oxo-octadecadienoate; RP, reversed phase; TAG, tria-
cylglyceride; TOF, time of flight.
This work was supported in part by the LIPIDMAPS Large Scale Collaborative
Grant GM-069338 from the General Medical Sciences Institute of the National
Institutes of Health. Its contents are solely the responsibility of the authors and
do not necessarily represent the official views of the National Institutes of Health
or other granting agencies.
1 To whom correspondence should be addressed.
Manuscript received 29 July 2011 and in revised form 26 August 2011.
e-mail: Robert.Murphy@ucdenver.edu
Published, JLR Papers in Press, August 31, 2011
DOI 10.1194/jlr.M019174
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of six figures and one table.
Copyright © 2011 by the American Society for Biochemistry and Molecular Biology, Inc.
2070
Journal of Lipid Research Volume 52, 2011
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Sigma-Aldrich or Fischer Scientific (Fair Lawn, NJ). Protocol
Production of oxCE and the consequences of its accu-
mulation are likely complex, especially in the milieu of a
multifactorial disease such as atherosclerosis. Certainly,
the diverse and ever-expanding array of CE oxidation
products has complicated efforts to investigate this phe-
nomenon. To more completely characterize the nature of
CE oxidation in atherosclerosis, we used HPLC-MS/MS to
structurally elucidate oxCE species isolated from human
atheromata. These analyses revealed a large array of abun-
dant oxCE species, including novel oxidation products. It
was immediately clear that this family of compounds was
both more diverse and more abundant than previously ap-
preciated. Interestingly, we found no evidence of triacylg-
lyceride (TAG) oxidation in lesion extracts, even though
polyunsaturated species were abundant and were shown
to colocalize with oxCEs by imaging mass spectrometry
(IMS). Note that several of the oxCE species identified in
femoral and popliteal lesions have also been detected in
iliac and carotid plaques and even in cholesterol-fed ze-
brafish (3–5, 16). These data suggest that the array oxCE
species reported here might be common to advanced le-
sions throughout the vasculature.
15-Lipoxygenase (15-LO) was named for its ability to
add an oxygen molecule to carbon fifteen of arachidonic
acid. This is perhaps a misnomer as cholesteryl linoleate
(18:2-CE) is the preferred substrate of 15-LO in vitro (17,
18). This enzymatic reaction results mainly in the forma-
tion of cholesteryl (S)-13-hydroxy-9Z,11E-octadecadieno-
ate ((S)13-(ZE)-HODE-CE). 15-LO is expressed in lesions
(19), and several studies have investigated its potential ac-
tivity by analyzing CEs for this chiral oxidation product.
These efforts have produced conflicting results (5, 20–22).
Here, chiral analysis of intact oxCE species in human le-
sions found no preponderance of the 15-LO-specific
product.
Hema 3® histology stain set was purchased from Fisher
Scientific.
Human samples
Data are presented from six atheromata samples originating
from four subjects. Three samples were generated during femo-
ral endarterectomy procedures on two individual patients. Three
unique lesions were recovered from the popliteal arteries of two
patients. Samples were immediately frozen (Ϫ80°C) after collec-
tion, and the elapsed time until extraction was kept to a mini-
mum, usually <24 h. These samples constitute discarded tissue
generated during medical procedures and were collected from
patients with informed consent. Additional approval from the
Colorado multi-institutional review board was granted for the
transfer of deidentified tissue samples to this laboratory for analy-
sis. Pooled human plasma (from 22 volunteer donors) was as-
sembled at the University of Texas Southwestern Medical Center
(Dallas, TX) and transferred to the laboratory at the University of
Colorado Denver with institutional approval.
Atheromata preparation
Samples were stored at Ϫ70°C before analysis. Once thawed,
atheromata were placed in a 1 ml etched glass tissue grinder con-
taining 1 ml of 1:1 MeOH:H2O (v/v) and homogenized over ice.
Certain samples were spiked with internal standard(s) prior to
homogenization. Homogenate was extracted twice using 500 ␮l
75:25 isooctane:ethyl acetate (v/v). The combined organic layers
were dried under N2, and then the residue was weighed, diluted
to 1 ␮g/␮l in isooctane, and stored at Ϫ20°C. Auto-oxidization
during homogenization/extraction was assessed. In certain ex-
periments, atheromata tissue was homogenized and extracted in
the presence of d4-18:2-CE at approximately equal concentration
as endogenous 18:2-CE. Oxidation products of endogenous 18:2-
CE were clearly detected; however, no oxidation of the d4-18:2-
CE was observed (supplementary Fig. II). To monitor oxidation
postextraction, identical analyses were performed on a single
sample immediately following extraction and again after 75 days
of storage at Ϫ20°C. The ratio of HODE-CE / 18:2-CE (by raw
peak area) was unchanged over time: 1.57 at day 0 and 1.54 at
day 75, indicating undetectable oxidation during this storage
period.
EXPERIMENTAL PROCEDURES
Chromatography
Materials
Normal-phase HPLC (NP-HPLC) was performed essentially as
previously reported (24). For most analyses, 1-10 ␮g of lipid resi-
due was injected. For preparative chromatography, up to 100 ␮g
of lipid was loaded onto a 250 × 4.6 mm, 5 ␮m, Ultramex Silica
column from Phenomenex. The same gradient and solvents were
used but at a flow of 1 ml/min, of which approximately 200 ␮l
was introduced into the mass spectrometer while the remainder
was diverted to a fraction collector.
Cholesteryl (+/Ϫ)-13-hydroxy-9Z,11E-octadecadienoic acid
(13-(ZE)-HODE-CE), (R)-13-(ZE)-HODE-CE, and (+/Ϫ)-9-
hydroxy-10E,12Z-octadecadienoic acid (9-(EZ)-HODE-CE) were
obtained from Cayman Chemical (Ann Arbor, MI). Also pur-
chased from Cayman Chemical were cholesteryl (+/Ϫ)-13-
hydroperoxy-9Z,11E-octadecadienoic acid (13-(ZE)-HpODE-CE)
and (+/Ϫ)-9-hydroperoxy-10E,12Z-octadecadienoic acid (9-(EZ)-
HpODE-CE) as a mixture. Cholesteryl 9Z-heptadecenoate was
obtained through Avanti Polar Lipids (Alabaster, AL). Custom-
synthesized d4-17,17,18,18-linoleic acid was supplied by Dr. Howard
Sprecher (23). The N,O-bis(trimethylsilyl)-trifluoroacetamide
(BSTFA) was purchased from Supelco (Bellefonte, PA). Meth-
oxyamine hydrochloride was obtained from MP Biomedicals
(Solon, OH). Polymer-supported carbodiimide beads were
purchased from Biotage (Uppsala, Sweden). The free fatty acids
13-oxo-9Z,11E-octadecadienoate (13-OxoODE), 9-oxo-10E,12Z-
octadecadienoate (9-OxoODE), and 11-hydroxy-5Z,8Z,12E,14Z-
eicosatetraenoate (11-HETE) were purchased from Cayman
Chemical, as was purified soybean 15-lipoxygenase. Free choles-
terol was obtained from Sigma-Aldrich (St. Louis, MO). All sol-
vents used were HPLC or Optima grade and were obtained from
Chiral separations
HPLC was performed on a 250 × 4.6 mm Chiracel® OD, cellu-
lose-tris (3,5-dimethylphenylcarbamate), 10 ␮m silica-gel column
from Chiral Technologies Inc. (West Chester, PA). 13-(ZE)-
HODE-CE was isolated from 25 ␮g of total lipid. (R)- and (S)13-
(ZE)-HODE-CE were separated using a gradient of methyl
tert-butyl ether (MTBE) in isooctane (700 ␮l/min), starting at
35% MTBE and ramping to 47% over 20 min.
Reversed-phase HPLC (RP-HPLC) was employed to separate
hydrolyzed acyl components as previously reported (25). Acyl
components derived from approximately 10-100 ␮g of total lipid
were analyzed. Cholesteryl esters were saponified according to
CE oxidation in human atheromata
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the method of Parks et al. using NaOH (5% w/w) in 10:1
were spiked with internal standard and analyzed by NP-HPLC-
MS/MS using MRM. The analysis included MRM transitions for
the internal standard, the three major CE species, and four fami-
lies of oxidation products for each major CE. These included CE +
oxygen (M + 16 amu), CE + oxygen – H2 (M + 14 amu), CE + O2
(M + 32 amu), CE + O2 – H2 (M + 30 amu) (supplementary Table I).
As reference standards were not available to generate calibration
curves for most oxCEs, an averaged calibration curve was used to
normalize all oxCEs.
ethanol:H2O (26). The solutions were then acidified and free
fatty acids were extracted with 2 × 500 ␮l 75:25 isooctane:ethyl
acetate. If acidification was undesirable, free fatty acids were iso-
lated by C18 solid phase extraction as previously described (25).
Oxidized cholesteryl ester derivatization and synthesis
Silylation of hydroxy- and hydroperoxy-CEs was done with
BSTFA as follows. Two microliters of BSTFA was added to ف10 ng
substrate in 50 ␮l ACN. The mixture was gently agitated for 1 min
before the solution was evaporated under a stream of N2 and di-
luted in 40 ␮l of isooctane. Ketones and aldehydes were con-
verted to methoxime species using gaseous methoxyamine in a
solvent-free reaction system as described previously (27). The de-
rivatized products were extracted and diluted as described
above.
For small-scale cholesteryl ester syntheses (>100 ␮g), various
free fatty acids were esterified to cholesterol using PS-carbodiim-
ide essentially as follows. One milligram of PS-carbodiimide
beads was added to 1 ␮g of free fatty acid in 1 ml dichloromethane
(DCM). After approximately 1 min of intermittent vortexing, 10 ␮g
of free cholesterol in 200 ␮l DCM were added, and the vial was
tumbled overnight at room temperature. The solution was then
decanted into a sample vial, dried under a stream of N2, and di-
luted in 100 ␮l isooctane. For larger-scale cholesteryl ester syn-
theses, solution-phase coupling chemistry was employed as previ-
ously described (24).
MALDI IMS
Artery segments (3-5 mm) from human subjects were cut from
still-frozen arteries to preserve gross morphology. Thawed sam-
ples were flushed with saline and embedded in modified OCT
(28). Cross-sectional slices (20 ␮m) were placed on cover slips
and lyophilized for 1 h. Slides were attached to the MALDI plate
and dihydroxybenzoic acid was sublimed onto the entire surface
as previously described (29). Images were acquired on a QSTAR
XL quadrupole-time of flight (Q-TOF) mass spectrometer
equipped with a MALDI ion source (Applied Biosystems/MDX
Sciex, Thornhill, Ontario, Canada). Instrument parameters and
data processing were described previously (28). For MALDI CID
spectra of authentic CEs and oxCEs, standards were dried onto
the MALDI plate from a 1 ng/␮l solution in isooctane. A solution
of NaCl (6 mM) in 1:1 MeOH:H2O (v/v) was then dried over the
resulting spot before the entire plate was coated with DHB by
sublimation.
IMS samples were stained with Hema 3® according to the man-
ufacturer’s guidelines following MALDI analysis. Adjacent sec-
tions were collected and fixed with 3% formaldehyde for 15 min,
then rinsed with 60% isopropanol in H2O before staining for
15 min with a filtered solution of Oil Red O (5% w/w in 60%
isopropanol:H2O) followed by rinsing and staining with the blue
solution of the Hema 3® stain set.
Mass spectrometry
Online NP-HPLC-MS/MS was performed on an AB Sciex API
3200 triple quadrupole mass spectrometer (AB Sciex, Toronto,
Canada). Eluent from all normal-phase chromatographic meth-
ods was modified with 10 mM ammonium acetate (NH4OAc) in
95:5 acetonitrile:H2O (v/v) introduced prior to the ion source at
a flow rate equal to one fifth that of the eluent. The mass spec-
trometer was operated in positive ion mode with an ion spray
voltage of 5.5 kV. For most experiments, the mass spectrometer
alternated between precursor ion scanning (precursors of m/z
369.3 or 385.3) and full mass Q3 scanning. Precursor ion data
from m/z 620 to 780 (or m/z 420 to 620), was collected over 5 s
periods with a collision energy of 18 V. Q3 scans from m/z 600 to
1000 were collected using no collision energy. Alternatively, CEs
and oxCEs were detected by multiple reaction monitoring
(MRM) in which 19 transitions were monitored for 200 msec
each using the same parameters as precursor ion experiments
(supplementary Table I). Collisionally induced decomposition
(CID) spectra, from m/z 100 to 700, were collected online using
parameters identical to precursor ion and MRM experiments.
CID spectra of hydrolyzed acyl components were also collected
online during RP-HPLC. These experiments were performed on
a Thermo Finnigan LTQ linear ion trap tandem mass spectrom-
eter (San Jose, CA) operating in negative ion mode using data-
dependent MS/MS. Eluent was introduced into the electrospray
ion source at 200 ␮l/min, where the voltage was set at 4.5 kV and
the capillary temperature was 150°C. The mass spectrometer al-
ternated between full mass scanning (m/z 150 to 350) and MS/
MS, using an isolation window of 1.8 amu and collision energy of
17 V with wideband activation.
Statistics and data analysis
Chromatographic peaks were integrated and area ratios were
generated using MultiQuant (ABSciex). Statistical analyses were
performed using GraphPad Prism 4 (GraphPad Software, Inc.).
RESULTS
NP-HPLC-MS/MS separation of cholesteryl esters of
human atheromata
Collisionally induced dissociation of ammoniated cho-
lesteryl esters, [M+NH4]+, yields a strong cholesteryl cation
at m/z 369, regardless of the acyl component (24). There-
fore scanning for precursor ions of the m/z 369 fragment
was a quasi-selective tandem mass spectrometry approach
for the detection of CE species. In these analyses, complex
mixtures of neutral lipids were extracted from homoge-
nates of human atheromata, separated by NP-HPLC, and
detected using this precursor ion method. As expected,
unoxidized cholesteryl esters showed little retention on
the silica column, with all species essentially coeluting just
after the solvent front (Fig. 1). CEs were exclusively ob-
served as ammoniated ions, the most abundant being 18:2-
CE, followed by cholesteryl arachidonate (20:4-CE), and
cholesteryl docosahexaenoate (22:6-CE). From 7 to 35
min, a series of increasingly polar oxCEs eluted. The m/z
values of these oxCEs were consistent with the addition of
one or two oxygen atoms to the most abundant CE species,
Quantitation of cholesteryl ester oxidation
Calibration curves were made for each of the three major CE
species and to commercially available oxCE species (9- and
13-HODE-CE as well as 9- and 13-HpODE-CE) using 7.8 pmol of
cholesteryl 9Z-heptadecenoate (17:1-CE) as a common internal
standard (supplementary Fig. I). Aliquots of atheroma extract
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Fig. 1. NP-HPLC-MS/MS analysis of cholesteryl esters extracted from a human femoral endarterectomy
atheroma sample. (A) Precursors of m/z 369.3 (m/z 620 to 780) total ion chromatogram. (B) Precursors of
m/z 369.3 extracted ion chromatograms of cholesteryl linoleate and major oxidized cholesteryl esters. Indi-
vidual extracted ion chromatograms are labeled with the m/z value extracted. All chromatograms are nor-
malized to the absolute signal for unoxidized cholesterol esters in panel A (set to 100% relative abundance),
except as noted. (C) Integrated mass spectral data, averaged over the elution time of unoxidized cholesteryl
ester species (2 to 4 min). CE species are labeled with m/z values and their acyl components by total acyl
carbons and double bonds. (D) Integrated mass spectral data, averaged over the elution time of oxidized
cholesteryl ester species (6 to 36 min). Oxidized CE species are labeled with m/z values.
18:2-CE and 20:4-CE (Fig. 1C, D). From the number of
chromatographic peaks, it was obvious that a complex ar-
ray of structurally diverse oxCEs was present. Isobaric, yet
chromatographically separable, species indicated that
there were likely several families of related isomers having
the same empirical formulae. Oxidized CEs were generally
observed as ammoniated ions; however, certain oxCEs ap-
peared to form both ammoniated and protonated ion spe-
cies (Fig. 1B, m/z 680, 663).
of four abundant peaks. Peaks 1a and 1c coeluted with
commercially available authentic standards of 13-(ZE)-
HODE-CE and 9-(EZ)-HODE-CE, respectively (data not
shown). Peaks 1b and 1d were likely the all-trans isomers
13-(EE)-HODE-CE and 9-(EE)-HODE-CE, respectively, as
previously determined (30). All group 1 peaks produced
identical product ion spectra that exactly matched both
HODE-CE standards. Isolated group 1 peaks were readily
trimethylsilylated with BSTFA, resulting in an increase in
mass (+72 amu) and a marked reduction in polarity (sup-
plementary Fig. III-A). Individually isolated group 1 peaks
were saponified, and the free acyl components were sub-
jected to RP-HPLC MS/MS. The retention times and CID
spectra of the carboxylate anions were consistent with the
authentic HODEs. Group 1 peaks were HODE-CE isomers
as indicated in Table 1.
The sections that follow describe the groups of peaks in
the extracted ion chromatograms (Fig. 1B).
Group 1 peaks
The most abundant ion, m/z 682, was consistent with the
addition of a single oxygen atom (+16 amu) to 18:2-CE
(Fig. 1B). The extracted ion chromatogram revealed a set
CE oxidation in human atheromata
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TABLE 1. Major oxidized cholesteryl esters of human atheromata
Group 2 peaks
m/z 698 revealed two sets of peaks. Group 3 consisted of
four peaks eluting from 19 to 22 min. Peaks 3a and 3b
coeluted with a authentic standard cholesteryl 13-(ZE)-
HpODE-CE and 9-(EZ)-HpODE-CE, respectively. On
the basis of previous separations of HpODE-CE mixtures,
peaks 3c and 3d were likely the corresponding all trans
isomers (30). Peaks 3a and 3b yielded positive ion CID
spectra that exactly matched that of the 13-HpODE-CE
standard, whereas the spectra of peaks 3c and 3d matched
the spectra of the 9-HpODE-CE standard. The CID of
ammoniated HpODE-CEs are rich with structural infor-
mation, and therefore, these spectra provided support
for their identification, as recently reported (31). These
oxCEs were readily trimethylsilylated upon treatment
with BSTFA (supplementary Fig. III-C). RP-HPLC-MS/
MS analyses of hydrolyzed acyl components (compared
with authentic HpODE standards) also supported the
identification of group 3 peaks as the HpODE-CE iso-
mers indicated in Table 1.
The early eluting peaks of m/z 682 were far less polar
than the isobaric HODE-CE family (group 1) (Fig. 1B).
The molecular weight and polarity of this oxCE family
were consistent with a series of epoxy-octadecenoate-CEs
(EpOME-CE). Group 2 peaks were collected individu-
ally, and the saponified acyl components were analyzed
by RP-HPLC-MS/MS. The retention times and CID spec-
tra of hydrolyzed peaks 2b and 2d matched authentic
12,13-(E)-EpOME and 9,10-(E)-EpOME, respectively.
Hydrolyzed peaks 2a and 2c produced the same spectra
as 2b and 2c, respectively, and were therefore hypothe-
sized to be the trans isomers. Group 2 peaks were likely a
series of EpOME-CEs as indicated in Table 1.
Group 3 peaks
The second most abundant ion, observed at m/z 698,
was consistent with addition a two oxygen atoms to 18:2-CE
(+32 amu) (Fig. 1B). The extracted ion chromatogram of
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Group 4 peaks
chromatogram of m/z 706 revealed a group of peaks with
similar retention times as the HODE-CEs. When group 7
peaks were isolated and treated with BSTFA, they readily
formed silylated products, m/z 778 (+72 amu) (supple-
mentary Fig. III-B). Group 7 peaks were hydrolyzed and
subjected to RP-HPLC-MS/MS. The retention times and
CID spectra of the acyl components were consistent with
authentic HETE isomers. Double-bond geometries were
not determined for this family. The HETE-CE identified
in atheromata are listed in Table 1.
Group 4 peaks of m/z 698 were consistent with the addi-
tion of two oxygen atoms to 18:2-CE (+32 amu) (Fig. 1B).
These compounds were significantly more polar than both
HpODE-CEs and HODE-CEs and consisted of a mixture of
isobaric compounds which all yielded identical product
spectra. When group 4 peaks were isolated and treated
with BSTFA, the products were exclusively monosilylated
(+72 amu), indicting these oxCEs contained only a single
hydroxyl (supplementary Fig. III-E). Group 4 peaks were
collected, hydrolyzed, and analyzed by RP-HPLC-MS/MS.
These experiments revealed a complex mixture of acyl
components whose product ion spectra exactly matched
previously published product ion spectra of epoxy-hydroxy
linoleate components (32). These results indicate that
group 4 peaks were hydroxy-epoxy-linoleate-CEs, as shown
in Table 1. Double bond geometry and stereochemistry
were not determined for this family.
Group 8 peaks
The ion at m/z 722 was consistent with the addition of
two oxygen atoms to 20:4-CE (Fig. 1B). The extracted
ion chromatogram of m/z 722 showed a group of peaks
with similar retention times as the HpODE-CE series.
Group 8 peaks were isolated and treated with BSTFA.
Like the silylation products of the HpODE-CE family, the
silylated products here were monosilylated and eluted
just after solvent front (supplementary Fig. III-D). Suffi-
cient quantities of material could not be isolated to allow
for RP-HPLC analysis of the saponified acyl compo-
nents. Group 8 peaks were likely a series of HpETE-CE
isomers similar to the HETE-CE family. Using highly sen-
sitive MRM detection provided evidence of a series of
corresponding oxoETE-CEs; however, these compounds
were at low abundance and could not be characterized
confidently.
Group 5 peaks
The ion at m/z 680 was consistent in mass and polarity
with an oxo containing 18:2-CE (Fig. 1B). The extracted
ion chromatogram revealed peaks at 13, 15, and 17 min.
In serial injection experiments, peaks 5a and 5b displayed
the similar retention times as synthetic cholesteryl 13-oxo-9Z,
11E-octadecadienoic acid (13-(ZE)-oxoODE) and 9-(EZ)-
oxoODE, respectively. All group 5 peaks formed both pro-
tonated, [M+H]+ (m/z 663), and ammoniated, [M+NH4]+
(m/z 680), molecular ion species as did the synthetic
oxoODE-CE standards. Group 5 peaks were isolated and
treated with methoxyamine and readily converted to
methoxime species, m/z 679 (+29 amu), confirming the
presence of an oxo moiety (supplementary Fig. IV-A).
Hydrolyzed acyl components from group 5 peaks were an-
alyzed by RP-HPLC-MS/MS. The results, when compared
with authentic standards, were consistent with the oxoODE
isomers. The oxoODE-CE species present in human ather-
omata are indicated in Table 1.
Group 9 peaks
Group 9 peaks were significantly more polar than both
the HETE-CE series and the HpETE-CE series and showed
similar retention times as the hydroxy-epoxy-linoleate-CEs
(Fig. 1B). Group 9 peaks were isolated from an atheroma
extract and treated with BSFTA. Like the silylation prod-
ucts of the hydroxy-epoxy-linoleate-CEs, the products were
exclusively monosilylated (+72 amu) (supplementary Fig.
III-F). Polarity was reduced but not eliminated, again sim-
ilar to the hydroxy-epoxy-linoleate-CEs. Sufficient material
was not available for RP-HPLC MS/MS of the acyl compo-
nents. On the basis of these results and the fact that
hydroxy-epoxy-arachidonates are known degradation
products of arachidonate peroxidation, group 9 was likely
a highly complex mixture of hydroxy-epoxy-arachidonate-
CE isomers (34).
Group 6 peaks
The ion at m/z 696 was consistent with a series of keto-
expoxy- or keto-hydroxy-linoleate-CEs (Fig. 1B). Meth-
oxyamine treatment resulted in conversion to the methoxime
species indicated by an increase in mass (+29 amu) and a
slight loss of polarity (supplementary Fig. IV-B). These results
confirmed the presence of an oxo moiety; however, BSTFA
treatment did not produce silylated species, suggesting the
second oxygen was not a hydroxyl. An epoxide moiety as
the second oxygen would be consistent with these observa-
tions. Attempts to hydrolyze these oxCEs and obtain negative
ion MS/MS spectra were unsuccessful. Indeed, a recent study
found such compounds are unstable during hydrolysis; and
therefore, analysis of the saponified acyl chain is not trivial
(33). The proposed keto-epoxy-linoleate-CEs observed in
human atheromata are listed in Table 1.
Chain-shortened ␻-oxidized cholesteryl esters
Scanning at lower mass (m/z 420 to 620) for precursors
of m/z 369.3 revealed a series of polar compounds eluting
during NP-HPLC-MS/MS analyses of atheromata extracts
(Fig. 2). The most abundant of these compounds, at m/z
558, was consistent with an ammoniated cholesteryl ester,
[M+NH4]+, with an acyl group having nine carbons, no
double bonds and a single oxo moiety. Treatment with
methoxylamine readily converted this compound to the
methoxime species (+29 amu), confirming the oxo moiety
(supplementary Fig. IV-C). Less abundant ions were con-
sistent with similar species having acyl components of
various chain lengths. Together these compounds were likely
Group 7 peaks
The ion at m/z 706 was consistent with the addition of
one oxygen atom to 20:4-CE (Fig. 1B). The extracted ion
CE oxidation in human atheromata
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Fig. 2. NP-HPLC-MS/MS analysis of cholesteryl esters of chain-shortened, ␻-oxidized acyl components
extracted from human atheroma. (A) Extracted ion chromatograms of the major species. Chromatographic
peaks are labeled with the m/z value extracted. (B) Integrated mass spectrum averaged over the period indi-
cated by the horizontal line. (C) General structure of chain shortened ␻-oxidized CEs. The table indicates
calculated nominal m/z values of ammoniated species, [M+NH4]+.
acid). The relatively high polarity of this family sug-
gested that the oxygen was likely present as a hydroxyl
although no further characterization was done. The
early-eluting compounds (Fig. 3B) were esterified to a
more saturated family of acyl components, with oleic
acid (18:1) and eicosatrienoic acid (20:3) being the
most abundant acyl species. A less polar oxygen moiety,
possibly an epoxy, was likely present on the cholesteryl
nucleus in this class of esters. Together, these species
had an absolute intensity <10% of unmodified 18:2-CE,
although no calibration curves were made for esters of
oxidized cholesterol.
a series of ␻-oxidized, chain-shortened fatty acyl cholesteryl
esters, also referred to as core aldehydes. Extracted ion
chromatograms of the major ions are shown in Fig. 2A.
Longer chain-length species eluted first, and shorter spe-
cies were eluted later; these results were consistent with
previously published reversed-phase separations which
showed the opposite elution profile (3, 35). There were
also several minor ion species with m/z values not consis-
tent with this series, e.g., m/z 554 and 568, although these
ions were typically of low abundance (Fig. 2B).
Esters of oxidized cholesterol
Scanning for precursors of m/z 385.3 (cholesteryl
cation+[O]) revealed two major groups of compounds.
The late-eluting group (Fig. 3C) had molecular weights
consistent with oxidized cholesterol esterified to the
typical acyl components esterified to unoxidized CE spe-
cies (i.e., linoleate, arachidonate, and docosahexaenoic
Quantitation of the specific 15-lipoxygenase product in
human atheromata
15-LO catalyzes the formation of (S)13-(ZE)-HODE-CE
from cholesteryl linoleate. The four HODE-CE isomers
were well separated by NP-HPLC, and their relative peaks
Fig. 3. NP-HPLC-MS/MS analysis of fatty acid esters of oxidized cholesterol. (A) Precursors of m/z 385.3
(m/z 620 to 780) total ion chromatogram. (B, C) Integrated mass spectral data averaged over the periods
indicated by the horizontal lines. CE species are labeled with m/z values and their acyl components by total
acyl carbons and double bonds.
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Fig. 4. Regio- and stereoselectivity of HODE-CEs
extracted from human atheromata. (A) NP-HPLC-
MS/MS separation of HODE-CE geometric isomers
from six human atheromata. (B) Chiral HPLC-MS/
MS separation of (R)- and (S)13-(ZE)-HODE-CE iso-
lated from the same human atheromata. HODE-CEs
were detected by multiple reaction monitoring (m/z
682.6 to 369.3). Roman numerals identify the indi-
vidual atheromata samples. The indicated abundance
ratios were calculated by peak area. Bottom trace
show typical chromatograms of authentic standards.
Abundance ratio averages SD are indicated for the
six samples and for intraday analyses of authentic
standards.
areas were determined by MRM (Fig. 4A). The average
peak area ratio of 13-(ZE)-HODE-CE / 9-(EZ)-HODE-CE
for six atheromata was 0.94 0.06 (mean SD). Equimolar
quantities of authentic standards produced a 13-/9- peak
area ratio of 1.02 0.07 (mean SD, n = 5 intraday mea-
surements). Chiral separations of 13-(ZE)-HODE-CE iso-
lated from each of the six atheromata each produced two
peaks that matched the retention times of authentic
(+/Ϫ)13-(ZE)-HODE-CE (Fig. 4B). Analysis of authentic
(R)13-(ZE)-HODE-CE revealed the individual enantio-
mers. The average S/R peak area ratio of the atheromata
was 0.99 0.05 (mean SD), whereas the S/R ratio of the
racemic standard was 1.00 0.05 (mean SD, n = 5 intra-
day measurements). These results indicated no significant
regio- or stereoselectivity in the HODE-CE profile of hu-
man atheromata.
showed very little oxidation; however, some of the more
abundant oxCE species, such as HODE-CEs and HpODE-
CEs, were detected (36).
IMS of human atherosclerotic plaques
To determine the localization of neutral lipid species
in plaques prior to extraction, 20 ␮m slices of human
atheromata where prepared and imaged by MALDI IMS.
These analyses revealed abundant ions with m/z values
consistent with sodium adducts of the major CEs and ox-
CEs within the plaques (Fig. 6K, L). MALDI-TOF prod-
uct ion spectra obtained directly from tissue slices or
from authentic standards confirmed the identity of ma-
jor CEs and oxCEs (Fig. 7). Interestingly, CID of the CE
sodium adducts yielded very different spectra than their
ammoniated counterparts. While ammoniated CEs
yielded an abundant m/z 369.3, sodiated CEs yielded lit-
tle m/z 369, instead showing strong ions corresponding
to sodiated acyl components, e.g., m/z 319 in the HODE-
CE spectra (Fig. 7C, G). The identities of major TAGs
and phospholipids were also confirmed by MALDI-TOF-
MS/MS (data not shown). All neutral lipid species were
exclusively observed as sodium adducts, whereas phos-
pholipids were observed as sodiated and protonated ion
species.
CEs and oxCEs were colocalized exclusively within
plaques, with 18:2-CE being the most abundant ion in
these regions (Fig. 6D, I, K, L). Phospholipids, mainly
palmitoyl-sphingomyelin and various glycerylphospho-
choline species, were also observed within the lesion, but
they were more intense in other areas, such as media, ad-
ventia, and extravascular adipose (Fig. 6A, F, D, I). Tria-
cylglycerides, as expected, were the most abundant ions
in extravascular adipose (data not shown), but they were
also present at lower levels in the lesion area. When spec-
tra obtained specifically from the plaque regions were in-
tegrated, TAGs and certain phospholipids were present;
however, CEs were consistently the most intense ions ob-
served (Fig. 6K, L).
Extent of cholesteryl ester oxidation
To more accurately determine the relative quantities of
the three major CE species (18:2-CE, 20:4-CE, and 22:6-
CE) and their oxidation products, human atheromata ex-
tracts were spiked with the internal standard 17:1-CE and
analyzed by NP-HPLC-MS/MS using MRM. After generat-
ing calibration curves, the relative abundances of the ma-
jor CEs and their oxidation products were determined for
each of the six human atheromata and pooled human
plasma. As expected, cholesteryl linoleate and its oxida-
tion products were by far the most abundant species,
followed by cholesteryl arachidonate and cholesteryl
docosahexaenoate and their oxidation products (Fig. 5).
Across the six samples, the 18:2-CE pool was an average of
23 13% oxidized, the 20:4-CE pool was 16 6% oxidized,
the 22:6-CE pool was 12 4% oxidized, and the total CE
pool was 21 11% oxidized (mean SD). Cholesteryl li-
noleate was the most oxidized CE species on a per-sample
basis (P = <0.001 compared with 20:4-CE; P < 0.005 com-
pared with 22:6-CE). The pooled plasma sample had a typ-
ical CE profile, in agreement with previous studies, and
CE oxidation in human atheromata
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Fig. 5. Extent of cholesteryl ester oxidation in human atheromata. Relative quantitation was assessed by
NP-HPLC-MS/MS as described in Experimental Procedures. Solid bars indicate the relative abundance of
the three major CE species; the summed oxidation products of each CE species are indicated by open bars.
Roman numerals indicate individual atheromata, the same samples shown in Fig. 4. The “i-vi” graph shows
the average SD of the six atheromata samples. Pooled plasma was derived from 22 healthy donors. Oxida-
tion levels in plasma were too low to appear clearly in this format; however, oxCE were detected at low levels
in pooled plasma.
produced different, but equally uninformative, product
ion spectra (Fig. 7). Therefore, negative ion MS/MS of the
hydrolyzed acyl anions were often critical for structural de-
termination. These experiments were particularly useful
because negative ion CID spectra of oxidized fatty acids
yield structurally relevant product ions, and these spectra
have been well studied (32, 37, 38). For example, the re-
gioisomers of the HETE-CE series could only be confi-
dently assigned after negative ion MS/MS of the acyl
components were obtained and compared with CID au-
thentic standards and previously published spectra. Nega-
tive ion MS/MS was also critical for the characterization of
the epoxy-hydroxy-linoleate-CE.
Derivatization approaches, such as trimethylsilylation,
were useful for determining the number and type of
functional groups present. For example, the hydroxy-
epoxy-linoleate-CEs were originally hypothesized to be
dihydroxy-linoleate-CEs; however, upon treatment with
the silylating reagent BSTFA, strictly monosilylated prod-
ucts were produced, indicating that only a single free hy-
droxylwaspresent(supplementaryFig.IV-E).Derivatization
with methoxyamine was critical for determining which
species contained an oxo moiety by observing their con-
version to the methoxime (supplementary Fig. IV). For
example, the keto-epoxy-linoleate-CEs were a complex
mixture of compounds, each present at very low abun-
dance. Therefore an approach which yielded information
regarding the entire family (i.e., that they all contained a
ketone) was an important step toward characterizing these
species.
DISCUSSION
Oxidized cholesterol esters in human peripheral vascular
lesions
Historically, oxCE analyses relied on HPLC-UV moni-
toring absorbance at 235 nm. This method is sensitive for
the detection of lipid species that contain a conjugated
diene (i.e., HODE-CEs); however, is not well suited for
other species (e.g., EpOME-CEs or unoxidized CEs (5,
22)). To detect a wider variety of species, these analyses
leveraged the predictable CID behavior of ammoniated
CEs. Regardless of the acyl chain, these species all produce
a strong ion at m/z 369 (cholesteryl cation) following CID.
Because of this common product ion, precursor ion scan-
ning (precursors of m/z 369) is selective for cholesteryl es-
ters and not significantly biased for any particular acyl
chain. This detection method revealed a complex array of
abundant oxCE species in extracts of vascular plaques
from human femoral and popliteal arteries. It was immedi-
ately apparent that the number of individual species and
their relative abundances were beyond that previously re-
ported. Although the amount CE oxidation varied be-
tween atheromata (Fig. 5), the array of species observed
(Table 1) was remarkably consistent. The oxCE pool was
composed of many families of isobaric compounds; in-
deed, every ion observed produced multiple peaks in the
chromatogram. The presence of tightly grouped peaks
suggested that some families were structurally related re-
gioisomers that likely contained the same oxygen moiety
[e.g., HODE-CEs (m/z 682)]. There were also indepen-
dent families, each containing several compounds, all of
which were isobaric [e.g., HODE-CEs and EpOME-CEs
(both m/z 682)].
Cholesteryl esters of ␻-oxidized, terminal-aldehyde fatty
acyl components, or core aldehydes, also produce the di-
agnostic m/z 369 ion upon CID. These species were de-
tected using essentially the same methods that were used
to detect longer chain fatty acyl groups. The m/z values
observed in these analyses were consistent with cholesteryl
With the notable exception of the HpODE-CE species,
positive ion CID spectra of ammoniated oxCEs were struc-
turally uninformative (31). CID of the sodium adducts
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Fig. 6. MALDI IMS analysis of CE and oxCE in human arteries with atherosclerotic lesions. Extracted pos-
itive ion MALDI images of sodiated (A, F) dipalmitoylphosphatidalcholine m/z 781.7, (B–G) HODE-CE m/z
687, and (C, H) 18:2-CE m/z 671. (D, I) Merged positive ion MALDI image of all three ions indicated above.
IMS ion intensities are indicated by color intensities, which were independently scaled for each panel. (E, J)
Adjacent slices stained with Oil Red O and Hema 3® blue as described in Experimental Procedures. (K, L)
Integrated MALDI-TOF spectra summed over the specific lesion areas within the arteries. The major ions are
labeled by total acyl carbons, total double bonds, and lipid class; all labeled species are sodium adducts,
[M+Na]+.
samples using sensitive MRM detection (data not shown).
Thus, a large number of very different oxidized fatty acyl-
cholesteryl esters are present in plaque that could be sub-
strates for esterases to release diverse oxylipins. Such free
fatty acids are known to express a host of activities, includ-
ing HODEs from linoleate oxidation causing pain (42),
anti-inflammatory epoxy-hydroxy products (i.e., hepoxil-
ins) (43, 44), EpOMEs (i.e., leukotoxins) from linoleate
(45, 46), products of 22:6 oxidation known as anti-inflam-
matory maresins (47), and perhaps most relevant, various
HETEs and 13-HODE as ligands for PPAR-␥ driving ex-
pression of CD36, a receptor for oxLDL (48–51).
esters of ␻-oxidized acyl components of various chain
lengths (Fig. 2B, C). Some of these species have been iden-
tified previously in human atheromata and have been
shown to form complexes with, and covalently bind to,
proteins (3, 39). Cholesteryl esters, oxidized in the choles-
terol nucleus, were also observed but in lower abundance
(Fig. 3).
Novel cholesteryl ester oxidation products
Various oxidized lipids, including many with the struc-
tural features found in these oxidized cholesteryl esters
isolated from human atherosclerotic plaques, are known
to have significant biological activities. Biological activity
has been found with intact cholesteryl esters as well as
oxidized fatty acids that would be derived following
ester hydrolysis. Experimentally oxidized 20:4-CE by 15-
lipoxygenase (immobilized soybean 15-LO) was found
to activate macrophages and cause release of proinflam-
matory cytokines (40). Cellular responses to oxCE may be
mediated through interactions with toll-like receptor-4
(41). This oxidized 20:4-CE was shown to contain many of
the products described in this study. Although present at
low abundance, singly and doubly oxidized products of
22:6-CE were also detected in the atherosclerotic plaque
Extent of cholesteryl ester oxidation in human
atheromata
Previous studies reported that up to 30% of the choles-
teryl linoleate in atheromata was present in oxidized form;
however, these studies were unable to detect many of the
oxCE species we observed by NP-HPLC-MS/MS (5). Initial
experiments suggested that a larger proportion of choles-
teryl linoleate was oxidized and that cholesteryl arachido-
nate and cholesteryl docosahexaenoate were also oxidized.
Because the precursor ion scanning experiment required
a relatively slow sampling rate, we used MRM for oxCE
CE oxidation in human atheromata
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Fig. 7. MALDI-TOF product ion spectra of CEs and oxCEs acquired from human atheromata and authentic standards. The species se-
lected for CID is indicated above the spectra; the source of the compound is indicted to the left. Upper panels: CID spectra acquired from
human atheromata tissue sections prepared as described in “Experimental Procedures.” Lower panels: CID spectra acquired from authen-
tic standards dried onto the MALDI plate, layered with NaCl and sublimed DHB matrix as described in Experimental Procedures An au-
thentic standard for oxoODE-CE was not available.
quantitation. After constructing calibration curves, it was
determined that up to 45% of the 18:2-CE pool in human
atheromata was present in oxidized form; however, most
samples showed closer to 25% 18:2-CE oxidation (23
13%, mean SD). Cholesteryl linoleate was the most oxi-
dized of the three major CE species; this observation was
consistent with cholesteryl linoleate being the preferred
substrate of 15-LO, although further analysis did not de-
tect a preponderance of the specific 15-LO oxidation
product (Fig. 4).
When the oxidized and unoxidized CEs were summed,
the relative amounts of the three most abundant CEs,
18:2-CE, 20:4-CE and 22:6-CE, were similar to the rela-
tive amounts detected in human plasma; however, the
level of CE oxidation in plasma was far less than in
atheromata. These results suggested either that oxCE
was selectively accumulated in the atheromata or that
CE from the blood was oxidized within the lesion. It has
been shown that HDL is the major carrier of oxCE in
the blood, not LDL, which is the particle associated with
lesion formation (52). Considering this, it is likely that
CE from the circulation is accumulated and oxidatively
modified within the lesion to some degree, perhaps
extensively.
13-HODE in linoleic acid-fed rabbits and in human ather-
omata reported a convincing preponderance of (S)13-
HODE in linoleate-fed rabbits at early time points (which
was reduced over time), but it found no stereospecificity
in human samples (20). Several investigations of the re-
gioisomers of HODE-CE in human atheromata and in hu-
man plasma found no excess oxygenation at carbon 13
and did not implicate 15-LO (5, 22, 53). No chiral separa-
tions of intact oxCE have been reported previously; there-
fore, direct analysis of oxCEs from human samples has not
been done. Here, normal-phase and chiral-phase HPLC-
MS/MS analysis of six human atheromata determined the
regio- and stereospecificity of the HODE-CEs as intact
species.
When the four regioisomers of HODE-CE were sepa-
rated by NP-HPLC-MS/MS, the peak areas of 13-(ZE)-
HODE-CE and 9-(ZE)-HODE-CE was essentially equal
(Fig. 4A). No sample had a 13-/9- ratio above the 95%
confidence interval (CI) of the authentic standards (95%
CI = 0.93 to 1.11). The 13-(ZE)-HODE-CE regioisomer was
then isolated by preparative NP-HPLC-MS/MS and sepa-
rated by chiral chromatography. The (S)- and (R)13-(ZE)-
HODE-CE peak areas were once again essentially equal
with only a single sample (Fig. 4B-ii) having an S/R ratio
above the 95% CI of the racemic standard (95% CI = 0.95
to 1.05). Over time, nonspecific products observed during
in vitro 15-LO-catalyzed CE oxidation are known to in-
crease relative to (S)13-(ZE)-HODE-CE, and this process
erodes the specificity of the product profile (17). In the
context of these observations, our results may not neces-
sarily rule out 15-LO as an initiating event leading to oxCE
in human atheromata, especially considering that human
vascular disease develops over many decades. It is likely
that nonenzymatic mechanisms (likely radical driven) pre-
dominate over time.
Relative quantitation of 15-LO-specific CE oxidation
product
Several studies have investigated the role of 15-LO in CE
oxidation by interrogating atheromata for the specific en-
zymatic oxidation product (S)13-(ZE)-HpODE-CE or its
reduction product (S)13-(ZE)-HODE-CE. These efforts
have yielded conflicting results. A large study of 80 human
atheromata reported a slight preponderance of (S)13-
HODE-CE after hydrolysis and methylation (S/R ratio of
1.12) (21). A study interrogating the stereospecificity of
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Localization of oxidized cholesteryl esters in human
In summary, cholesteryl ester oxidation in human ather-
omata resulted in an array of oxCEs more abundant and
diverse than previously appreciated. This family of lipids
included oxidation products of the major CE species pres-
ent in human plasma, and several novel oxCE species were
identified. Quantitative measurements determined that,
on average, 21 11% (mean SD) of the CE pool was
present in oxidized form, with one sample showing over
40% oxidation. These analyses also revealed that 18:2-CE
was oxidized to a greater extent than other CE species.
IMS revealed that oxCEs were localized entirely within the
lesion area and had a nearly identical spatial distribution
as unmodified CEs. Triacylglycerides were also present
within the lesion; however, polyunsaturated TAG species
showed no evidence of oxidative modification. Selective
oxidation of 18:2-CE and an absence of TAG oxidation
were observations consistent with 15-LO activity; however,
further analyses of HODE-CEs indicated no preponder-
ance of the specific 15-LO oxidation product. This result
suggested that either 15-LO is not a major source of oxCE
in human atheromata or the generation nonspecific prod-
ucts overwhelmed the oxCE profile. This situation could
occur through “seeding,” in which peroxide products of
15-LO initiate further oxidation, or by intermolecular
isomerization and racemization of direct 15-LO oxidation
products; both mechanisms would invoke nonspecific rad-
ical chemistry.
atheromata
Homogenization and extraction of whole atheromata,
the most common technique for lipid analysis, destroys in-
formation regarding the in situ distribution of plaque
components. An advantage of IMS is its ability to provide
spatial information about molecules that cannot be visual-
ized by other techniques. This is particularly true for lip-
ids, where IMS has revealed the distributions of various
lipids in different tissues (54, 55). Surprisingly, IMS studies
of atheromata are exceedingly scarce, and none has re-
ported the detection of oxCEs (56, 57). Thus, it remained
unclear whether oxCEs were concentrated in specific ar-
eas of atheromata, such as the necrotic core, or the shoul-
der regions where there is more cellularity.
To determine the localization of oxCE species in
human atheromata, we analyzed atherosclerotic human
arteries by MALDI-IMS. These studies revealed the
distribution of CEs, oxCEs, and many other lipid species.
The lipids observed and their relative abundance was
highly dependent on the anatomical region examined. In
areas composed mainly of calcified material, few ions were
detected and no lipids were observed. Triacylglycerides
were mainly observed in the extravascular adipose but also
within the plaque, although at lower abundances. Phos-
pholipids (e.g., dipalmitoyl-phosphocholine) were ob-
served throughout the arteries and were therefore useful
as anatomical landmarks when interpreting IMS. Choles-
teryl esters and oxCEs were completely localized within
the plaque (Fig. 6B, C, G, H, K, L). Sodium adducts of
these species, [M+Na]+, were by far the most abundant
ions within the lesions, but they were not observed at all in
other anatomical regions. Extracted ion images of 18:2-CE
and HODE-CE indicated these species had essentially
identical spatial distributions exclusively within the plaque
(Fig. 6D, I). These results suggested that CE oxidation oc-
curred throughout the lesion because oxCEs were not
concentrated in any particular region of the plaques.
It was also notable that TAGs were observed in the
plaque as rather abundant ions (Fig. 6K, L). This was a
curious observation as no oxidized TAGs were detected in
atheromata extracts by MALDI-IMS or by HPLC-MS/MS
(supplementary Fig. V). This was the case even for athero-
mata that showed over 40% oxidation of the CE pool. In
control experiments, oxTAG species, formed by treating
plaque extracts with ozone, were readily observed by
HPLC-MS/MS, confirming that oxTAGs, when present,
are detectable by these methods (supplementary Fig. VI).
These results indicate that TAGs and CEs, while both pres-
ent in the lesion, were oxidized differently and suggest
that CE oxidation is somewhat specific. If oxidative pro-
cesses in the atheromata were purely nonselective, one
would reasonably expect to find evidence of polyunsatu-
rated TAG oxidation in plaque samples where oxCEs were
clearly evident. Mechanisms of selective oxCE accumula-
tion include enzymatic CE oxidation (e.g., 15-LO), selec-
tive uptake of oxCE, incomplete remodeling of oxCE
(compared with oxTAG), and physical separation of CE
and TAG at a scale unresolved by IMS.
The authors thank Dr. David W. Russel for providing the pooled
human plasma sample. The authors also thank Drs. Max
Wohlauer and Jeffery Harr for assisting in the collection of
human atheromata samples.
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