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INTRODUCTION |
Atherosclerosis is a devastating disease responsible for profound
human morbidity and mortality (1, 2). The precursor of the
atherosclerotic lesion, the fatty streak, begins to develop in the
first decade of life and is characterized by the accumulation of
monocyte/macrophages within the intimal layer of the blood vessel (3,
4). There is evidence that oxidized lipids, primarily derived from low
density lipoproteins (LDL),1
contribute to all stages of atherosclerotic development (5-8). Initially, they facilitate monocyte deposition within the
subendothelial space by stimulating endothelial cells to express
monocyte-specific adhesion molecules (9, 10) and secrete monocyte
chemoattractants (11, 12). Later, highly oxidized lipids such as
malondialdehyde and 4-hydroxynonenal modify the protein component of
LDL so that it is recognized by the macrophage scavenger/oxidized LDL
receptor rather than the native LDL receptor (13-15). Uptake of
oxidized LDL by macrophages generates foam cells that reside in the
subendothelial space. It is the lipid-laden foam cells that are the
hallmark of the fatty streak lesion.
We have previously demonstrated that mildly oxidized LDL, which we have
termed "minimally modified" (MM-LDL), stimulated human aortic
endothelial cells to bind human monocytes in vitro (9). By
separating the components of MM-LDL it was found that the phospholipid fraction contained nearly all of the biological activity (9). When the
phospholipids from MM-LDL and native LDL were compared it was found
that phospholipids containing arachidonic acid were preferentially
oxidized compared to phospholipids containing other polyunsaturated
fatty acids (16). This lead us to suspect that the biologically active
phospholipids were oxidized derivatives of arachidonic acid-containing
phospholipids. We then found that autoxidized
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) had identical biological properties as MM-LDL, and we began
using Ox-PAPC as a surrogate for MM-LDL (17).
Recently, our group has described three compounds present in MM-LDL,
Ox-PAPC, and rabbit atherosclerotic lesions that stimulated endothelial
cells to bind monocytes in vitro (18). All were derived from
the oxidation of arachidonic acid-containing phospholipids in LDL (16).
Interestingly, we found that antibodies to these lipids were
spontaneously produced in vivo by apolipoprotein E knockout
mice that were genetically predisposed to develop atherosclerosis (18).
Two of the biologically active compounds were produced by oxidative
fragmentation of the arachidonic acid moiety in the sn-2
position of PAPC and were identified as
1-palmitoyl-2-(5)oxovaleryl-sn-glycero-3-phosphocholine (POVPC) and
1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC).
The molecular structure of the third molecule, which gave a signal at
m/z 828.5 by electrospray ionization-mass
spectrometry (ESI-MS), was not determined at that time. In this study
we provide evidence that this molecule contained an epoxyisoprostane in
the sn-2 position of the phospholipid (Scheme
1A) and this molecule undergoes dehydration to form a structurally similar molecule with a
mass of 810.5 [M + H+] (Scheme 1B).
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EXPERIMENTAL PROCEDURES |
Materials--
Tissue culture media, serum, and supplements were
obtained from Irvine Scientific and Hyclone Laboratories, Inc.
Acetonitrile, chloroform, methanol, ethyl acetate, and water (all
Optima grade) were obtained from Fisher Scientific, Pittsburgh, PA.
Gelatin (endotoxin-free, tissue culture grade), porcine liver esterase, calcium chloride, methoxylamine hydrochloride, ammonium acetate, sodium
borohydride, platinum(IV) oxide, phospholipase A2
(naja naja), and butylated hydroxytoluene (BHT) were
obtained from Sigma. Authentic
L-
-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL) or
Sigma. Deuterated chloroform (99.99% D), deuterated methanol (99+%
D), and deuterium oxide (99.9% D) were obtained from Aldrich Chemical
Co. Sodium borodeuteride was obtained from Cambridge Isotope
Laboratories, Inc.
Endothelial Cell Cultures--
Human aortic endothelial cells at
passages 4-7 were cultured as described in medium 199 supplemented
with 10% fetal bovine serum (9, 12).
Monocyte Adhesion Assay--
These studies were performed
essentially as described previously (9). Blood monocytes were isolated
from a large pool of healthy human blood donors by a modification of
the Recalde procedure (19). For monocyte adhesion assays, human aortic
endothelial cells were incubated with test medium for 4 h at
37 °C. The test medium was removed, the endothelial cells were
washed, and a suspension of human monocytes was added for 12-15 min
after which nonadherent monocytes were removed. Bound monocytes were
counted and expressed as monocytes/microscopic field.
Lipid Oxidation--
PAPC was oxidized by transferring 1 mg in
100 µl of chloroform to a clean 16 × 125-mm glass test tube and
evaporating the solvent under a stream of nitrogen. The lipid residue
was allowed to autoxidize while exposed to air for 24-72 h at room
temperature. The extent of oxidation was monitored by flow
injection ESI-MS.
High Performance Liquid Chromatography--
Normal phase high
performance liquid chromatography (NP-HPLC) was performed by injecting
oxidized phospholipid preparations (resuspended in chloroform) onto a
silica column (Adsorbosphere, 250 × 22-mm, 5 µm; Alltech
Associates, Inc.) and eluting isocratically with a mobile solvent of
acetonitrile/methanol/water (77:8:15, v/v/v, pH 5.0 with formic acid)
at a flow rate of 18.0 ml/min. Typically, Ox-PAPC produced from 25-35
mg of PAPC was applied for each run. Reverse phase HPLC (RP-HPLC) of
oxidized phospholipids was performed with a C8 column
(Betasil, C8, 250 × 10-mm, 5 µm, Keystone
Scientific, Inc.). Phospholipids were eluted with a mobile phase of
80% methanol that was changed linearly over a period of 60 min to
100% methanol at 5 ml/min. Fractions containing oxidized phospholipids
of interest were collected by monitoring ultraviolet absorbance and
ESI-MS (LC/MS). Oxidized free fatty acids were separated by RP-HPLC
using a C18 column (Betasil, C18, 250 × 10-mm, 5 µm, Keystone Scientific, Inc.). A mobile phase of 60%
methanol containing 1 mM ammonium acetate changed linearly
over 60 min to 100% methanol containing 1 mM ammonium
acetate was used. When isolating lipids for NMR analysis, solvents
without ammonium acetate were used. UV absorbance was detected with a
diode array detector (L-3000, Hitachi, Ltd., Tokyo, Japan) scanning
from 200 to 350 nm at 2.5 nm resolution.
Phospholipase A2 Hydrolysis--
Phospholipid
fractions collected by HPLC were dried under argon to a lipid residue
and resuspended in 1 ml of phosphate-buffered saline containing 5 mM CaCl2. To this solution was added 5 units of
phospholipase A2. The solution was mixed and incubated at
37 °C for 45 min. After incubation, the lipids were extracted with 1 ml of ethyl acetate containing 0.01% BHT after acidification with
formic acid to pH 3.0.
Methoxylamine Derivatization--
Phospholipid fraction
containing 828.5 (i2) was isolated by sequential NP-HPLC and RP-HPLC
from 5 mg of Ox-PAPC. The fraction was dried under argon, and 1 ml of
0.92 mM methoxylamine hydrochloride in 1×
phosphate-buffered saline was added. The solution was mixed thoroughly
and incubated for 45 min at 37 °C. After incubation the lipids were
extracted with CHCl3/MeOH + BHT and analyzed by positive
ion ESI-MS.
Lipid Reduction--
Chemical reduction of lipids was achieved
by addition of 600 µl of a 70 mM solution of sodium
borohydride or sodium borodeuteride in acetonitrile at room temperature
for 30 min. Following incubation, 1 ml of ethyl acetate containing
0.01% BHT and 1 ml of water was added. The solution was mixed
thoroughly and centrifuged at 2,000 × g for 5 min. The
ethyl acetate phase was transferred to a clean glass tube, and 30 µl
of formic acid was added to displace sodium from phospholipid sodium salts.
Lipid Hydrogenation--
Lipids were hydrogenated by exposure to
hydrogen gas in the presence of platinum(IV) oxide (20). Oxidized
lipids were resuspended in 300 µl of ethyl acetate and transferred to
a 25-ml round bottom flask. Platinum(IV) oxide (1 mg) was added and the
flask was covered with a rubber septum. Two 18-gauge hypodermic needles
were placed through the septum, and the flask was flushed by
introduction of hydrogen through one of the needles. After flushing,
one of the needles was removed and a balloon containing hydrogen gas was attached to the other. The samples were incubated with constant stirring at room temperature for 45 min. The reaction mixture was
transferred to a 13 × 100-mm glass test tube and dried under argon gas. The lipid residue was resuspended in 1 ml of
chloroform/methanol (2:1, v/v), 400 µl of water, and 20 µl of
concentrated formic acid and then the lipids were recovered from the
chloroform phase after mixing and centrifugation.
Carboxylic Acid 18O
Labeling--
18Oxygen exchange experiments were performed
by incubation of the free fatty acids with porcine liver esterase in
H218O (21). To the oxidized fatty acid residue
was added 100 µl of H218O and 23 units of
porcine liver esterase. The contents were mixed thoroughly and
incubated for 60 min at 37 °C with occasional mixing. Lipids were
extracted by addition of 300 µl of chloroform/methanol (2:1, v/v) to
the reaction mixture.
Mass Spectrometry--
ESI-MS was performed using an API
III triple-quadrupole biomolecular mass analyzer
(Perkin-Elmer Sciex Instruments, Norwalk, CT) fitted with an
articulated, pneumatically assisted nebulization probe and an
atmospheric pressure ionization source. Details of calibration and
tuning have been described previously (18). Phospholipids were
introduced into the mass spectrometer by direct flow injection analysis
(FIA) in acetonitrile/water/formic acid (50:50:0.1, v/v/v) or via
liquid chromatography (LC/MS) and analyzed as the protonated molecule
[M + H+] in positive ion mode. The mass spectrometer was
set to scan from m/z 450 to 950 with an orifice
voltage of +65, a step size of 0.3, a dwell time of 3 msec, and a scan
speed of ~4 s. Fatty acids were analyzed as carboxylate anions
[M
] by FIA in methanol/water (50:50, v/v) with 1 mM ammonium acetate or by LC/MS in chromatography solvent.
For negative ion ESI-MS/MS, a solvent of 100% methanol with 1 mM ammonium acetate was used, and daughter ion spectra were
obtained by colliding the Q1 selected ion of interest with argon in Q2,
and scanning Q3 to analyze the fragment ion products. Reconstructed
selected ion chromatograms were produced by software supplied by PE Sciex.
High Resolution-Fast Atom Bombardment/Mass
Spectrometry--
High resolution-fast atom bombardment/MS spectra
were obtained using a VG ZAB-SE fast atom bombardment mass spectrometer
(Micromass, Manchester, UK) equipped with a 11/250 data system. HPLC
fractions containing oxidized phospholipids of interest were dried
under argon and resuspended in an aqueous solution of 0.1%
trifluoroacetic acid. To the static fast atom bombardment probe
containing 1-2 µl of liquid matrix
(m-nitrobenzylalcohol/thioglycerol/trifluoroacetic acid,
50:50:0.5, v/v/v) was added 1-2 µl of the oxidized phospholipid solution. Spectra were recorded using a 8 kV accelerating potential, cesium bombardment at 22 kV and 1-2 µA, and a mass resolution of
3,000 (10% valley, M/
M). The mass spectrometer was set to scan from
m/z 200-1,000, and ~10 scans were collected
into a multichannel analyzer. The data were smoothed, centroided, and
mass measured using cesium iodide ion clusters for calibration.
Nuclear Magnetic Resonance Spectroscopy--
All proton nuclear
magnetic resonance (1H NMR) spectra were recorded on a
Bruker ARX-500 MHz spectrometer using a microprobe (2.5 mm) NMR tube.
Proton chemical shifts were reported in parts per million (ppm) on the
scale with reference to CHCl3 ( 7.24). 1H NMR spectral data were tabulated in terms of
multiplicity of proton absorption (s, singlet; d, doublet; dd, doublet
of a doublet; dt, doublet of a triplet; t, triplet; q, quartet; m,
multiplet; br, broad), coupling constants (Hz), and number of protons.
Purified lipids were dried under argon, resuspended in
CDCl3 (90 µl) and transferred to a microprobe NMR tube
for analysis. Proton-proton homodecoupling experiments were performed
using a power level of 55 dB.
UV-visible Spectrophotometry--
Absorbance spectra in the
190-500 nm range for various oxidized phospholipids were measured
using a Shimadzu Biospec-1601 UV-visible spectrophotometer (Shimadzu
Scientific Instruments, Inc, Columbia, MD). For extinction coefficient
determination, the isomers of 828.5 and 810.5 were isolated by
sequential normal phase and reverse phase HPLC and quantified by
FIA-ESI-MS using dimyristoyl phosphatidylcholine as an internal
standard (22). The UV-visible absorbance was scanned for
m/z 828.5 and 810.5 isomers in methanol (1 ml) at
three different concentrations. Using these absorbance values, molar
extinction coefficients were calculated (23) for isomers of
m/z 828.5 and 810.5.
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RESULTS |
Effect of Isomers of m/z 828.5 and 810.5 on Monocyte-Endothelial
Interactions--
We have previously separated the phospholipid
components of Ox-PAPC by NP-HPLC and found that the second peak
enriched in m/z 828.5 induced endothelial cells
to bind monocytes in vitro (18). We repeated these
experiments and collected the active fraction between 16.5 and 18.0 min
(Fig. 1A) that contained
mostly m/z 828.5 and 810.5 (mass spectrum not
shown). The lipids in this fraction were then applied to a reverse
phase column, which effectively separated several isomers of
m/z 828.5 and 810.5 (Fig. 1B). Each major peak was collected, dried under argon, resuspended in tissue culture medium, and tested for the ability to induce endothelial cells
to bind monocytes. The only peak that showed significant biological
activity above control was the second of the peaks containing
m/z 828.5 (Fig. 1C). This isomer
caused a dose-dependent increase in monocyte binding
reaching a statistically significant increase over control as low as
380 ng/ml. The fatty acid hydrolyzed from the sn-2 position
of the biologically active isomer did not induce monocyte-endothelial
interactions (data not shown). As a convention throughout this article,
the five major isomers of m/z 828.5 resolved by
RP-LC/MS will henceforth be abbreviated 828.5 (i1-5) and the three
major isomers of m/z 810.5 will be abbreviated
810.5 (i1-3).
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Fig. 1.
Electrospray ionization-liquid
chromatography/mass spectrometry (ESI-LC/MS) of oxidized
1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine
(Ox-PAPC). Ox-PAPC produced by autoxidation of 25 mg of PAPC
was applied to a preparative NP HPLC column and eluted isocratically
with acetonitrile/methanol/water (77:8:15, v/v/v) at 18 ml/min
(A). A fraction (~1/50th) of the eluant was diverted to an
electrospray mass spectrometer, and the balance was recovered in a
fraction collector. The fraction shaded in panel A was
collected, evaporated to dryness, resuspended in methanol, and analyzed
by reverse phase ESI-LC/MS (B). Lipids were eluted with a
linear gradient of 80% methanol to 100% methanol over 60 min.
Reconstructed selected ion chromatograms of m/z
828.5 (solid line) and m/z 810.5 (dashed line) are shown during NP-LC/MS (A) and
RP-LC/MS (B). Numbered peaks in panel B
were collected and analyzed for the ability to induce endothelial cells
to bind monocytes (C). LPS (1 ng/ml) was used as a
positive control.
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UV-visible Spectrophotometry--
All isomers with
m/z 828.5 possessed nearly identical UV maxima at
252 nm, and all isomers of the m/z 810.5 possessed identical UV maxima at 257 nm (Fig.
2). These UV maxima were consistent with
a specific conjugated system within all of these molecules. Extinction
coefficients (
) at room temperature in methanol for the various
isomers were calculated to be: 828.5 (i1) = 24,070; 828.5 (i2) = 18,632; 828.5 (i3) = 17,975; 828.5 (i4) = 22,275; 828.5 (i5) = 15,917; 810.5 (i1) = 20,503; 810.5 (i2) = 19,572; and 810.5 (i3) = 14,950.
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Fig. 2.
Ultraviolet spectroscopy of
m/z 828.5 and 810.5. Ultraviolet
spectra were obtained by scanning between 200 and 350 nm during
RP-LC/MS analysis of the NP-LC/MS fraction containing
m/z 828.5 and 810.5. These measurements were made
in approximately 90% methanol and 10% water.
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Fast Atom Bombardment-High Resolution-Mass Spectrometry--
To
confirm the molecular formula of m/z 828.5 we
analyzed the molecule by high resolution-fast atom bombardment/MS. The
experimental mass of the ion was determined to be 828.5391, which
closely matched the mass of a molecule with the elemental composition
of C44H79NO11P (calculated
mass = 828.5381). Because unoxidized PAPC has an elemental composition of C44H81NO8P, we
concluded that during oxidation this molecule acquired three oxygen
atoms and lost two hydrogen atoms. Based on the mass of the products
observed after phospholipase A2 hydrolysis of this
molecule, we further concluded that the only oxidized part of the PAPC
molecule was the arachidonic acid in the sn-2 position of
PAPC. Thus, the molecular formula of this oxidized fatty acid in the
sn-2 position was
C20H30O5, compared with the
elemental formula of arachidonic acid,
C20H32O2.
Dehydration of m/z 828.5 to 810.5--
Periodically, positive ion
ESI-MS was performed on stored preparations of Ox-PAPC and it was
noticed that the relative ratio of 828.5 to 810.5 decreased over time,
suggesting that 810.5 may be a decomposition product of 828.5. To test
this, we isolated isomers of 828.5 by RP-LC/MS and allowed each of them
to undergo spontaneous dehydration. After 48 h at 4 °C in
chloroform, we reanalyzed the sample by RP-LC/MS using the same
chromatographic conditions that were used for original isolation. Two
isomers of 828.5 were collected, the biologically active isomer, 828.5 (i2), and a biologically inactive isomer, 828.5 (i5), (Fig.
3A). After 48 h at
4 °C, 828.5 (i2) had partially decomposed to a molecule that
co-migrated with 810.5 (i2) eluting at 31.5 min (Fig. 3B). In contrast, the tube containing 828.5 (i5) contained a mixture of two
isomers of 810.5 that co-eluted at 30.0 min and 32.5 min with 810.5 (i1) and 810.5 (i3), respectively (Fig. 3C). This experiment showed that 828.5 (i2) underwent dehydration to form 810.5 (i2) and
that 828.5 (i5) underwent dehydration to form 810.5 (i1) and 810.5 (i3). In addition, this suggested that the molecular structure of 810.5 (i2) was similar to 828.5 (i2). Derivatization with
bis(trimethylsilyl)trifluoroacetamide demonstrated the presence of one
hydroxyl group in m/z 828.5, which was lost in
m/z 810.5 (data not shown).
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Fig. 3.
Specific dehydration of
m/z 828.5 to 810.5. Isomers of
m/z 828.5 (solid lines) and 810.5 (dotted lines) were separated by RP-LC/MS (A).
The peak containing 828.5 (i2) was collected, allowed to undergo
spontaneous dehydration, and analyzed by identical RP-LC/MS conditions
as described above (B). In a similar experiment, the peak
containing 828.5 (i5) was collected, allowed to undergo spontaneous
dehydration, and analyzed by RP-LC/MS (C).
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Methoxylamine Hydrochloride Derivatization--
As a measure of
the number of carbonyl or epoxide groups we examined the derivatization
of the molecule with methoxylamine hydrochloride, which adds 47 mass
units to carbonyl group and reactive epoxide groups. Treatment of 828.5 (i2) with methoxylamine hydrochloride yielded several compounds (Fig.
4). An ion at m/z 857.4 was produced by the addition of a methoxylamine group and subsequent loss of water ([M + H+] + 47 
18). An
ion at m/z 875.7 was produced by the addition of
a methoxylamine without the loss of water ([M + H+] + 47). An ion at m/z 886.5 was produced by the
addition of two methoxylamine groups with subsequent loss of two waters
{[M + H+] + 2(47) 2(18)}. An ion at
m/z 904.5 was produced by the addition of two
methoxylamines with the loss of one water. When the reaction with
methoxylamine hydrochloride was allowed to proceed, the most abundant
ion was m/z 904.5 (data not shown). These data
indicate the presence of two groups on m/z 828.5, which react with methoxylamine hydrochloride.
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Fig. 4.
Methoxylamine derivatization of 828.5 (i2). By sequential NP-HPLC and RP-HPLC, 828.5 (i2) was isolated
from 5 mg of Ox-PAPC. 1 ml of 0.92 mM methoxylamine
hydrochloride in 1× phosphate-buffered saline was added to the dry
lipid residue. The solution was mixed thoroughly and incubated for 45 min at 37 °C. After incubation the derivatized lipids were extracted
with CHCl3/MeOH + BHT and analyzed by positive ion
ESI-MS.
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Sodium Borohydride and Sodium Borodeuteride
Reduction--
Reduction of the molecules (m/z
810.5 and 828.5) by sodium borohydride and sodium borodeuteride was
used to confirm the number of reducible oxygen groups. Sodium
borohydride can effectively reduce hydroperoxides, ketones, aldehydes,
and some epoxides to hydroxyl groups, thereby altering the molecular
weight of the molecule in a predictable manner. Individual isomers of
828.5 and 810.5 were isolated by RP-LC/MS and then treated with sodium borohydride, re-extracted, and analyzed by positive ion FIA-ESI-MS. Each reactive group adds two hydrogens. After reduction with sodium borohydride, the molecular weight of each isomer of 828.5 was increased
by 4 Da to m/z 832.5. This suggested that all
isomers of 828.5 possessed two reducible oxygen-containing functional groups such as aldehydes, ketones, and/or reactive epoxides. Fig. 5 shows the positive ion ESI-MS of the
purified biologically active isomer, 828.5 (i2), before (Fig.
5A) and after (Fig. 5B) sodium borohydride
reduction. Some 1-palmitoyl-lysophosphatidylcholine (m/z 496.2) and its corresponding sodium salt
(m/z 518.1) were produced by partial
saponification of the phospholipid during the reduction procedure. The
reduction was likely incomplete because of the presence of a signal at
m/z 830.4.
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Fig. 5.
Electrospray ionization-mass spectrometry of
m/z 828.5 (i2) reduced with sodium
borohydride. Purified 828.5 (i2) obtained by sequential NP-LC/MS
and RP-LC/MS was distributed evenly between two tubes and treated with
no additions (A) or sodium borohydride (B) in
acetonitrile for 30 min at room temperature. Lipids were extracted with
ethyl acetate/water with formic acid and analyzed by positive ion
ESI-MS.
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Treatment with sodium borodeuteride, in addition to reducing an
aldehyde, ketone, or reactive epoxide, will simultaneously add two
deuterium atoms to the group, one associated with oxygen. Because
ESI-MS was performed in a solvent containing H2O, the deuterium bound to the oxygen will undergo rapid exchange with protons
in the solvent. Indeed, when 828.5 (i2) was treated with sodium
borodeuteride the major product was an ion at m/z
834.5 rather than m/z 832.5 as seen when the
molecule was treated with sodium borohydride (data not shown). These
deuterium additions are consistent with the presence of two reducible
oxygen functionalities. When 810.5 (i2) was treated with sodium
borohydride, the mass was increased by six mass units to 816.6 (data
not shown), and with sodium borodeuteride, the mass was increased by
nine mass units to 819.6 (Fig.
6A). Because this molecule
only possessed two reducible oxygen groups, a reactive double bond of
the enone was also reduced. It is known that treatment of reactive
enones with sodium borohydride results in the reduction of both
carbonyl groups as well as double bonds (24). Furthermore, we
hypothesize that the reduced double bond of 819.6 contained one
deuterium and one hydrogen. We then analyzed m/z
819.6 by positive ion FIA-ESI-MS/MS (Fig. 6B). The spectrum
showed the expected sequential losses of water from the parent
(m/z 819.6 to 801.5 to 783.4) in addition to two
daughter fragments at m/z 594.2 and 609.3, which
correspond to a 5-carbon aldehyde and a 6-carbon deuterium-labeled
aldehyde at the sn-2 position, respectively (Fig.
6B, inset). The production of these two ions can
best be explained by the reduction of a molecule with an epoxide at the
5,6 position and the alternate association of the hydroxyl group with
the 5 or 6 position.
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Fig. 6.
ESI-MS and MS/MS of sodium
borodeuteride-treated 810.5 (i2). Purified 810.5 (i2) was treated
with sodium borodeuteride as described in Fig. 4 and analyzed by
positive ion flow injection ESI-MS (A). The
m/z 819.6 ion was then analyzed by positive ion
ESI-MS/MS (B).
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Hydrogenation--
Hydrogenation with platinum(IV) oxide and
hydrogen gas was used to determine whether the oxidized fatty acid in
the sn-2 position was linear or cyclic. Based on high
resolution-fast atom bombardment/MS data, the molecular formula of the
fatty acid in the sn-2 position of 810.5 (i2) was predicted
to be C20H28O4 containing four
oxygen atoms and six double bond equivalents. In addition, sodium
borohydride data had shown that the oxygen-containing groups were
reducible. Therefore, if the oxidized fatty acid was linear we would
expect hydrogenation of the four double bonds (addition of eight
protons) plus reduction of the two oxygen-containing groups (addition
of four protons), which would be reflected by an increase of 12 mass units from m/z 331.2 to 343.2. If the oxidized
fatty acid contained a cycle, we would expect that this cycle would not
be hydrogenated, and the fatty acid would maintain one double bond
equivalent and, therefore, give a signal at m/z
341.2 rather than m/z 343.2 expected with a
linear molecule.
We isolated the fatty acid from 810.5 (i2) by hydrolysis with
phospholipase A2 and subsequent RP-ESI/MS. The oxidized
free fatty acid was then incubated in the presence of hydrogen gas either with or without platinum(IV) oxide and then analyzed by negative
ion FIA-ESI-MS. The oxidized fatty acid that was incubated in the
absence of platinum(IV) oxide contained the unaltered fatty acid with
m/z 331 (Fig.
7A) and the oxidized fatty
acid incubated in the presence of platinum contained major ions at
m/z 339 and 341 (Fig. 7B). Obtaining a
mixture of major ion at m/z 341 implied hydrogenation of five reducible functional groups, e.g.
double bonds or reducible oxygens. Presence of an ion at
m/z 339 implied incomplete hydrogenation of the
molecule and no peak was found at m/z 343, which
is expected for a linear molecule. The retention of a double-bond
equivalent suggested that the molecule possessed a ring structure and
was not a linear molecule.
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Fig. 7.
Hydrogenation of the fatty acid obtained from
the dehydration product of the biologically active
m/z 828.5 isomer. The
dehydration product of the biologically active isomer of
m/z 828.5 was isolated by sequential NP and
reverse phase chromatography and treated with phospholipase
A2. The free fatty acid was isolated by reverse phase HPLC
and treated with platinum(IV) oxide under hydrogen. The
m/z of the carboxylate anions (M)
of the untreated (A) and
PtO2/H2-treated (B) fatty acid were
then measured by negative ion ESI-MS.
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ESI-MS/MS of m/z 828.5 (i2) and m/z 810.5 (i2) Fatty
Acids--
Purified isomers of m/z 828.5 (i2)
and 810.5 (i2) were treated with phospholipase A2, and the
released fatty acids purified by RP-LC/MS. The free fatty acid had
identical UV absorbance characteristics compared with the esterified
form (data not shown). The isolated oxidized fatty acids were then
analyzed by negative ion FIA-ESI-MS/MS (Fig.
8A). The fatty acid isolated
from 810.5 (i2) was incubated with porcine liver esterase in the
presence of H218O, which resulted in
replacement of the 16O on the carboxyl end of the fatty
acid with 18O. The exchange did not go to completion and
the fraction of molecules that contained zero, one, and two
18O atoms was approximately 6, 41, and 53%, respectively
(data not shown). The doubly labeled fatty acid
(m/z 353) was analyzed by negative ion ESI-MS/MS
and the fragmentation pattern was compared with the unlabeled molecule
(Fig. 8, A and B). Because of the exchange of two
16O atoms with two 18O atoms, the labeled
molecule was 4 Da larger than the unlabeled molecule. Daughter ions
that possessed the carboxylic acid portion of the molecule maintained
this 4 Da difference (Fig. 8B, arrows). Conversely, many of the daughter ions of both compounds had identical m/z and were presumably formed by loss of the
carboxyl end of the molecule. The fragmentation profile obtained was
consistent with the structure of an oxidized fatty acid with an epoxide
at the 5,6 position (m/z 115 for
16O-labeled molecule and m/z 119 for
18O labeled molecule), a covalent bond between the 8 and 12 carbon (m/z 191 and 220 for
16O-labeled molecule and m/z 195 and
224 for 18O-labeled molecule), a ketone at the 9 position,
and a double bond at the 10, 11 position (m/z 97 for both 16O- and 18O-labeled molecules). This
structure was confirmed by 1H NMR spectroscopy.
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Fig. 8.
Electrospray ionization tandem mass
spectrometry of the fatty acid released from 810.5 (i2). The
dehydration product of 828.5 (i2) was isolated by sequential NP-LC/MS
and RP-LC/MS and treated with phospholipase A2. The fatty
acid was isolated by a second RP-LC/MS run, collected, and analyzed by
ESI-MS/MS in the negative mode (A). The free fatty acid
obtained by a second RP (C18)-LC/MS run was incubated with
porcine liver esterase in H218O. The contents
were mixed thoroughly and incubated for 60 min at 37 °C with
occasional mixing. Lipids were extracted by addition of 300 µl of
chloroform/methanol (2:1, v/v), dryed, and analyzed by ESI-MS/MS in the
negative ion mode (B).
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Proton NMR Spectra of m/z 828.5 (i2), 810.5 (i2), and Free Fatty
Acid from 810.5 (i2)--
The 1H NMR spectra of
m/z 828.5 (i2) and 810.5 (i2) provided some
structural information on these molecules. One of the major differences
between the 1H NMR spectra of m/z
828.5 (i2) and 810.5 (i2) was the presence of an ,
-unsaturated
carbonyl group in 810.5 (i2) as shown by proton resonances at 7.53 and 6.34 (data not shown). The 1H NMR spectrum of the free
fatty acid obtained from m/z 810.5 (i2) (332 (i2)) suggested the presence of an ,-unsaturated carbonyl group
( 7.53 and 6.34), a trisubstituted alkene ( 6.16), a
cis-alkene ( 5.49 and 5.32), and an epoxide ( 3.39 and
2.99) (Fig. 9). Chemical shift values
from 1H NMR spectra of known molecules, prostaglandin
A2, prostaglandin E2, prostaglandin
J2, 12-prostaglandin J2, and
(±)-5,6-EET, which are structurally similar to 332 (i2) were useful in
assigning proton resonances. The structure of these molecules are
provided for reference (Fig. 10).
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Fig. 9.
1H NMR of the fatty acid obtained
from m/z 810.5. The 500 MHz
1H NMR spectrum of the 332 (i2) in CDCl3
indicates the presence of an ,-unsaturated carbonyl group ( 7.53 and 6.34), a tri-substituted alkene ( 6.16), a
cis-alkene ( 5.49 and 5.32), and an epoxide ( 3.39 and 2.99).
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The connectivity between various functional groups were assigned based
on 1H-1H homodecoupling experiments for 332 (i2). Irradiation of proton resonances at 7.53 (dd,
C11-H) resulted in the conversion of the dd
(J = 1.7, 6 Hz, C10-H) at 6.34 to a d
(J = 1.7 Hz) suggested that these two protons
(C11-H and C10-H) were coupled to each other.
This irradiation also showed some changes on the multiplet at 3.65 (C12-H). Irradiation of proton resonances at 6.34 (dd,
C10-H) resulted in the conversion of the dd
(J = 2, 6 Hz) at 7.53 (C11-H) to a d
(J = 2 Hz) suggesting these two protons were coupled to
each other (Fig. 11A).
Irradiation of proton resonances at 6.16 (d, J = 8.4 Hz, C7-H) resulted in the conversion of dd
(J = 1.9, 8.4 Hz) at 3.39 (C6-H) to a d
(J = 1.9 Hz), which suggested these two protons are
coupled to each other. Irradiation of proton resonances at 5.49 (m,
C15-H) resulted in changes in 5.32 (m,
C14-H) and vice versa. This suggested that these two proton resonances correspond to cis-alkene hydrogens. In
addition to the above changes, irradiation of proton resonances at 5.49 (m, C15-H) resulted in the conversion of an apparent q
(J = 7.4 Hz) at 1.94 (C16-H) to a
triplet. This suggested the presence of allylic hydrogens ( 1.94, C16-H) adjacent to a cis-alkene hydrogen
(C15-H). Irradiation of proton resonances at 5.32 (m, C14-H) resulted in changes in the proton resonances at 2.80 and 2.65 (two C13-H). Irradiation of proton resonances
at 3.65 (m, C12-H) resulted in the conversion of the dd
(J = 2.0, 6.0 Hz) at 7.53 (C10-H) to a
d (J = 6 Hz) and the dd (J = 1.7, 6.0 Hz) at 6.34 (C11-H) to a d (J = 6 Hz)
suggesting these three protons were coupled to each other. This
irradiation also resulted in changes in the proton resonances at 2.80 and 2.65 (C13-Hs). The protons at 2.80 and 2.65 were assigned as the diastereotopic allylic hydrogens adjacent to both
C14-H and C12-H.
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Fig. 11.
Representative 1H-1H
Homodecoupling spectra of 332 (i2). Irradiation of proton
resonances at 6.34 (dd, C10-H) resulted in the
conversion of the dd at 7.53 to a d suggests these two protons are
coupled to each other (panel A). Irradiation of proton
resonances at 3.39 resulted in the conversion of the doublet at 6.16 to a singlet and some minor changes in the multiplet at 2.99 (C5-H) (panel B).
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Irradiation of proton resonance at 3.39 (dd, J = 1.9, 8.4 Hz) resulted in the conversion of the d (J = 8.4 Hz) at 6.16 to a singlet (Fig. 11B) and some minor
changes in the multiplet at 2.99 (C5-H). This result
suggested that the dd at 3.39 was the hydrogen on C6 in
the 5,6-epoxide group. The coupling constant value for the 5,6-epoxide
hydrogen coupling (J = 2.1 Hz) suggested the
trans-epoxide. Irradiation of the proton resonances at 2.80 and 2.65 (C13-Hs) resulted in changes in the proton resonances at 5.32 and 3.39. Proton resonance at 2.44 (dt, J = 3.2, 7.1 Hz) was assigned as C4-H. The
proton resonance at 2.33 were characteristic for C2
methylene hydrogens and a triplet at 0.87 (t, 6.5 Hz) was
characteristic of a terminal methyl group. The proton resonances at 1.24 were characteristic of C17-C19 and
C3 hydrogens. The proton resonances corresponding to
C4-Hs were not clearly seen due to the broad signals
from water at 1.8-1.4.
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DISCUSSION |
These studies have demonstrated that the biologically active
molecule of m/z 828.5 (i2) is
1-palmitoyl-2-(5,6)-epoxyisoprostane E2-sn-glycero-3-phosphocholine (PEIPC). This
molecule was 1 of the 5 isomers of m/z 828.5 present in Ox-PAPC (Fig. 1). Accurate mass measurements by high
resolution fast atom bombardment/MS analysis demonstrated that
m/z 828.5 was an arachidonic acid derivative with
the addition of three oxygens and the loss of two hydrogens. The
identification of the active isomer was made by performing structural
analysis on m/z 828.5 (i2), its dehydration
product m/z 810.5 (i2) (Fig. 3), and the fatty
acids liberated from these phospholipids. It was necessary to use both
the native molecule and dehydration product because of the difficulty
of recovering the liberated fatty acid from m/z
828.5 (i2). Dehydration of a -hydroxy ketone
(m/z 828.5 (i2)) to an ,-unsaturated enone (m/z 810.5 (i2)) was demonstrated by
1H NMR analysis of the intact phospholipids (data not
shown) and has been described previously for several prostaglandins
(i.e. conversion of prostaglandin E2 to
prostaglandin A2) (25). Derivatization with
bis(trimethylsilyl)trifluoroacetamide also demonstrated the presence of
one hydroxyl group in m/z 828.5 (i2), which was
lost in m/z 810.5 (i2) (data not shown). Studies
with sodium borohydride where four hydrogens were added to the molecule
suggested the presence of two reducible oxygens (Fig. 5). This was also
consistent with addition of two methoxylamine to the molecule (Fig. 4).
Previous studies have shown that both epoxide and carbonyl groups add
amines (26, 27). ESI-MS/MS analysis of the fatty acid isolated from m/z 810.5 (i2) (Fig. 8) and
m/z 828.5 (i2) (data not shown) with O18 labeling demonstrated that there was a reactive oxygen
at the 5 position. Further evidence for reactive oxygen at this
position was the ability of m/z 810.5 (i2) and
828.5 (i2) to form POVPC under MS/MS conditions. MS/MS analysis of
m/z 810.5 (i2) reduced with sodium borohydride
and sodium borodeuteride suggested that this oxygen was an 5,6-epoxide.
This was suggested by the association of the reduced oxygen with either
the 5 or 6 carbon during fragmentation (Fig. 6). The fact that the
fatty acids liberated from m/z 810.5 (mass 331)
was cyclic, was shown by the addition of only 10 hydrogens during
hydrogenation rather than 12 hydrogens, which would be expected if the
molecule were linear (Fig. 7).
The detailed structure of the liberated fatty acid was determined by
proton NMR analysis (Fig. 9). The 1H NMR spectrum of the
332 (i2) showed the presence of ,-unsaturated carbonyl group ( 7.53 and 6.34), a trisubstituted alkene ( 6.16), a
cis-alkene ( 5.49 and 5.32), and an epoxide ( 3.38 and
2.99). The chemical shift and coupling constant values of 332 (i2)
indicated that it was a cyclopentenone containing an exocyclic allylic
epoxide. The connectivity between various functional groups in 332 (i2) was obtained based on 1H-1H homodecoupling
experiments (Fig. 11). All isomers with m/z 828.5 possessed nearly identical UV maxima at 252 nm (mean = 19,774) and all isomers of m/z 810.5 possessed an
identical UV maxima at ~257.5 nm (mean = 18,342), which was
attributable to the presence of an 
,-epoxy, ,-unsaturated
enone, or ,-epoxy, ,-unsaturated dienone. These values were
comparable with the structurally related
12-PGJ2 (
max = 248 nm,
= 17,000), which contains a -hydroxy, ,-unsaturated
enone (28) (Fig. 10).
A number of groups have identified isoprostanes as a major product of
autoxidation of arachidonic acid (Refs. 29 and 30; reviewed in Ref.
31). F2 isoprostanes are produced during in vitro oxidation of LDL by a variety of free radical generating systems including copper (32), peroxynitrite (33), and endothelial cell
cultures (34). F2 isoprostanes were shown to be initially formed from arachidonic acid esterified to phospholipids, and free
isoprostanes can then be released by hydrolysis (35). Increased levels
of isoprostanes have been found in atherosclerotic lesions (36).
Recently, association of F2 isoprostanes with
atherosclerosis has been examined. In patients with
hypercholesterolemia the levels of 8-iso-PGF2
were
approximately twice those of age matched controls (37). In another
study, a nearly 50-fold increase in 8-iso-PGF2 and
IPF2-I levels were found in atherectomy specimens from
lesions as compared with nonlesion vascular tissue (38).
More than 64 isoprostanes have been shown to be randomly produced by
arachidonic acid oxidation (31). The amount of
D2/E2 and F2 isoprostanes differ
considerably depending upon the type of oxidative stress (39). Although
large numbers of isoprostanes have been described, no epoxyisoprostanes
have previously been identified. PEIPC was one of the most prominent
products during the autoxidation of PAPC (18). The lack of previous
identification of epoxyisoprostanes may be attributed to the harsh
conditions typically used for the release of fatty acids from
phospholipids. Base hydrolysis and acidification during extraction can
alter or destroy the reactive allylic epoxide functionality present in
these molecules. It is known that molecules containing allylic epoxide
functionality can add nucleophiles such as methanol and amines (26, 40)
and undergo rearrangements (41). We observed addition of
CD3OD to PEIPC when lipids were extracted with
CHCl3/(CD3)3OD under certain
conditions (data not shown). Autoxidation of unesterified arachidonic
acid also generated ions with m/z 349 and
m/z 331. However, UV and HPLC characteristics of
the m/z 349 and m/z 331 derived from arachidonic acid autoxidation were very different than
that of the free fatty acids released from m/z
828.5 or m/z 810.5. This suggested that the
mechanism by which arachidonic acid and arachidonoyl phospholipids
autoxidize were not identical.
The proposed mechanism for the generation of
epoxyisoprostane during autoxidation of
PAPC is shown in Fig. 13. Oxidation of arachidonoyl phospholipids were
shown to generate four regioisomers of F2 or
E2/D2 isoprostanes via the proposed
prostaglandin endoperoxide phospholipid intermediates
(G2-IsoP-PC and H2-IsoP-PC) (42). It is known
that allylic hydroperoxides can undergo dehydration to generate allylic
epoxides (43, 44). Decomposition of endoperoxide nucleus and
rearrangement of allylic hydroperoxide of 5-G2-IsoP-PC is
expected to form an allylic epoxide containing 5,6-epoxyisoprostane E2 phospholipid (m/z 828.5).
Dehydration of 5,6-epoxyisoprostane E2-PC generates an
epoxycyclopentenone isoprostane (m/z 810.5). Alternatively, reduction of allylic hydroperoxide and decomposition of
endoperoxide nucleus in 5-G2-IsoP-PC has been shown to
generate 5-E2-IsoP-PC. Dehydration of
5-E2-IsoP-PC is expected to generate the corresponding
cyclopentenone isoprostane 5-A2-IsoP-PC. It is likely that
the proposed hydroperoxide rearrangements of other regioisomers of
G2-Iso-PC could generate different regioisomers of
epoxyisoprostane phospholipids.
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Fig. 12.
Proposed mechanism by which
epoxyisoprostane-containing phospholipids are produced from arachidonic
acid-containing phospholipids.
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§¶,
,
TOP
ABSTRACT
INTRODUCTION
