A 13-oxo-9,10-epoxytridecenoate phospholipid analogue of the genotoxic 4,5-epoxy-2E-decenal: Detection in vivo, chemical synthesis, and adduction with DNA

Article abstract of DOI:10.1021/tx9002484

Often guided by analogy with nonphospholipid products from oxidative cleavage of polyunsaturated fatty acids, we previously identified a variety of biologically active oxidatively truncated phospholipids. Previously, 4,5-epoxy-2(E)-decenal (4,5-EDE) was found to be produced by oxidative cleavage of 13-(S)-hydroperoxy-9,11-(Z,E)-octadeca-dienoic acid (13-HPODE). 4,5-EDE reacts with deoxy-adenosine (dAdo) and deoxy-guanosine (dGuo) to form mutagenic etheno derivatives. We hypothesized that a functionally similar and potentially mutagenic compound, that is, 13-oxo-9,10-epoxytridecenoic acid (OETA), would be generated from 9-HPODE through an analogous fragmentation. We expected that an ester of 2-lysophosphatidylcoline (PC), OETA-PC, would be produced by oxidative cleavage of 9-HPODEPC in biological membranes. An efficient, unambiguous total synthesis of trans-OETA-PC was first executed to provide a standard that could facilitate the identification of this phospholipid epoxyalkenal that was shown to be produced during oxidation of the linoleic acid ester of 2-lysoPC. Finally, trans-OETA-PC was detected in a lipid extract from rat retina. The identity of the naturally occurring oxidatively truncated phospholipid was further confirmed by derivatization with methoxylamine that produced characteristic mono and bis adducts. The average amount of trans-OETA-PC in rat retina, 0.33 pmol, is relatively low as compared to other oxidatively truncated PCs, for example, the 4-hydroxy-7-oxohept-5-enoic acid PC ester (2.5 pmol) or the 4-keto-7-oxohept-5- enoic acid PC ester (1.7 pmol), derived from the docosahexaenoic acid ester of 2-lysoPC. This, most likely, is because docosahexaenoate PCs are particularly abundant in the retina as compared to the linoleate PC ester precursor of OETA-PC. As predicted by analogy with 4,5-EDE, OETA-PC reacts with dAdo and dGuo, as well as with DNA, to form mutagenic etheno adducts.

Full text of DOI:10.1021/tx9002484

5
16
Chem. Res. Toxicol. 2010, 23, 516–527
A 13-Oxo-9,10-epoxytridecenoate Phospholipid Analogue of the
Genotoxic 4,5-Epoxy-2E-decenal: Detection in Vivo, Chemical
Synthesis, and Adduction with DNA
†
†
†
‡
†
Clementina Mesaros, Bogdan G. Gugiu, Rong Zhou, Seon Hwa Lee, Jaewoo Choi,
†
‡
,†
James Laird, Ian A. Blair, and Robert G. Salomon*
Department of Chemistry, Case Western ReserVe UniVersity, 2074 Adelbert Road, Millis 212, CleVeland,
Ohio 44106, Centers for Cancer Pharmacology and Excellence in EnVironmental Toxicology, Department of
Pharmacology, UniVersity of PennsylVania School of Medicine, Philadelphia, PennsylVania 19104-6160
ReceiVed July 21, 2009
Often guided by analogy with nonphospholipid products from oxidative cleavage of polyunsaturated
fatty acids, we previously identified a variety of biologically active oxidatively truncated phospholipids.
Previously, 4,5-epoxy-2(E)-decenal (4,5-EDE) was found to be produced by oxidative cleavage of 13-
(
(
S)-hydroperoxy-9,11-(Z,E)-octadeca-dienoic acid (13-HPODE). 4,5-EDE reacts with deoxy-adenosine
dAdo) and deoxy-guanosine (dGuo) to form mutagenic etheno derivatives. We hypothesized that a
functionally similar and potentially mutagenic compound, that is, 13-oxo-9,10-epoxytridecenoic acid
OETA), would be generated from 9-HPODE through an analogous fragmentation. We expected that an
(
ester of 2-lysophosphatidylcoline (PC), OETA-PC, would be produced by oxidative cleavage of 9-HPODE-
PC in biological membranes. An efficient, unambiguous total synthesis of trans-OETA-PC was first
executed to provide a standard that could facilitate the identification of this phospholipid epoxyalkenal
that was shown to be produced during oxidation of the linoleic acid ester of 2-lysoPC. Finally, trans-
OETA-PC was detected in a lipid extract from rat retina. The identity of the naturally occurring oxidatively
truncated phospholipid was further confirmed by derivatization with methoxylamine that produced
characteristic mono and bis adducts. The average amount of trans-OETA-PC in rat retina, 0.33 pmol, is
relatively low as compared to other oxidatively truncated PCs, for example, the 4-hydroxy-7-oxohept-
5
-enoic acid PC ester (2.5 pmol) or the 4-keto-7-oxohept-5-enoic acid PC ester (1.7 pmol), derived from
the docosahexaenoic acid ester of 2-lysoPC. This, most likely, is because docosahexaenoate PCs are
particularly abundant in the retina as compared to the linoleate PC ester precursor of OETA-PC. As
predicted by analogy with 4,5-EDE, OETA-PC reacts with dAdo and dGuo, as well as with DNA, to
form mutagenic etheno adducts.
Introduction
The polyunsaturated fatty acyl (PUFA) chains of membrane
phospholipids (PLs) are susceptible to oxidative cleavages that
generate oxidized phospholipids (oxPLs) with abbreviated acyl
chains containing aldehydes, the corresponding acids, as well
as hydroxyl, hydroperoxyl, and epoxy groups. OxPLs remain
anchored in the membrane. However, because of their polar
functionality, the oxidatively truncated acyl chains protrude from
the membrane into the aqueous phase (1) where they can interact
with proteins. They can act as receptor ligands, for example,
for CD36 receptor-mediated endocytosis of oxidized low-density
lipoprotein (oxLDL) by macrophage cells (2) that contributes
to the pathogenesis of cardiovascular disease. Similar oxPL-
induced CD36-mediated endocytosis of photoreceptor cell outer
segment tips by retinal pigmented endothelial cells contributes
to rapid turnover of photoreceptor cells under conditions of
oxidative stress (3). Binding of oxPL with CD36 receptors in
platelet membranes promotes platelet activation and thrombosis
1
*
To whom correspondence should be addressed. Tel: 216-368-2592.
(
4). Binding of certain oxPL with the scavenger receptor (SR)
Fax: 216-368-3006. E-mail: rgs@cwru.edu.
†
Case Western Reserve University.
University of Pennsylvania School of Medicine.
Abbreviations: BHT, butylated hydroxytoluene; dAdo, deoxy-adenosine;
B1 prevents binding to SR-B1 of its physiological ligand, high-
density lipoprotein (5). Furthermore, oxPLs interfere with SR-
B1-mediated selective uptake of cholesteryl esters in hepatocytes
and thus may have an inhibitory effect on reverse cholesterol
transport. Some oxPLs covalently modify proteins, for example,
generating carboxyalkylpyrroles that incorporate the ε-amino
group of lysine residues (6). For example, oxidative cleavage
of PLs incorporating docosaheaxenoic acid (DHA), the most
abundant PUFA in photoreceptor cell disk membranes, generates
the 4-hydroxy-7-oxohept-5-enoic acid phosphatidylcoline ester
(HOHA-PC). This bis electrophile reacts with proteins to
produce carboxyethylpyrroles (7) that are angiogenic and
immunogenic. These biological activities contribute, respec-
tively, to the pathogenesis of choroidal neovascularization (8)
‡
1
DCC, dicyclohexylcarbodiimide; dGuo, deoxy-guanosine; DHA, docosa-
heaxenoic acid; DHP, dihydropyran; DMAP, N,N-dimethylaminopyridine;
DM-PE, 1,2-dimyristoyl-sn-glycero-phosphatidyl-3-ethanoalmine; DTPA,
diethylenetriaminepentaacetic acid; 4,5-EDE, 4,5-epoxy-2(E)-decenal; HNE,
-hydroxynon-2-enal; HOHA, 4-hydroxy-7-oxohept-5-enoic acid; 9-H-
PODE, 9-hydroperoxy-(Z,E)-9,11-octadecadienoic acid; 13-HPODE, 13-
hydroperoxy-(Z,E)-9,11-octadecadienoic acid; KOHA, 4-keto-7-oxohept-
4
5
-enoic acid; MPO, myeloperoxidase; MRM, multiple reaction monitoring;
NMO, 4-methylmorpholine N-oxide; OETA, 13-oxo-9,10-epoxytridecenoic
acid; oxLDL, oxidized low-density lipoprotein; oxPLs, oxidized phospho-
lipids; PA-PC, 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine;
PC, phosphatidylcoline; PPTS, pyridinium p-toluenesulfonate; PUFA,
polyunsaturated fatty acyl; SPE, solid-phase extraction; SR, scavenger
receptor; TLC, thin-layer chromatography; TPAP, tetrapropylammonium
perruthenate.
1
0.1021/tx9002484  2010 American Chemical Society
Published on Web 02/04/2010

A 13-Oxo-9,10-epoxytridecenoate Phospholipid
Chem. Res. Toxicol., Vol. 23, No. 3, 2010 517
Scheme 1. Formation of HNE and HOOA-PC from PA-PC
to cause substantial amounts of DNA damage in vivo through
the known reaction of 4,5-EDE with DNA (18). 4,5-EDE reacts
with deoxy-adenosine (dAdo) and deoxy-guanosine (dGuo) to
form etheno derivatives (19) (Scheme 2). This chemistry
provides a significant correlation between a product of lipid
peroxidation and a mutagenic DNA lesion. The etheno adducts
of DNA, when formed in vivo, are normally repaired. However,
in diseased individuals, these lesions are not repaired and
subsequent DNA replication leads to mutations (20) or apoptosis
(
21). Mutations in protooncogenes and tumor suppressor genes
have been directly implicated in human cancer (20). Mutagenic
etheno adducts have been detected in human tissue samples (22).
We hypothesized that a functionally similar and potentially
mutagenic compound, that is, 13-oxo-9,10-epoxytridecenoic acid
Scheme 2. Formation of 4,5-EDE from 13-HPODE and
Postulated Formation of OETA from 9-HPODE
(
OETA), would be generated from 9-HPODE through an
analogous fragmentation (Scheme 2). We expected that an ester
OETA-PC of 2-lyso-PC would be produced by oxidative
cleavage of 9-HPODE-PC in biological membranes. We now
report an unambiguous total synthesis of trans-9,10-OETA-PC
as well as its generation by oxidation of the linoleic acid ester
LA-PC of 2-lysoPC. The natural occurrence of OETA-PC in
vivo was established by detection in a lipid extract from rat
retina. As predicted by analogy with 4,5-EDE, OETA-PC reacts
with dAdo and dGuo, as well as with DNA, to form mutagenic
etheno adducts (Scheme 2).
Materials and Methods
Materials. 1-Palmitoyl-2-linoleyl- sn-glycero-3-phosphatidyl-
choline (PL-PC) and 1,2-dimyristoyl-sn-glycero-phosphatidyl-3-
ethanoalmine (DM-PE) were obtained from Avanti Polar Lipids
(
Alabaster, AL), and myeloperoxidase (MPO) was supplied by
and retinal degeneration (9), the “wet” and “dry” forms,
respectively, of age-related macular degeneration.
Analogy with “mirror image” nonphospholipid products from
oxidative cleavage of PUFAs has guided our efforts to identify
naturally occurring oxPLs. Previously, we postulated the forma-
tion of a 5-hydroxy-8-oxooct-6-enoic acid PC ester HOOA-PC
Calbiochem (La Jolla, CA). Dry pyridine and chloroform were
supplied by ACROS (New Jersey). Ammonium acetate, dGuo, and
dAdo were purchased from Sigma-Aldrich. (St. Louis, MO).
Supelclean LC-18 solid-phase extraction (SPE) columns were from
Supelco (Bellefonte, PA). Chelex-100 chelating ion-exchange resin
(
100-200 mesh size) was from Bio-Red Laboratories (Hercules,
CA). HPLC grade water and acetonitrile were obtained from Fisher
Scientific Co. (Fair Lawn, NJ). ACS grade ethanol was obtained
from Pharmco (Brookfield, CT). Gases were supplied by BOC
Gases (Lebanon, NJ).
(Scheme 1) from 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phos-
phocholine (PA-PC) by analogy with the well-known formation
of 4-hydroxynon-2-enal (HNE). An authentic sample prepared
by unambiguous total synthesis (10) confirmed that HOOA-PC
is formed from PA-PC (11) and subsequently allowed the
1
General Methods. H NMR spectra were recorded on Varian
Gemini spectrometers operating at 200 or 300 MHz using CHCl3
(δ 7.24 ppm) as an internal standard. All chemical shifts are reported
in parts per million (ppm) on the δ scale relative to the solvent
used. H NMR spectra are presented as follows: Multiplicity is given
as s, singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet;
qAB, quartet AB; and m, multiplet; coupling constants (Hz), number
of protons. C NMR spectra were recorded on Varian Gemini
spectrometers operating at 50 and 75 MHz using CDCl (δ 77.0)
as an internal standard. Signal multiplicities were established by
DEPT experiments. High-resolution mass spectra were recorded
on a Kratos AEI MS25 RFA high-resolution mass spectrometer at
20 eV.
Thin-layer chromatography (TLC) was performed on glass plates
precoated with silica gel (Kieselgel 60 F254, E. Merck, Darmstadt,
Germany); R values are quoted for plates of thickness 0.25 mm.
f
demonstration of its presence in oxLDL (2) and its CD36
-
mediated biological activities (12). Total synthesis of more
oxidized derivatives of HOOA-PC (13) similarly allowed their
identification in oxLDL and the demonstration of their biological
activities.
1
1
3
“Mirror image” hydroperoxides 9- and 13-hydroperoxy-(Z,E)-
3
9
,11-octadecadienoic acid (9- and 13-HPODE) are major
primary oxidation products (Scheme 2) from linoleic acid (LA),
the most abundant PUFA in the human body (14). Cell injury
can initiate lipid peroxidation, leading to generation of these
hydroperoxides through nonenzymatic reactions, induced by free
radicals. Various lipoxygenases enantioselectively transform LA
into specific enantiomers of 9- or 13-HPODE (15). 9(S)-HPODE
is produced by the action of cyclooxygenases 1 and 2 on LA
The plates were visualized by viewing the plates under short-
wavelength UV light or by exposure to iodine vapor. PL spots were
visualized using a molybdenum-based detection spray (23). Flash
chromathography was performed using Silica Gel 60A, 32-63 µM
from Sorbent Technologies (Atlanta, GA) or ICN SiliTech 32-63
D 60A from ICN Biomedicals Gmbh Eschwege (Germany).
(
16). Free radical-induced oxidation delivers a racemic mixture
of these hydroperoxides. Previously, 4,5-epoxy-2(E)-decenal
4,5-EDE) was shown to be produced by oxidative cleavage of
3(S)-HPODE (Scheme 2), a fragmentation that is promoted
(
1
by vitamin C in vitro under oxidative conditions (17). Further-
more, vitamin C levels comparable to the intracellular concen-
tration reached with a dose of 200 mg per daysthe recom-
mended daily dose of vitamin C for healthy adultsswas found
2-(8-Bromo-octyloxy)-tetrahydropyran. Pyridinium p-toluene-
sulfonate (PPTS, 217 mg, 0.86 mmol) was added to a solution of
the alcohol 8-bromo-octanol (1942 mg, 8.67 mmol) and dihydro-
pyran (DHP, 1.1 g, 13 mmol, freshly distilled) in dry methylene

5
18 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Mesaros et al.
chloride (40.0 mL) (24). The resulting solution was stirred overnight
at room temperature and then diluted with water, and the resuting
mixture was extracted with ethyl acetate, washed with brine, and
dried on sodium sulfate. The solvent was removed with a rotary
evaporator. The crude product was purified by flash chromatography
on a silica gel column (10% ethyl acetate in hexanes) to afford
mL), and then, the washes were added to the separatory funnel.
The mixture was then shaken to give two clear phases that were
separated. The organic layer was washed with water (3 mL), 1%
NaHCO (3 mL), and brine and then dried on Na SO . The solvent
3
2
4
was removed with a rotary evaporator, and the residue was purified
by flash chromatography on a silica gel column with 30% ethyl
acetate in hexanes to afford the title compound (1.64 g, 96%): TLC
2
-(8-bromo-octyloxy)-tetrahydropyran (2.5 g, 98%): TLC (8% ethyl
1
1
acetate in hexanes) R
f
) 0.28. H NMR (CDCl
3
, 300 MHz): δ 4.55
(ethyl acetate/hexanes 3:7) R ) 0.37. H NMR (300 MHz, CDCl ):
f
3
(
J
t, J ) 3 Hz, 1H), 3.81-3.88 (m, 1H), 3.67-3.74 (tt, J
2
1
) 6 Hz,
δ 5.48-5.58 (m, 1H), 5.69-5.79 (m, 1H), 4.48 (d, J ) 6 Hz, 2H),
3.58-3.64 (m, 2H), 2.03 (s, 3H), 1.98-2.04 (m, 2H), 1.23-1.56
(14H).
1
3
) 6 Hz, 1H), 3.51-3.32 (m, 2H), 1.30-1.87 (18H). C NMR
): 98.92 (CH), 67.65 (CH ), 62.43 (CH ), 34.06
), 32.84 (CH ), 30.83 (CH ), 29.74 (CH ), 29.30 (CH ), 28.74
), 28.15 (CH ), 26.18 (CH ), 25.54 (CH ), 19.76 (CH ).
(
(
(
75 MHz, CDCl
CH
CH
3
2
2
2
2
2
2
2
1
1-Hydroxy-undec-9-enoic Acid (6). A 250 mL round-bottom
flask was charged with Jones reagent (8 mL, 2.67 M Na Cr in
) and acetone (40 mL). A solution of acetic acid and
1-hydroxy-undec-2-enyl ester (1.2 g, 5.2 mmol) in acetone (40
2
2
2
2
2
2 7
O
2
11-(Tetrahydropyran-2-yloxy)-undec-2-en-1-ol (2). Liquid am-
4
1
2 4
M H SO
monia (33 mL) and iron(III) nitrate (20 mg) were placed in a 100
mL three-necked flask equipped with a magnetic stirrer, dry ice/
acetone-cooled condenser, and drying tube with potassium hydrox-
ide for protection from moisture. To this mixture, lithium (200 mg,
mL) was added dropwise at 0 °C from an addition funnel over 2 h.
The color changed from orange to dark brown. The mixture was
stirred at room temperature for an additional 4 h, and then, the
organic solution was decanted from an orange residue. The residue
was dissolved in water (30 mL), and the color changed to green-
blue. This was extracted with ethyl acetate (3 × 10 mL). The
organic extracts were added to the decanted solution. Solvents were
then removed by rotary evaporation to give a brown residue. This
was dissolved in water (30 mL) and extracted with ethyl acetate (3
2
8.8 mmol) was added in portions allowing the blue color to
dissipate between additions. Propargyl alcohol (1 mL, 17 mmol)
in tetrahydrofuran (8 mL, freshly distilled) was added over 25 min,
and the reaction mixture was then allowed to boil under reflux for
100 min. 2-(8-Bromo-octyloxy)-tetrahydropyran (2.5 g, 8.5 mmol)
in tetrahydrofuran (7 mL) was then added over 35 min, and the
mixture was allowed to boil under reflux for 135 min. Lithium wire
×
20 mL). The organic solution was washed with water (20 mL)
to remove residual chromium species (26) and then extracted with
0% NaOH (20 mL). The aqueous phase was acidified with 6 M
(
50 mg, 7.2 mmol) was added resulting in a persistent blue color
in the mixture (25). After 30 min, sufficient ammonium chloride
was added to dispel the blue color. Most of the ammonia was the
evaporated under a gentle stream of nitrogen, and the residue was
poured onto ice (50 g). The resultant mixture was extracted with
diethyl ether (3 × 30 mL) and then ethyl acetate (2 × 50 mL).
The combined organic extract was dried and concentrated by rotary
evaporation to give a residue that was purified by flash chroma-
tography on a silica gel column with 25% ethyl acetate in hexanes
1
HCl to pH 1 and then extracted with ethyl acetate (4 × 10 mL).
The combined organic extracts were washed with water (10 mL)
2 4
and brine (10 mL) and then dried on Na SO . The yellow-green
color of the organic phase gradually faded to become colorless.
The solvent was removed with a rotary evaporator, and the white
fluffy solid residue was purified by flash chromatography on a silica
gel column with 70% ethyl acetate in hexanes to afford 11-hydroxy-
to afford 11-(tetrahydropyran-2-yloxy)-undec-2-en-1-ol (2.1 g,
undec-9-enoic acid (1.04 g, 86%): TLC (ethyl acetate/hexanes 1:1)
1
9
1%): TLC (ethyl acetate/hexanes 1:4) R
f
) 0.3. H NMR (300
1
R
4
f
) 0.28. H NMR (300 MHz, CDCl
3
): δ 5.55-5.72 (m, 2H),
MHz, CDCl
3
3
3
): δ 5.55-5.72 (m, 2H), 4.55 (t, J ) 3 Hz, 1H),
.81-3.88 (m, 1H), 3.67-3.74 (tt, J ) 6 Hz, J ) 6 Hz, 1H),
.51-3.32 (m, 2H), 1.98-2.04 (m, 2H), 1.30-1.87 (18H).
): 133.52 (CH), 128.9 (CH), 98.89 (CH),
.06 (d, J ) 3 Hz, 2H), 2.32 (t, J ) 6 Hz, 2H), 2.0-2.03 (m, 2H),
1.58-1.63 (m, 2H), 1.21-1.35 (8H).
-(3-Hydroxymethyl-oxiranyl)octanoic Acid (7). A solution of
1
2
1
3
C
8
NMR (75 MHz, CDCl
3
dioxirane in acetone (27) (8.8 mL, 60 mM) was added dropwise to
a solution of 11-hydroxy-undec-9-enoic acid (120 mg, 0.6 mmol)
in chloroform (4 mL) at room temperature. The resulting mixture
was stirred for an additional 15 min at room temperature. The
6
2
1
7.71 (CH
9.77 (CH
9.74 (CH
Acetic Acid, 11-(Tetrahydro-pyran-2-yloxy)-undec-2-enyl
2
), 63.86(CH
), 29.43 (CH
).
2
), 62.39 (CH
2
), 32.22 (CH
2
), 30.82 (CH
2
),
),
2
2
), 29.12 (CH
2
), 26.24 (CH
2
), 25.54 (CH
2
2
solvents were removed by rotary evaporation. The title compound
Ester (4). A solution of acetic anhydride from a newly opened
bottle (∼1.3 g, 12.4 mmol) in pyridine (5 mL, freshly distilled)
was cooled in an ice bath. A solution of 11-(tetrahydropyran-2-
yloxy)-undec-2-en-1-ol (2.2 g, 8 mmol) in pyridine (4 mL, freshly
distilled) was added in one portion, and the mixture was stirred for
1
was obtained in quantitative yield. H NMR (300 MHz, CDCl
3
,):
δ 3.91-3.56 (2H), 2.96-2.89 (m, 2H), 2.32 (t, 2H, J ) 7.3 Hz),
1
3
1
.63-1.31 (12H). C NMR (75 MHz, CDCl
CH ), 58.98 (CH), 56.39 (CH), 34.00 (CH ), 31.45 (CH
CH ), 29.08 (CH ), 28.90 (CH ), 25.83 (CH ), 24.63 (CH ). HRMS
FAB): m/z calculated for C11 [(M - H) ], 217.1440; found,
17.1445.
-(3-Formyl-oxiranyl)-octanoic Acid (8). 8-(3-Hydroxymethyl-
3
): 179.3 (C), 61.6
(
(
(
2
2
2
), 29.11
2
2
2
2
2
3
h at 0 °C and then for 3 more hours while it was warmed to
room temperature (26). It was then poured onto a mixture of ice
30 g) and hexanes (20 mL) and shaken, and then, the layers were
+
20 4
H O
2
(
8
separated. The aqueous layer was extracted with hexanes (3 × 10
oxiranyl)octanoic acid (130 mg, 0.6 mmol) was dissolved in
dichloromethane (60 mL, freshly distilled) containing 4 Å molecular
sieves and 4-methylmorpholine N-oxide (NMO, 105 mg, 0.9 mmol).
Solid tetrapropylammonium perruthenate (TPAP, 32 mg, 0.091
mmol, 0.15 equiv) was then added under argon (28), and the
resulting green mixture was stirred at room temperature for 2 h,
until TLC analysis with 40% ethyl acetate in hexanes showed the
complete disappearance of the starting material and the appearance
mL). The combined organic layers were washed with water and
2 4
then brine and then dried over Na SO . The extract was concentrated
by rotary evaporation to give a residue that was purified by flash
chromatography on a silica gel column with 12% ethyl acetate in
hexanes to afford the title compound (2.37 g, 95%): TLC (ethyl
1
acetate/hexanes 1:9) R
f
) 0.32. H NMR (300 MHz, CDCl
3
): δ
5
3
3
.48-5.57 (m, 1H), 5.69-5.79 (m, 1H), 4.55 (t, J ) 3 Hz, 1H),
.81-3.88 (m, 1H), 3.67-3.74 (tt, J ) 6 Hz, J ) 6 Hz, 1H),
.51-3.32 (m, 2H), 2.00 (s, 3H), 1.98-2.04 (m, 2H), 1.30-1.87
1
2
of a new spot (R ) 0.26). The molecular sieves were washed with
f
(
18H).
Acetic Acid, 11-Hydroxy-undec-2-enyl Ester (5). A solution
four times the initial volume of the ethyl acetate to recover the
entire product. The solution was filtered through a 1 cm layer of
silica gel on a 60 mL separatory funnel. The solvent was removed
of acetic acid, 11-(tetrahydro-pyran-2-yloxy)-undec-2-enyl ester (2.3
g, 7.5 mmol), and PPTS (237 mg, 0.75 mmol) in absolute ethanol
by rotary evaporation to afford the title compound (123 mg, 96%
1
(
12.5 mL, freshly distilled over CaH
2
) was heated for 6 h at 50 °C.
yield) in high purity. H NMR (75 MHz, CDCl
3
,): δ 8.99 (d, 1H,
The solution was then concentrated by rotary evaporation, and the
residue was taken up in ethyl acetate (10 mL) to give a milky
suspension that was transferred to a separatory funnel (24). The
reaction flask was rinsed with water (5 mL) and ethyl acetate (5
J ) 6.2), 3.20 (dt, 1H, J ) 6.9, J ) 1.9), 3.11 (dd, 1H, J ) 6.3, J
13
) 1.9), 2.33 (t, 2H, J ) 7.4), 1.63-1.31 (12H). C NMR (300
MHz, CDCl ): 198.56 (CH), 179.83 (C), 59.17 (CH), 56.79 (CH),
33.97 (CH ), 31.19 (CH ), 29.04 (CH ), 29.01 (CH ), 28.87 (CH ),
3
2
2
2
2
2

A 13-Oxo-9,10-epoxytridecenoate Phospholipid
Chem. Res. Toxicol., Vol. 23, No. 3, 2010 519
2
5.73 (CH
[(M - H) ], 215.1283; found, 215.1292.
-[3-(3-Oxo-propenyl)-trans-oxiranyl]-octanoic Acid (trans-
2
), 24.59 (CH
2
). HRMS (FAB): m/z calculated for
Table 1. Calibration Equations, Detection Limits, and
Extraction Efficiency
+
11 19 4
C H O
8
detection
OETA, 1). To a stirred suspension of formylmethylene triph-
enylphosphorane (150 mg, 0.495 mmol) (29) in 4 mL of dry toluene
at 0 °C, the above aldehyde (70 mg, 0.327 mmol) was added. The
resulting mixture was stirred for 3 days at 4 °C under argon. The
solvent was then removed by rotary evaporation. The residue was
extracted with ethyl acetate (4 × 5 mL). The aqueous phase was
acidified with 2 M HCl to pH 4 and then extracted with ethyl acetate
limit
(fmol)
extraction
efficiency
compound
calibration equation
2
OETA-PC
PL-PC
y ) 2.1835 × (R ) 0.9993)
9
2
37.6 ( 6.7
58.8 ( 1.6
2
y ) 0.920 × (R ) 0.9913)
for each sample. LC-MS analysis of oxidized lipids was performed
on a Quattro Ultima mass spectrometer (Micromass, Wythenshawe,
United Kingdom) equipped with an electrospray ionization (ESI)
probe interfaced with a Waters 2790 (Waters, Milford, MA) HPLC
system. OETA-PC (50 µg, determined by aliquoting a solution of
a known concentration (determined by microphosphorous assay)
was dissolved in 1 mL of chloroform/methanol (1:1). The solution
(
4 × 10 mL). The combined organic extracts were washed with
water (10 mL) and brine (10 mL) and then dried on Na SO . The
2
4
sample was concentrated by rotary evaporation to give a residue
that was purified by flash chromatography on a silica gel column
with 75% ethyl acetate in hexanes to afford OETA (70 mg, 90%):
1
TLC (ethyl acetate/hexanes 1:1) R
CDCl
f
) 0.3. H NMR (300 MHz,
(
100 µL) was diluted with 400 µL of chloroform/methanol (1:1).
3
,): δ 9.53 (d, 1H, J ) 7.6), 6.52 (dd, 1H, J ) 15.8, J ) 6.8)
To this solution (50 µL) the DM-PE standard (50 µL, 5-15 ng)
was added, and the mixture was chromatographed on a Prodigy
ODS C18 column (150 mm × 2 mm, 5 µm, Phenomenex, Torrance,
CA.) with a binary solvent gradient, starting from 50% water in
methanol with a linear gradient to 100% methanol over 10 min
and then 100% methanol for 20 min at a flow rate of 0.2 mL/min.
All of the mobile phase solvents contained 0.2% formic acid to
enhance the MS signal. The total ion current was obtained in the
mass range of m/z 200-1000 at 30 V of cone energy in the positive
ion mode. Three kilovolts was applied to the electrospray capillary.
All compounds were monitored in the positive ion mode for the
parent ion and the major daughter ion, previously determined by
direct injection using ESI. The optimal collision energy determined
to obtain the most intense daughters was 20 eV. In Table 1 are
given the calibration equation for OETA-PC and PL-PC, as well
as the extraction efficiencies determined as follows. Liposomes
containing 10 µg of OETA-PC and the corresponding precursors
6
2
.35 (dd, 1H, J ) 15.8, J ) 7.4) 3.29 (dd, 1H, J ) 6.9, J ) 1.9),
.92 (dt, 1H, J ) 5.6, J ) 1.9), 2.33 (t, 2H, J ) 7.4), 1.63-1.31
(
12H). 13C NMR (75 MHz, CDCl
3
): 192.64 (CH), 179.83 (C),
1
3
2
53.12 (CH), 133.57 (CH), 61.91 (CH), 56.21 (CH), 33.90 (CH
1.87 (CH ), 29.09 (CH ), 28.90 (CH ), 28.87 (CH ), 25.73 (CH
4.59 (CH ). HRMS (FAB): m/z calculated for C13
2
),
),
2
2
2
2
2
2
H
21
O
4
[(M -
+
H) ], 241.1440; found, 241.1447.
-Palmitoyl-2-(13-oxo-9,10-trans-epoxy-octadeca-11-enoyl)-sn-
1
glycero-3-phosphatidylcholine (trans-OETA-PC). A mixture of
the acid (OETA, 9 mg, 0.037 mmol) and 2-lyso-phosphatidylcholine
(
lyso-PC, 9.2 mg, 0.018 mmol) was dried overnight on a vacuum
pump (0.1 mmHg) equipped with a dry ice-acetone trap, at room
temperature, and then was dissolved in dry CHCl (1 mL, shaken
with P for 0.5 h and distilled). Dicyclohexylcarbodiimide (DCC,
8.5 mg, 0.09 mmol) and N,N-dimethylaminopyridine (DMAP, 2.2
3
2 5
O
1
mg, 0.018 mmol) were added. The mixture was stirred for 10 days
under argon. The mixture was then concentrated on a rotary
evaporator, and the residue was purified by flash chromatography
(
PL-PC) and 130 µg (50% of the total lipid content of liposomes)
of DP-PC were prepared as described previously (vide infra). After
Bligh and Dyer extraction, the amounts recovered were determined
by LC-MS relative to DM-PE added prior to analysis and compared
to a mixture composed of 10 µg from the OETA-PC and precursors
and the same amount of DM-PE. The extraction efficiency for
OETA-PC was 37.6 ( 6.7%.
on a silica gel column with CHCl
0.27) to produce the PL, OETA-PC (10 mg, 87%). H NMR
300 MHz, CDCl ,): δ 9.53 (d, J ) 7.6, 1H), 6.52 (dd, J ) 15.8,
J ) 6.8, 1H) 6.35 (dd, J ) 15.8, J ) 7.4, 1H), 5.17 (m, 1H),
.29-4.40 (m, 2H), 3.80-4.12 (m, 4H), 3.35 (s, 9H), 3.29 (dd, J
6.9, J ) 1.9, 1H), 2.92 (dt, J ) 5.6, J ) 1.9, 1H), 2.33 (t, J )
3 2 f
/MeOH/H O (16/9/1, TLC: R
1
)
(
3
4
)
Derivatization of PLs. Methoxime derivatives of lipids were
prepared by suspending the lipid (50 µg), previously dried in a
stream of nitrogen in 200 µL of freshly dried pyridine with 10%
methoxylamine hydrochloride. The reaction mixture was incubated
for 2 h at room temperature. Solvents and volatile byproducts were
removed under a nitrogen stream, and residues were resuspended
in chloroform:methanol (1:1). Nonlipid components were removed
by passing the solution through a C18 cartridge (Supelclean, 3 mL),
as follows. The reaction mixture dissolved in 50% methanol (1 mL)
was loaded onto an S-PE cartridge, which was previously equili-
brated by passing 10 mL of water. The column then was eluted
with a gradient of water and methanol: 50% methanol (10 mL),
60% methanol (5 mL), 75% methanol (5 mL), 90% methanol (10
mL), and then pure methanol. The derivatized lipids were eluted
with 90-100% methanol. Lipids were dried again under a nitrogen
stream. Derivatives of standard were characterized by ESI-MS/MS
and chromatographed on the same Prodigy ODS C18 column (150
mm × 2 mm, 5 µm) using the same HPLC conditions described
above for oxidized lipids. The LC/ESI-MS/MS was operated in
the positive ion mode. The analytes were detected by multiple
reaction monitoring (MRM). Mass transitions, m/z 747.9f184.5
and m/z 794.7f184.5 for OETA-PC methoxime derivatives were
monitored.
13
7
.4, 2H), 1.22-1.61 (42H), 0.85 (t, J ) 6,3H). C NMR (300 MHz,
CDCl ): 192.64 (CH), 179.83 (C), 153.12 (CH), 133.57 (CH), 61.91
CH), 56.21 (CH), 33.90 (CH ), 31.87 (CH ), 29.09 (CH ), 28.90
), 25.73 (CH ), 24.59 (CH ). HRMS (FAB): m/z
68NO10P [(M - H) ], 718.4660; found, 718.4664.
3
(
(
2
2
2
CH
2
), 28.87 (CH
2
2
2
+
calculated for C37
H
Oxidative Generation of OETA-PC from PL-PC. Lipid
Oxidation. The dry lipid, PL-PC (1 mg), was hydrated at 37 °C
for 1 h in phosphate buffer (1 mL) supplemented with DTPA to
minimize transition metal-induced autoxidation. Then, the hydrated
PL-PC was made into unilamellar vesicles by extrusion using an
Avanti Mini-Extruder (Avanti Polar Lipids, Inc.). For the Cu(II)
2
oxidation, 10 µL of a solution of 500 µM CuCl was added to 1.99
mL of vesicles. For the UV-induced oxidation, the vesicles (2 mL)
were kept in a quartz tube placed in the center of a Rayonet
photochemical reactor with three 80 W low pressure mercury UV
(
350 nm) Rayonet lamps (Southern New England Ultraviolet,
Midtown, CT). The temperature was maintained at about 28 °C.
The MPO oxidation was performed with the MPO-NO -glucose/
2
-
glucose oxidase system (7). Sampling (200 µL) was performed at
various times, and the reaction was stopped by adding butylated
hydroxytoluene (BHT, 2 µL, 40 µM) and catalase (7 µL, 300 µM)
(7). For quantification purposes, the lipid extraction was performed
immediately using a slightly modified Bligh and Dyer method (30),
and the samples were dried in a stream of nitrogen at room
temperature. The dried samples were stored in vials sealed under
argon at -80 °C.
LC/ESI-MS/MS Analysis. A standard curve was generated by
incorporating a fixed amount of internal standard (1,2-myristoyl-
sn-glycero-3-phosphatidylethanolamine, DM-PE), varying the levels
of the analyte, and plotting peak area ratio versus analyte mol ratio
Detection and Quantification of trans-OETA-PC in Rat
Retina. Extraction of PLs from Retina. Rat retinas were harvested
from five normal albino rats by Mary Rayborn and Dr. Xiaorong
Gu. To prevent contamination by blood, or in vitro oxidation, the
retinas were rinsed with saline antioxidant cocktail (saline PBS pH
7.4, containing 40 µM DTPA, and 4 nM BHT). The retinas were
immediately homogenized manually in a plastic vial using a
stainless steel pestle coated with Teflon, and lipid extraction was

5
20 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Mesaros et al.
reaction mixture was incubated at 60 °C for 48 h. Samples were
placed on ice for 30 min, and the DNA was precipitated by adding
ice-cold ethanol (1050 µL) followed by ice-cold 2.9 M sodium
acetate (35 µL). The samples were centrifuged at 4000g for 15
min, and the supernatant was removed. The DNA pellet was washed
with ethanol/water (1 mL, 7:3 v/v), and residual solvent was
removed by evaporation under nitrogen.
Enzymatic Hydrolysis of OETA-PC-Modified Calf
Thymus DNA. The modified DNA pellet was dissolved in 1 mL
of 10 mM MOPS containing 100 mM NaCl (pH 7.0) by sonication
for 5 min. DNase I (556 units) dissolved in 200 µL of 10 mM
Figure 1. Mass spectrometric fragmentation of OETA-PC.
then performed immediately after homogenization using the method
of Bligh and Dyer (30). The extract was dried under a stream of
nitrogen, sealed in a vial under argon, and kept at -80 °C for 24 h
before being analyzed by LC-MS.
2
MOPS containing 120 mM MgCl (pH 7.0) was added, and the
sample was incubated for 90 min at 37 °C. At the end of this
incubation, 50 µL of 10 mM MOPS containing 100 mM NaCl (pH
Quantitative Analysis of PLs. The same LC-MS system used
for qualitative analysis was used here. The MRM transitions used
to detect the oxPL were the mass to charge ratio (m/z) for the
7
.0) and 15.5 units of nuclease P1 was added followed by 25 mM
ZnCl
2
(50 µL). The incubation was then continued for 2 h at 37
+
°C. Finally, 30 units of alkaline phosphatase in 0.5 mL of 0.4 M
MOPS (pH 7.8) was added, and this was followed by an additional
molecular cation [M + H] and their respective daughter ion. Mass
transitions, m/z 718.3f 184.5 for OETA-PC (Figure 1) and m/z
1
-h incubation.
759.1f184 for PL-PC, were monitored simultaneously. Calibration
Isolation of DNA Adducts from Modified Calf Thymus
curves for quantitative analysis of OETA-PC and PL-PC were
constructed by adding a fixed amount of internal standard (DM-
PE) into various amounts of authentic samples. The chromatograms
are presented in Figure 2 (vide infra). Equations describing the
calibration curves are presented in Table 1 (vide infra). Although
we did not have the authentic standard, we also monitored
theoretical ion pairs parent/daughter for the corresponding acid of
OETA-PC, that is, in which the carboxaldehyde has been oxidized
to a carboxyl group: m/z 734.5f184. When standard become
available, these data can be used to provide identification and
quantification of the acid. Calculation of trans-OETA-PC levels in
DNA. The hydrolysate was applied directly to an SPE cartridge (1
g, 6 mL, Supelclean) that had been prewashed with acetonitrile
(
15 mL) followed by water (15 mL). The cartridge was washed
with water (4 mL) and methanol/water (1 mL; 5:95, v/v). Etheno-
dGuo and etheno-dAdo were eluted in acetonitrile/water (6 mL;
5
0:50, v/v). The elutes were evaporated to dryness under nitrogen
and dissolved in water (100 µL). LC/MS analysis was conducted
on a 20 µL aliquot of this solution using gradient system 1.
Results
rat retina is as follows: n
A
) y/(nst)(a)(EE), where n ) mole
A
analyte, y ) peak area ratio, a ) from calibration curves (y ) ax),
Synthesis of trans-OETA-PC. To facilitate studies of the
generation, chemistry, and biology of OETA-PC, we engaged
in a synthetic program targeting this product. The route
established is short and utilizes straightforward steps that are
easy to execute. The synthetic target was envisioned to be
available from the acid 1 via esterification of 2-lyso-phosphati-
dylcholine. Intermediate 1 could be generated from an allylic
alcohol 2 by epoxidation of the E double bond and chain
homologation (Scheme 3). In turn, intermediate 2 might be
assembled through alkylation of an acetylide nucleophile,
derived from propargyl alcohol, with a hydroxyl-protected
derivative of the commercially available bromohydrin 3.
Previously, Goerger and Hudson (26) reported a total
synthesis of parinaric acid, starting from 9-decen-1-ol. In five
rather elaborate steps, they obtained the intermediate 2, starting
from the bromo-alcohol 3. We arrived at the same intermediate
in only two steps in 92% overall yield. The very convenient,
one-pot reaction developed by Patterson (25) for the conversion
of terminal acetylenic alcohols into (E)-olefinic alkenols,
succeeded when we used a 2:1 ratio of propargyl alcohol to the
electrophile 3 to generate the allylic alcohol 2. This protocol
produced exclusively the requisite E isomer of 2 (Scheme 4)
as expected.
n
st ) moles of internal standard used, and EE ) extraction efficiency
(
from Table 1).
Reaction of OETA-PC with dAdo, dGuo, and Calf
Thymus DNA. Liquid Chromatography. Chromatography for LC/
MS/UV experiments was performed using a Waters Alliance 2690
HPLC system (Waters Corp.) and a Hitachi L-4200 UV detector
at 231 nm. Gradient system 1 consisted of a Synergi Polar RP
column (250 mm × 4.6 mm i.d., 4 µm; Phenomenex) at a flow
rate of 1 mL/min. Solvent A was 5 mM ammonium acetate in water,
and solvent B was 5 mM ammonium acetate in acetonitrile. The
linear gradient (gradient 1) was as follows: 6% B at 0 min, 6% B
at 2 min, 10% B at 15 min, 80% B at 25 min, 80% B at 35 min,
6
% B at 37 min, and 6% B at 45 min. Separations were performed
at room temperature.
Mass Spectrometry. Mass spectrometry was conducted using
a Thermo Finnigan LCQ ion trap mass spectrometer (Thermo
Finnigan, San Jose, CA) equipped with an APCI source in the
positive ion mode. The LCQ operating conditions were as follows:
vaporizer temperature at 450 °C, heated capillary temperature at
1
50 °C, with a discharge current of 5 µA applied to the corona
needle. Nitrogen was used as the sheath (80 psi) and auxiliary (5
arbitrary units) gas to assist with nebulization. Full scanning
analyses were performed in the range of m/z 100 to m/z 800.
Collision-induced dissociation (CID) experiments coupled with
n
multiple tandem mass spectrometry (MS ) employed helium as the
The allylic hydroxyl of 2 was protected as an acetate and the
primary alcohol was deprotected to 5 via established procedures
(24). The alcohol 5 was then oxidized and deprotected during
workup to provide the acid 6 in one step, as the only product
collision gas. The relative collision energy was set at 20% of the
maximum (1 V).
Reaction of OETA-PC with DNA Bases. A solution of OETA-
PC (1.44 mg, 2 µmol) in ethanol (36 µL) was added to dGuo (2.67
mg, 10 µmol) or dAdo (2.52 mg, 10 µmol) in Chelex-treated 100
mM phosphate buffer (pH 7.4, 314 µL). After sonication for 15
min, the reaction mixture was incubated at 60 °C for 48 h. The
samples were filtered through a 0.2 µm Costar cartridge prior to
analysis of a portion of the sample (20 µL) by LC/MS using gradient
system 1.
Preparation of OETA-PC-Modified Calf Thymus DNA. Calf
thymus DNA (500 µg, 1.68 µmol) was dissolved in 100 mM
phosphate buffer (pH 7.4, 314 µL) and treated with OETA-PC (1.44
mg, 2 µmol) in ethanol (36 µL). After sonication for 15 min, the
(
by NMR). The conversion of the intermediate acetoxy acid to
the hydroxy acid 6 was the serendipitous result of acidifying
the reaction mixture to pH 1 using 6 M HCl. This catalyzed
solvolysis of the allylic acetate group. In anticipation of the
Wittig homologation, we had envisioned a route involving
protection of the acid with simultaneous hydrolysis of the allylic
acetate (13). We decided, however, to carry on with the acid 6,
thus saving two steps. Protection of the acid functionality as an
ester proved indeed to be unnecessary (31). Epoxidation of

A 13-Oxo-9,10-epoxytridecenoate Phospholipid
Chem. Res. Toxicol., Vol. 23, No. 3, 2010 521
Figure 2. LC/ESI-MS/MS analysis of OETA-PC from oxidized PL-PC liposomes. (A) Synthetic OETA-PC standard (parent m/z 718.3), MRM
chromatogram (daughter m/z 184.5). (B) OETA-PC detected in oxidized PL-PC liposomes, MRM chromatogram (718.3f184.5).
Scheme 3. Strategy for Synthesis of trans-OETA-PC
-80 °C until they were analyzed (usually not more than 24 h).
The extracts were analyzed by LC/ESI-MS/MS. Preliminary
identification of product peaks was achieved by comparing
retention times with authentic samples. The authentic sample
of OETA-PC, synthesized as described above, was characterized
by high-resolution mass spectrometry and multinuclear NMR.
Positive ion ESI-MS/MS analysis of the authentic lipid yielded
both parent and daughter ions that were used as the specific
mass transition ion pairs in LC/ESI-MS/MS analysis. A common
daughter ion m/z 184 was produced in all samples, as found
for all other phosphatidylcholines detected previously in our
lab. The retention time and the parent/daughter ion pair were
collectively used to identify a peak in the oxidation reaction
product mixture chromatograms.
Oxidized PL-PC liposomes from all three experiments
produced the expected oxidatively truncated OETA-PC (Figure
2
) in various yields. A single peak with a retention time that is
allylic alcohol 6 with a freshly prepared solution of dimethyl-
dioxirane in acetone (27) was completed in 30 min at room
temperature and gave pure trans-epoxide 7 quantitatively after
removal of the solvents and volatile byproducts under reduced
pressure. Oxidation of the free alcohol in 7 with TPAP/NMO
identical to the trans-OETA-PC standard (Figure 2A) is present
in the MRM chromatogram of PL-PC oxidation product
mixtures (Figure 2B, reconstructed MRM chromatograms),
demonstrating the generation of OETA-PC in these oxidation
experiments.
(
28) proceeded in excellent yield to afford the aldehyde 8.
To further confirm the identity of the peak detected, the
product mixtures of oxidation of PL-PC liposomes were treated
with methoxylamine hydrochloride to derivatize the aldehyde
functional group. Methoxime derivatization of an aldehyde
carbonyl results in a net mass increase of 29 Da. Methoxime
derivatives of pure OETA-PC were prepared to provide
authentic samples for analyzing product mixtures from PL-PC
oxidation. Each derivative was purified using a C18 mini-column
and then analyzed by LC-MS in the positive ion mode.
Methoxime derivatization of the synthetic standard of OETA-
PC resulted in the expected m/z 747.9, which corresponds to a
methoxime modification (29 Da) of the aldehyde group of
OETA-PC (m/z 718.3) (Figure 3A). In addition, an unexpected
bis-methoxylamine derivative (m/z 794.7) was produced, in
which both the aldehyde and the epoxy group of OETA-PC
are modified by methoxylamine (Figure 3C,D).
Epoxyalkenal acid 1 was obtained in good yield from the
aldehyde-epoxy-acid 8 by a Wittig reaction (29). The DMAP/
DCC-promoted coupling of the acid 1 with lyso-PC gave trans-
OETA-PC in 87% yield.
Production of OETA-PC in Vitro from PL-PC. To
demonstrate that OETA-PC can be produced by oxidation of
unsaturated PLs in vitro, PL-PC liposomes were oxidized under
various reaction conditions. In one experiment, the liposomes
were irradiated with 350 nm UV light in phosphate-buffered
saline (PBS) at room temperature in the air. In parallel
experiments, PL-PC liposomes were oxidized with copper(II)
-
or peroxynitrate produced from a MPO-NO
2
-glucose/glucose
oxidase system (7). To minimize transition metal-induced
oxidation, the buffer was supplemented with a metal chelator,
diethylenetriaminepentaacetic acid (DTPA). The oxidation reac-
tion mixture was sampled at various times. Catalase was added
to quench the reactions by removing hydroperoxides from the
samples. To prevent further autoxidation of the samples during
processing and storage, BHT was also added. Lipids were
extracted using a slightly modified Bligh and Dyer method (30)
as described in the experimental part, dried under a nitrogen
stream at room temperature, stored under argon in the dark at
Both derivatives display a common daughter ion m/z 184.5
(C H15NO P). The bis-methoxylamine derivative of OETA-PC
5 4
is presumably generated through opening of the epoxy ring.
Typical MRM chromatograms of oxidized PL-PC liposomes
after derivatization are presented in Figure 2. Before derivati-
zation, no peaks corresponding to derivatized standards were

5
22 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Mesaros et al.
Scheme 4. Synthesis of OETA-PC
observed (data not shown). After derivatization, new peaks
corresponding to derivatized standards appeared with a parallel
disappearance of the peak corresponding to OETA-PC.
Evolution Profiles of OETA-PC from Oxidation of
PL-PC. Quantification of OETA-PC in reaction product mix-
tures from oxidation of PL-PC was achieved by LC/ESI-MS/
MS in the positive ion mode using the MRM function.
Appropriate [M + H]+ and a common daughter ion m/z 184
were monitored. Calibration curves were produced using an
authentic sample of OETA-PC prepared by the unambiguous
total synthesis described above and commercially available PL-
PC. Accurate determination of the amounts of these PLs was
achieved by a microphosphorous assay. Standard curves were
generated by incorporating a fixed amount of DM-PE as internal
standard and various amounts of the analyte and plotting the
peak area ratio versus analyte mole ratio. Trace amounts of
OETA-PC were detected in the commercial PL-PC. The
amounts of OETA-PC detected in the commercial PL-PC were
subtracted from the initial values of the oxidation product. The
consumption of PL-PC was greatest under UV light (Figure 4),
Figure 3. LC/ESI-MS/MS analysis of OETA-PC derivatives. (A) The methoxime of OETA-PC standard (parent m/z 747.9), MRM chromatogram
daughter m/z 184). (B) The methoxime of OETA-PC detected in derivatized oxidized PL-PC liposomes, MRM chromatogram (747.9f184.5). (C)
(
The bis-methoxylamine adduct from OETA-PC standard (parent m/z 794.7), MRM chromatogram (daughter m/z 184). (D) The bis-methoxylamine
adduct of OETA-PC detected in derivatized oxidized PL-PC liposomes, MRM chromatogram (794.7f184.5).

A 13-Oxo-9,10-epoxytridecenoate Phospholipid
Chem. Res. Toxicol., Vol. 23, No. 3, 2010 523
PUFAs in retina, we expected that the peroxidation of PL-PC
in retina would produce OETA-PC as found in our in vitro
experiments. Other oxidized PCs (oxPCs), derived from PL-
PC, were identified previously in rat retina (3). To examine if
OETA-PC is present in intact retinas, retinas harvested from
albino rats were analyzed by LC-ESI/MS/MS. The retinas from
five rats were harvested. To prevent contamination by blood,
or in vitro oxidation, the retinas were rinsed with a saline
antioxidant cocktail (PBS, DTPA, and BHT). The retinas were
immediately homogenized manually in a plastic vial using a
stainless steel pestle coated with Teflon. Lipid extraction using
the procedure of Bligh and Dyer (30) was then performed
immediately after homogenization. The extracted lipids were
analyzed by LC-ESI/MS/MS in the positive ion mode. After
the solvents were removed, the residue was redissolved in
chloroform:methanol (1:1), one-fifth of the sample was injected
into the LC-MS. OETA-PC, and the standard DM-PE was
monitored simultaneously. Figure 6B presents the reconstructed
MRM chromatogram showing the presence of OETA-PC in rat
retina. The retention time (17.1 min), parent ion, and daughter
ion were collectively used to identify LC-ESI/MS/MS peak by
comparison with the authentic standard (Figure 6A) as described
above for monitoring the generation of OETA-PC during in vitro
oxidation of PL-PC.
Figure 4. Consumption of PL-PC under various oxidation conditions.
Data are the average of two sets of independent experiments.
Multiple peaks are present in the chromatogram of the rat
retina sample (Figure 6B). A peak at 17.1 min has the same
retention time and MRM as the authentic OETA-PC (Figure
6B). Because they produce a 184.5 Da fragment, the other peaks
in Figure 6B apparently correspond to PCs that are isomeric
with OETA-PC. To further verify the structure of the HPLC
peak presumed to be OETA-PC, the sample from the rat retina
was derivatized with methoxylamine hydrochloride (Figure S20
of the Supporting Information).
Before reaction with methoxylamine, no peaks corresponding
to the mono and bis methoxylamine derivatives appeared (Figure
S21 of the Supporting Information). After derivatization, new
peaks appeared, corresponding to the mono and bis methoxy-
lamine adducts with a concomitant disappearance of the peak
corresponding to OETA-PC. The chromatogram of a methoxime
derivative of pure OETA-PC, prepared as described in the
previous paragraph, is presented in Figure S20A of the Sup-
porting Information. A poorly resolved peak in the chromato-
gram of the derivatized rat retina extract (Figure S20B of the
Supporting Information) exhibited the same net increase of 29
Da and retention time in the MRM as the derivatized standard.
This corresponds to the addition of one molecule of methoxy-
lamine and loss of water to generate a methoxime derivative of
the aldehyde group. A well-resolved peak in the chromatogram
of the derivatized rat retina extract (Figure 7D) exhibited the
same additional increase of 47 Da and retention time MRM as
the standard bis adduct (Figure S20C of the Supporting
Information). This corresponds to the addition of a second
molecule of methoxylamine by nucleophilic attack on the
epoxide group.
Figure 5. Evolution profile for production of OETA-PC from aerial
oxidation of PL-PC liposomes promoted by Cu(II), UV, or MPO at 37
°
C. Quantification was achieved with LC/ESI-MS/MS. The yields were
calculated by dividing the amount of each analyte by the amount of
starting PL-PC. Data are the average of two sets of independent
experiments.
a
Table 2. Amounts of OETA-PC Per Rat Retina
animal
OETA-PC (pmol) average*
1
2
3
4
5
0.27 ( 0.01
0.42 ( 0.32
0.47 ( 0.30
0.20 ( 0.30
0.30 ( 0.08
a
Amounts are the average of four measurements from both eyes of
same animal.
but the yield of OETA-PC was higher when the vesicles were
-
oxidized with the MPO-NO
Figure 5).
The product evolution profiles for oxidation promoted by the
2
-glucose/glucose oxidase system
(
-
The absolute amounts of OETA-PC detected in five rats are
presented in Table 2. The values were calculated employing a
calibration curve and extraction coefficient determined by LC/
ESI-MS/MS as described in the experimental part.
2
MPO-NO -glucose/glucose oxidase system and UV are similar,
rising steadily in the first 2 h and more slowly afterward. The
Cu(II) oxidation resulted in a much lower yield of OETA-PC.
This may be because Cu(II) not only catalyzes the oxidation of
PL-PC (6) but also promotes further oxidation of OETA-PC to
other products, for example, the corresponding carboxylic acid.
Table 2 shows the maximum yields for OETA-PC produced
by different oxidation methods.
The average amount of OETA-PC in rat retina, 0.33 pmol,
is relatively low, as compared to oxPCs derived from DHA-
PC, for example, HOHA-PC (2.5 pmol) or 4-keto-7-oxohept-
5-enoic acid (KOHA)-PC (1.7 pmol). In part, this is because
PL-PC is present in lower amounts (3) than DHA-PC in the rat
retina (see Table 3).
Quantification of OETA-PC in Rat Retina. Because many
reports indicated that light damage induces the peroxidation of

5
24 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Mesaros et al.
Figure 6. LC/ESI-MS/MS detection of OETA-PC (718.3f184.5) from rat retina. (A) Synthetic standard OETA-PC. (B) Rat retina extract.
Figure 7. LC-APCI/MS analysis of reaction between OETA-PC and dGuo at 60 °C for 48 h.
a
Table 3. Amounts of Some PLs Detected Per Rat Retina (3)
together with residual dAdo at a retention time of 12.8 min
Figure 8). The LC/MS characteristics of this adduct were
(
PL
absolute amount (nmol)
6
identical to authentic 1,N -etheno-dAdo (19). Its mass spectrum
PA-PC
PL-PC
DHA-PC
HOHA-PC
KOHA-PC
2.64 ( 0.32
2.06 ( 0.32
+
+
exhibited an intense MH at m/z 276, together with a BH
2
ion
2
+
7.0 ( 0.24
at m/z 160. MS analysis of MH m/z 276 gave rise to exclusive
+
0.0025 ( 0.0023
0.0017 ( 0.0012
formation of the BH
2
product ion at m/z 160.
Formation of Etheno-dGuo and Etheno-dAdo in Calf
Thymus DNA. Calf-thymus DNA was treated with an ap-
proximately equimolar amount of OETA-PC. The DNA was
then hydrolyzed with a mixture of DNase I, nuclease P1, and
alkaline phosphatase in MOPS buffer. These hydrolysis condi-
tions were shown to give essentially quantitative recovery of
normal DNA bases. Modified DNA bases were separated from
normal bases using the SPE procedure described in the Materials
and Methods. Etheno-dGuo and etheno-dAdo were detected in
the DNA hydrolysate (Figure 9). On the basis of a 71% recovery
through the extraction and hydrolysis procedure, the signals for
etheno-dGuo and etheno-dAdo corresponded to 3.5 and 5.5
adducts/10 (7) normal bases, respectively.
a
Amounts are the average of four measurements from both eyes of
the same animal.
Nucleotide Etheno Adduct Formation by OETA-PC.
Reaction with dAdo. A solution of OETA-PC was added to
dGuo or dAdo in Chelex-treated phosphate buffer, and the
mixture was incubated for 48 h at 60 °C. LC/APCI/MS analysis
of the reaction product mixture from dGuo after 48 h revealed
the presence of one major adduct together with residual dGuo.
The dGuo adduct eluted at a retention time of 15.5 min in system
(Figure 7). Its APCI mass spectrum revealed an intense MH
at m/z 292, together with a BH
MH (m/z 292) resulted in a BH
LC/MS characteristics are identical to those for authentic1,N -
etheno-dGuo (19).
Reaction with dGuo. LC/APCI/MS analysis of the reaction
mixture from dAdo after 48 h using gradient system 1 revealed
the presence of one dAdo adduct (retention time, 21.0 min)
+
1
+
+
2
2
ion at m/z 176. MS analysis
+
2
product ion at m/z 176. These
2
Discussion
An efficient, practical, total synthesis was accomplished for
OETA and OETA-PC starting from a commercially available

A 13-Oxo-9,10-epoxytridecenoate Phospholipid
Chem. Res. Toxicol., Vol. 23, No. 3, 2010 525
Figure 8. LC-APCI/MS analysis of reaction between OETA-PC and dAdo at 60 °C for 48 h.
Figure 9. LC-APCI/MS analysis of reaction of OETA-PC with calf thymus DNA at 60 °C for 48 h.
2
Scheme 5. Plausible Mechanism for Formation of 1,N -Etheno-dGuo
bromo-alcohol. This synthesis provides ready access to adequate
amounts of the pure lipids needed for biological studies. The
pure OETA-PC served as a standard that enabled the identifica-
tion of this epoxyaldehyde in the oxidation product mixture from

5
26 Chem. Res. Toxicol., Vol. 23, No. 3, 2010
Mesaros et al.
PL-PC initiated via three different methods: Cu(II), MPO, and
UV light. The MPO treatment gave the highest yield (3.4%),
while the Cu(II) oxidation produced OETA-PC in the lowest
amount (0.2%). Because the yield obtained by UV induction
was higher (2.4%) than the one by Cu(II), we considered that
OETA-PC might be formed in vivo in UV-exposed organs, for
example, the eye. OETA-PC was also identified in vivo in the
lipid extract of the rat retina. The presence of numerous lipids
in this extract that apparently react with 1 and 2 equiv of
methoxylamine (see Figure S20B,D of the Supporting Informa-
tion) is the reasonable consequence of the formation of a mixture
of stereo- and regioisomers during the free radical-induced
oxidation of LA-PC in vivo. Our synthetic standard is the pure
trans-9,10-epoxide. Both cis- and trans-3,5-EDE are formed
upon oxidative fragmentation of LA hydroperoxides (32). It is
highly likely that cis-OETA-PC is also present among the
oxidatively truncated PLs present in retina.
R01EY016813, and R01CA91016 and by a State of Ohio Third
Frontier BRTT Award TECH05-064.
Supporting Information Available: 1H and 13C NMR
spectra of new compounds and a description of the statistical
data analysis for rat retina MS quantification of OETA-PC. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(
1) Greenberg, M. E., Li, X. M., Gugiu, B. G., Gu, X., Qin, J., Salomon,
R. G., and Hazen, S. L. (2008) The lipid whisker model of the structure
of oxidized cell membranes. J. Biol. Chem. 283, 2385–2396.
2) Podrez, E. A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M.,
Finton, P. J., Shan, L., Gugiu, B., Fox, P. L., Hoff, H. F., Salomon,
R. G., and Hazen, S. L. (2002) Identification of a novel family of
oxidized phospholipids that serve as ligands for the macrophage
scavenger receptor CD36. J. Biol. Chem. 277, 38503–38516.
3) Sun, M., Finnemann, S. C., Febbraio, M., Shan, L., Annangudi, S. P.,
Podrez, E. A., Hoppe, G., Darrow, R., Organisciak, D. T., Salomon,
R. G., Silverstein, R. L., and Hazen, S. L. (2006) Light-induced
oxidation of photoreceptor outer segment phospholipids generates
ligands for CD36-mediated phagocytosis by retinal pigment epithelium:
A potential mechanism for modulating outer segment phagocytosis
under oxidant stress conditions. J. Biol. Chem. 281, 4222–4230.
4) Podrez, E. A., Byzova, T. V., Febbraio, M., Salomon, R. G., Ma, Y.,
Valiyaveettil, M., Poliakov, E., Sun, M., Finton, P. J., Curtis, B. R.,
Chen, J., Zhang, R., Silverstein, R. L., and Hazen, S. L. (2007) Platelet
CD36 links hyperlipidemia, oxidant stress and a prothrombotic
phenotype. Nat. Med. 13, 1086–1095.
(
(
(
OETA-PC forms mutagenic etheno adducts upon reaction
with dAdo and dGuo, as well as with the DNA. We did not
look for the DNA adducts in the rat retina. The DNA adducts
are not formed in high yields from OETA-PC and DNA (3.5
7
or 5.5 adducts/10 normal bases). OETA-PC is also not very
abundant in the retina, so we did not expect to be able to detect
these adducts. Future studies will look for the DNA adducts
extracted from blood or epithelial cell lines, which endocytose
lipoproteins, since PL-PC is one of the major constituents of
LDL. Furthermore, the detection of the DNA adducts in vivo
will most likely require additional enhancement in the sensitiv-
ity, possibly by immunoafinity purification. A plausible mech-
anism for the reaction of OETA-PC with dAdo or dGuo to give
(
5) Ashraf, M. Z., Kar, N. S., Chen, X., Choi, J., Salomon, R. G., Febbraio,
M., and Podrez, E. A. (2008) Specific oxidized phospholipids inhibit
scavenger receptor b1-mediated selective uptake of cholesteryl esters.
J. Biol. Chem. 283, 10408–10414.
(
6) Kaur, K., Salomon, R. G., O’Neil, J., and Hoff, H. F. (1997)
(Carboxyalkyl)pyrroles in human plasma and oxidized low-density
lipoproteins. Chem. Res. Toxicol. 10, 1387–1396.
6
2
the unsubstituted etheno-dAdo adduct 1,N -etheno-dAdo or 1,N -
etheno-dGuo (see Scheme 2) is presented in Scheme 5 for the
case of dGuo.
(
7) Gu, X., Sun, M., Gugiu, B., Hazen, S., Crabb, J. W., and Salomon,
R. G. (2003) Oxidatively truncated docosahexaenoate phospholipids:
total synthesis, generation, and peptide adduction chemistry. J. Org.
Chem. 68, 3749–3761.
Condensation of the aldehyde group of OETA-PC with the
primary amino group of dGuo generates the corresponding
Schiff base. Then, S i attack of a nitrogen nucleophile in the
N
(
8) Ebrahem, Q., Renganathan, K., Sears, J., Vasanji, A., Gu, X., Lu, L.,
Salomon, R. G., Crabb, J. W., and Anand-Apte, B. (2006) Carboxy-
ethylpyrrole oxidative protein modifications stimulate neovascular-
ization: Implications for age-related macular degeneration. Proc. Natl.
Acad. Sci. U.S.A. 103, 13480–13484.
corresponding imidic acid tautomer opens the allylic epoxide
to generate an allylic alcohol. A Grob fragmentation (a
vinylogous retroaldol reaction) of this intermediate delivers
etheno-dGuo, 9-oxononanoyl-PC, and acetylene. The energy
released because of aromatization of the tricyclic purine-derived
(9) Hollyfield, J. G., Bonilha, V. L., Rayborn, M. E., Yang, X., Shadrach,
K. G., Lu, L., Ufret, R. L., Salomon, R. G., and Perez, V. L. (2008)
Oxidative damage-induced inflammation initiates age-related macular
degeneration. Nat. Med. 14, 194–198.
10) Deng, Y., and Salomon, R. G. (1998) Total synthesis of γ-hydroxy-
R,ꢀ-unsaturated aldehydic esters of cholesterol and 2-lysophosphati-
dylcholine. J. Org. Chem. 63, 7789–7794.
11) Subbanagounder, G., Deng, Y., Borromeo, C., Dooley, A. N., Berliner,
J. A., and Salomon, R. G. (2002) Hydroxy alkenal phospholipids
regulate inflammatory functions of endothelial cells. Vasc. Pharmacol.
(
(
2
base in 1,N -etheno-dGuo and the favorable entropy of this
fragmentation reaction,that is, decomposition of the allylic
alcohol intermediate into three molecules, facilitate the cleavage
of two C-C bonds (19). The proposed pathway is in agreement
with the finding that 4,5-EDE is stable under the reaction
conditions in the absence of dGuo or dAdo (19).
3
8, 201–209.
(
12) Podrez, E. A., Poliakov, E., Shen, Z., Zhang, R., Deng, Y., Sun, M.,
Finton, P. J., Shan, L., Febbraio, M., Hajjar, D. P., Silverstein, R. L.,
Hoff, H. F., Salomon, R. G., and Hazen, S. L. (2002) A novel family
of atherogenic oxidized phospholipids promotes macrophage foam cell
formation via the scavenger receptor CD36 and is enriched in
atherosclerotic lesions. J. Biol. Chem. 277, 38517–38523.
13) Sun, M., Deng, Y., Batyreva, E., Sha, W., and Salomon, R. G. (2002)
Novel bioactive phospholipids: Practical total syntheses of products
from the oxidation of arachidonic and linoleic esters of 2-lysophos-
phatidylcholine. J. Org. Chem. 67, 3575–3584.
As noted above, the relatively polar acyl chains of oxidatively
truncated PLs protrude from membranes into the aqueous phase.
This may contribute to their uniquely high susceptibility to
lipolysis, for example, by platelet-activating factor-acetylhy-
drolase, which hydrolyzes the truncated acyl groups of oxPCs
in addition to platelet-activating factor (33, 34). Therefore, it
can be anticipated that the formation of OETA-PC in vivo will
be accompanied by the release of the free acid OETA that is
also expected to react with DNA to produce mutagenic etheno
derivatives. Linoleic-containing PC is the major PL in lipopro-
teins, and thus, in cells that endocytose oxidized lipoproteins,
OETA-PC may be an important DNA-damaging agent.
(
(
14) Spiteller, G. (1998) Linolei acid peroxidatin- the dominant lipid
peroxidation process in low density lipoprotein and its relationship to
chronic diseases. Chem. Phys. Lipids 95, 105–162.
(15) Kamitani, H., Geller, M., and Eling, T. (1998) Expression of
5-lipoxygenase by human colorectal carcinoma Caco-2 cells during
1
apoptosis and cell differentiation. J. Biol. Chem. 242, 21569.
16) Hamberg, M., and Samuelson, B. (1967) Oxygenation of unsaturated
fatty acids by the vesicular gland of sheep. J. Biol. Chem. 242, 5366.
17) Lee, S. H., Oe, T., and Blair, I. A. (2001) Vitamin C-induced
decomposition of lipid hydroperoxides to endogenous genotoxins.
Science 292, 2083–2085.
(
(
Acknowledgment. We thank Mary Rayborn and Xiarong Gu
for harvesting rat retinas. We are grateful for support of this
work by National Institutes of Health Grants R01GM21249,
(
18) Levine, M., Conry-Cantilena, C., Wang, Y., Welch, R. W., Washko,
P. W., Dhariwal, K. R., Park, J. B., Lazarev, A., Graumlich, J. F.,

A 13-Oxo-9,10-epoxytridecenoate Phospholipid
Chem. Res. Toxicol., Vol. 23, No. 3, 2010 527
King, J., and Cantilena, L. R. (1996) Vitamin C pharmacokinetics in
healthy volunteers: Evidence for a recomended dietary allowance.
Proc. Natl. Acad. Sci. U.S.A. 93, 3704–3709.
(27) Adam, W., and Hadjiarapoglou, L. (1993) Dioxiranes: Oxidation
chemistry made easy. Top. Curr. Chem. 164, 45–62.
(28) Kobierski, M. E., Kim, S., Murthi, K. K., Iyer, R. S., and Salomon,
R. G. (1994) Synthesis of a pyrazole isostere of pyrroles formed by
the reaction of the amino groups of protein lysyl residues with
levuglandin E2. J. Org. Chem. 59, 6044–6050.
(
19) Lee, S. H., Oe, T., and Blair, I. A. (2002) 4,5-Epoxy-2(E)-decenal-
induced formation of 1,N6-etheno-2′-deoxyadenosine and 1,N2-etheno-
2
′-deoxyguanosine adducts. Chem. Res. Toxicol. 15, 300–304.
(
29) Katsuki, T., Lee, A. W. M., Ma, P., Martin, V. S., Masamune, S.,
Sharpless, K. B., Tuddenham, D., and Walker, F. J. (1982) Synthesis
of saccharides and related polyhydroxylated natural products. 1. Simple
alditols. J. Org. Chem. 47, 1373–1378.
(
(
20) Marnett, L. (2000) Oxyradicals and DNA damage. Carcinogenesis
1, 361–370.
2
21) Johnson, T. M., Yu, Z., Ferrans, V. J., Lowenstein, R. A., and Finkel,
T. (1996) Reactive oxygen species are downstream mediators of p53-
dependent apoptosis. Proc. Natl. Acad. Sci. U.S.A. 93, 11848–11852.
22) Burcham, P. C. (1998) Genotoxic lipid peroxidation products: their
DNA damaging properties and role in formation of endogenous DNA
adducts. Mutagenesis 13, 287–305.
23) Dittmer, J. C., and Lester, R. L. (1964) A simple, specific spray for
the detection of phospholipids on thin-layer chromatograms. J. Lipids
Res. 5, 126–127.
24) Miyashita, M., Yoshikoshi, A., and Grieco, P. (1977) Pyridinium
p-toluenesulfonate. A mild and efficient catalyst for the tetrahydro-
pyranylation of alcohols. J. Org. Chem. 42, 3772–3774.
25) Patterson, J. (1984) A convenient one-pot procedure for the conversion
of terminal acetylenic alcohols (and O-derivatives) into (E)-olefinic
alcohols (or derivatives). Synthesis 337.
26) Goerger, M. M., and Hudson, B. S. (1988) Synthesis of all trans-
parinaric acid-d8 specifically deuterated in the vinyl positions. J. Org.
Chem. 53, 3148–3153.
(
(
(
30) Bligh, E., and Dyer, W. (1959) Lipids extraction. Can. J. Biochem.
Physiol. 37, 911–917.
31) Nicolaou, K. C., Roschangar, F., and Vourloumis, D. (1998) Chemical
biology of epothilones. Angew. Chem. Int. Ed. 37, 2014–2045.
32) Kumazawa, K., Wada, Y., and Masuda, H. (2006) Characterization
of epoxydecenal isomers as potent odorants in black tea (Dimbula)
infusion. J. Agric. Food Chem. 54, 4795–4801.
(
(
(
(
(
(
33) Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre,
T. M. (1996) Mammalian platelet-activating factor acetylhydrolases.
Biochim. Biophys. Acta 1301, 161–173.
(
34) Subramanian, V. S., Goyal, J., Miwa, M., Sugatami, J., Akiyama, M.,
Liu, M., and Subbaiah, P. V. (1999) Role of lecithin-cholesterol
acyltransferase in the metabolism of oxidized phospholipids in plasma:
Studies with platelet-activating factor-acetyl hydrolase-deficient plasma.
Biochim. Biophys. Acta 1439, 95–109.
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