An 1O2 route to γ-hydroxyalkenal phospholipids by vitamin E-induced fragmentation of hydroperoxydiene-derived endoperoxides

Article abstract of DOI:10.1021/tx200093m

Biologically active phospholipids that incorporate an oxidatively truncated acyl chain terminated by a γ-hydroxyalkenal are generated in vivo. The γ-hydroxyalkenal moiety protrudes from lipid bilayers like whiskers that serve as ligands for the scavenger receptor CD36, fostering endocytosis, e.g., of oxidatively damaged photoreceptor cell outer segments by retinal pigmented endothelial cells. They also covalently modify proteins generating carboxyalkyl pyrroles incorporating the ε-amino group of protein lysyl residues. We postulated that γ-hydroxyalkenals could be generated, e.g., in the eye, through fragmentation of hydroperoxy endoperoxides produced in the retina through reactions of singlet molecular oxygen with polyunsaturated phospholipids. Since phospholipid esters are far more abundant in the retina than free fatty acids, we examined the influence of a membrane environment on the fate of hydroperoxy endoperoxides. We now report that linoleate hydroperoxy endoperoxides in thin films and their phospholipid esters in biomimetic membranes fragment to γ-hydroxyalkenals, and fragmentation is stoichiometrically induced by vitamin E. The product distribution from fragmentation of the free acid in the homogeneous environment of a thin film is remarkably different from that from the corresponding phospholipid in a membrane. In the membrane, further oxidation of the initially formed γ-hydroxyalkenal to a butenolide is disfavored. A conformational preference for the γ-hydroxyalkenal, to protrude from the membrane into the aqueous phase, may protect it from oxidation induced by lipid hydroperoxides that remain buried in the lipophilic membrane core.

Full text of DOI:10.1021/tx200093m

ARTICLE
pubs.acs.org/crt
An 1O2 Route to γ-Hydroxyalkenal Phospholipids by Vitamin
E-Induced Fragmentation of Hydroperoxydiene-Derived
Endoperoxides
Xiaodong Gu, Wujuan Zhang, Jaewoo Choi, Wei Li, Xi Chen, James M. Laird, and Robert G. Salomon*
Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106, United States
S Supporting Information
b
ABSTRACT: Biologically active phospholipids that incorpo-
rate an oxidatively truncated acyl chain terminated by a
γ-hydroxyalkenal are generated in vivo. The γ-hydroxyalkenal
moiety protrudes from lipid bilayers like whiskers that serve as
ligands for the scavenger receptor CD36, fostering endocytosis,
e.g., of oxidatively damaged photoreceptor cell outer segments
by retinal pigmented endothelial cells. They also covalently
modify proteins generating carboxyalkyl pyrroles incorporating
the ε-amino group of protein lysyl residues. We postulated that
γ-hydroxyalkenals could be generated, e.g., in the eye, through
fragmentation of hydroperoxy endoperoxides produced in the retina through reactions of singlet molecular oxygen with
polyunsaturated phospholipids. Since phospholipid esters are far more abundant in the retina than free fatty acids, we examined
the influence of a membrane environment on the fate of hydroperoxy endoperoxides. We now report that linoleate hydroperoxy
endoperoxides in thin films and their phospholipid esters in biomimetic membranes fragment to γ-hydroxyalkenals, and
fragmentation is stoichiometrically induced by vitamin E. The product distribution from fragmentation of the free acid in the
homogeneous environment of a thin film is remarkably different from that from the corresponding phospholipid in a membrane. In
the membrane, further oxidation of the initially formed γ-hydroxyalkenal to a butenolide is disfavored. A conformational preference
for the γ-hydroxyalkenal, to protrude from the membrane into the aqueous phase, may protect it from oxidation induced by lipid
hydroperoxides that remain buried in the lipophilic membrane core.
’ INTRODUCTION
The retina is especially vulnerable to oxidative damage owing
to its high proportion of polyunsaturated fatty acyls (PUFAs),
high concentration of oxygen, and chronic exposure to light.
Exposure of rats to intense visible light results in the consump-
tion of PUFAs in the retina and the production of oxidatively
truncated phosphatidylcholines (oxPCs)2 and phosphatidyletha-
nolamines (oxPEs).11 Lipid oxidation can involve free radical,
enzymatic, or singlet oxygen pathways. Ample evidence supports
the premise that photo generated singlet oxygen contributes to
oxidative injury in the eye. Light initiates an action potential by
inducing isomerization of an 11-cis to an all-trans retinal-protein
Schiff base in rhodopsin. This photosensitive receptor is reset
through hydrolysis of the Schiff base releasing all-trans retinal
that is reduced to all-trans retinol, and, through isomerization,
oxidation, and, condensation with opsin, the initial Schiff base is
regenerated.12 However, before it is reduced to retinol, especially
under conditions of oxidative stress where NADH levels are
depleted, all-trans retinal can be excited to its triplet state that can
transfer energy to molecular oxygen to give singlet oxygen.13,14
Phospholipids that incorporate an oxidatively truncated acyl
chain terminated by a γ-hydroxyalkenal functional array are
generated in vivo by oxidative fragmentation of polyunsaturated
phospholipids. The γ-hydroxyalkenal moiety protrudes from lipid
bilayers like whiskers1 that serve as ligands for the scavenger receptor
CD36 (for a figure summarizing the structures of phospholipids and
fatty acids mentioned in this article, see Supplementary In-
formation), fostering endocytosis of oxidatively damaged photo-
receptor cell outer segments by retinal pigmented endothelial cells.2
The γ-hydroxyalkenal moiety also covalently modifies proteins
generating carboxyalkyl pyrroles (Figure 1) that incorporate the
ε-amino group of protein lysyl residues.3,4 Carboxyethyl pyrroles
(CEPs) are especially abundant in retinas from individuals with age-
related macular degeneration (AMD).5 They trigger toll-like recep-
tor-mediated angiogenesis into and destruction of the retina, known
as wet AMD, causing rapid loss of vision.6ꢀ8 They also trigger an
immune-mediated destruction of the retina known as dry AMD.
Thus, mice immunized with CEP-modified mouse albumin develop
a dry AMD-like phenotype that includes subretinal pigment epithe-
lium (RPE) deposits and RPE lesions mimicking geographic
atrophy.9 Apparently γ-hydroxyalkenal-derived oxidative protein
modifications, e.g., CEPs, participate in the pathogenesis of AMD.10
Received: March 1, 2011
Published: May 13, 2011
r
2011 American Chemical Society
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Figure 1. Oxidative cleavage of polyunsaturated fatty acyl (PUFA) phosphatidylcholines generates γ-hydroxyalkenal phosphatidylcholines that react
with proteins to deliver carboxyalkyl pyrroles.
A reaction of singlet oxygen with linoleic acid (LA) generates
the unconjugated hydroperoxyoctadecadienoate (10- and 12-
HPODE) regioisomers and the conjugated hydroperoxydienes
9- and 13-HPODE (Figure 2). Through further reaction with
singlet oxygen, 10- and 12-HPODE are transformed into the
dihydroperoxydienes 9,12- and 10,13-diHPODE15 that can
undergo fragmentation to give γ-hydroxyalkenals.15ꢀ17 A reac-
tion of singlet oxygen with 9- and 13-HPODE delivers hydro-
peroxy endoperoxides (9- and 13-HP-Endo).18 The present
study was undertaken to determine if PUFA hydroperoxy
endoperoxides undergo fragmentation to γ-hydroxyalkenals.
Furthermore, because phospholipid esters are far more abundant
than free fatty acids, it seemed pertinent to examine the influence
of a membrane environment on the fate of hydroperoxy
endoperoxides. We now report that linoleate hydroperoxy
endoperoxides in thin films and their phospholipid esters in
biomimetic membranes fragment to give γ-hydroxyalkenals that
can be oxidized further to the corresponding butenolides (vide
infra). Vitamin E promotes the fragmentation, and the product
distribution from fragmentation of the free acid in the homo-
geneous environment of a thin film is remarkably different from
that from the corresponding phospholipid in a model membrane.
’ EXPERIMENTAL PROCEDURES
Figure 2. Singlet molecular oxygen reaction pathways to γ-hydroxyalk-
enals. Further oxidation produces butenolides in competition with
adduction to proteins to generate carboxyalkyl pyrroles.
General Methods. See Supporting Information.
Liqid Chromatograph/Mass Spectrometry. LC/ESI/MS/MS
analysis of hydroperoxy endoperoxide fragmentation was performed on
a Quattro Ultima (Micromass, Wythenshawe, UK) connected to a
Waters 2690 solvent delivery system with an autoinjector. The source
temperature was maintained at 100 °C, and the desolvation temperature
was kept at 200 °C. The drying gas (N2) was maintained at ca. 450 L/h,
and the core flow gas was kept at ca. 50 L/h. The multiplier was set at an
absolute value of 500. Optimized parameters for hydroperoxy endoper-
oxides and their fragmentation products were obtained with authentic
samples. MS scans at m/z 80ꢀ1000 were obtained for standard
compounds (Table S1 in Supporting Information).
Argon was used as collision gas at a pressure of 5 psi for MS/MS
analysis. For MS/MS analysis, the collision energy was optimized for
each compound. For selected reaction monitoring (SRM) experiments,
the optimum collision energy (giving the strongest signal) was deter-
mined for each m/z parentꢀdaughter ion pair (Table S2 in Supporting
Information). FOA (8-(furan-2-yl)octanoic acid) did not give daughter
fragments during collision and, therefore, was monitored by selected ion
recording (SIR, m/z = 209).
Online chromatographic separation was achieved using a 150 ꢁ
2.0 mm i.d. Prodigy ODS-5 μm column (Phenomenex, Torrance, CA),
using binary solvent gradients, water/acetonitrile or water/methanol
supplemented with 0.2% formic acid or 2 mM NH4OAc. For the
carboxylic acids, the water/acetonitrile gradient started with 35%
acetonitrile for 10 min, then rose to 60% in 0.5 min, and linearly rose
to 75% in 15 min. The gradient was then reversed to 35% acetonitrile in
0.5 min and held for 6 min. For HODA methoxime derivatives, the
methanol/water gradient, supplemented with 2 mM NH4OAc, started
with 5% methanol and rose to 100% methanol in 15 min, then was held
at 100% methanol for another 15 min. The gradient was then reversed to
5% methanol in 0.5 min and held at 5% methanol for 4.5 min before the
next run. For ODFO-PFB ester derivatives, the methanol/water gradi-
ent, supplemented with 2 mM NH4OAc, started with 50% methanol and
rose to 100% methanol in 10 min, then was held at 100% methanol for
another 15 min. The gradient was then reversed to 50% methanol in
0.5 min and held for 4.5 min before the next run. For the phospholipids,
the methanol/water gradient, supplemented with 0.2% formic acid,
started with 85% methanol and rose to 100% in 15 min, then was held at
100% methanol for another 15 min. The gradient was then reversed to
85% methanol in 0.5 min and held at 85% methanol for 4.5 min before
the next run. The solvents were all delivered at 200 μL/min.
(9Z,11E)-13-(2-Methoxypropan-2-ylperoxy)octadeca-9,11-
dienoic Acid (13-HP-MiP). 13-HPODE (120 mg, 0.38 mmol) and
pyridinium p-toluene sulfonate (PPTs, 10 mg) were dissolved in dry
CH2Cl2 (10 mL). Then 2-methoxypropene (100 μL, excess) was added
dropwise via a syringe. The resulting mixture was stirred for 0.5 h under
argon. Then solvents were removed by rotary evaporation, and the crude
product was then flash chromatographed on silica gel eluting with hexane
and ethyl acetate (2/1) to afford the pure 13-HP-MiP (114 mg, 30 mmol,
yield = 78%); Rf = 0.38 with hexane and ethyl acetate (1/1). 1H NMR
(400 MHz, CDCl3): δ 6.41 (dd, J = 15.6, 10.8, 1H), 5.92 (t,
J = 10.8, 1H), 5.53 (dd, J = 15.2, 8, 1H), 5.37 (dt, J = 10.8, 7.6, 1H),
4.33 (m, 1H), 3.23 (s, 3H), 2.27 (t, J = 7.6 Hz, 2H), 2.11 (m, 2H),
1.50ꢀ1.65 (2H), 1.10ꢀ1.40 (22H), 0.81 (t, J = 6.8, 3H). 13C NMR
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(400 MHz, CDCl3): δ 180.3, 133.1, 133.0, 128.2, 128.1, 104.8, 84.9, 49.4,
34.3, 33.3, 32.0, 29.7, 29.3, 29.2, 27.9, 25.3, 24.8, 23.2, 23.0, 22.7, 14.3.
HRMS (FAB) m/z [M þ Na]þ calcd for C22H40O5Na, 407.2774; found,
407.2783.
158.4, 120.4, 84.4, 33.7, 32.9, 29.1, 29.0, 28.9, 24.8, 24.8. HRMS (FAB)
m/z [M þ H]þ calcd for C12H19O4, 227.1283; found, 227.1274. ESI-
MS/MS analysis yielded the following diagnostic fragments: m/z 225.2
[M ꢀ H]ꢀ; m/z 207, 181, 136, 110, 97 (see Figure 6B).
1-Hexadecanoyl-2-(8-(6-(1-(2-methoxypropan-2-ylpero-
xy)hexyl)-3,6-dihydro-1,2-dioxin-3-yl)octanoyl)-sn-glycero-
3-phosphocholine (13-HP-Endo-PC-MiP). A solution of 13-HP-
PC-MiP (24 mg, 0.027 mmol) and tetraphenylporphine (TPP, 4 mg) in
CH2Cl2 (50 mL) was cooled to 0 °C in a pyrex photoreaction apparatus.
Oxygen was bubbled through the solution through a gas dispersion tube,
and the mixture was illuminated by an internal tungsten lamp (500 W)
inside an internal annular cold finger for 96 h. Solvent was removed
by rotary evaporation, and the crude product was purified by HPLC
using a 250 ꢁ 10.00 mm luna 5 C18(2) semipreparative column
(Phenomenex, Torrance, CA). The column was eluted with metha-
nol/water (100:5, v/v) containing 5 mM NH4OAc at 5 mL/min. The
fractions were collected and analyzed by ESI-MS on a Thermo Finnigan
LCQ DecaXP instrument in the positive mode using nitrogen as the
sheath and auxiliary gas. The heated capillary temperature was 200 °C,
the source voltage was 4.50 kV, and the capillary voltage was 20.00 V.
13-HP-PC-MiP (9 mg, 0.01 mmol) was recovered, and the title
compound 13-HP-Endo-PC-MiP was obtained (9 mg, 0.01 mmol, yield =
8-(6-(1-(2-Methoxypropan-2-ylperoxy)hexyl)-3,6-dihydro-
1,2-dioxin-3-yl)octanoic Acid (13-HP-Endo-MiP). A solution of
13-HP-MiP (142 mg, 0.37 mmol) and tetraphenylporphine (TPP, 9 mg)
in CH2Cl2 (60 mL) was cooled to 0 °C in a pyrex photoreaction
apparatus. Oxygen was bubbled through the solution through a gas
dispersion tube and the mixture illuminated by an internal tungsten lamp
(500 W) inside an internal cold finger condenser for 72 h. Then the solvent
was removed by rotary evaporation, and the crude product was flash
chromatographed on silica gel eluting with hexane and ethyl acetate (1/1)
to get 13-HP-Endo-MiP (102 mg, 0.25 mmol, yield = 68%); Rf = 0.36. 1H
NMR (400 MHz, CDCl3): δ 5.90ꢀ6.10 (m, 2H), 4.40ꢀ4.90 (2H),
4.00ꢀ4.20 (1H), 3.14 (s, 3H), 2.32 (t, J = 7.2 Hz, 2H), 1.20ꢀ1.70 (26H),
0.80ꢀ0.90 (3H). HRMS (FAB) m/z [M þ Na]þ calcd for C22H40O7Na,
439.2672; found, 439.2674.
8-(6-(1-Hydroperoxyhexyl)-3,6-dihydro-1,2-dioxin-3-yl)-
octanoic Acid (13-HP-Endo). 13-HP-Endo-MiP (20 mg, 0.048 mmol)
and pyridinium p-toluene sulfonate (PPTs, 2 mg) were dissolved in dry
methanol (5 mL). The mixture was stirred under argon for 24 h. The
solvent was removed by rotary evaporation, and the crude product purified
by flash chromatography on silica gel eluting with hexane and ethyl acetate
(1/1) to give 13-HP-Endo (9 mg, 0.032 mmol, yield = 67%); Rf = 0.31. 1H
NMR (400 MHz, CDCl3): δ 5.90ꢀ6.05 (2H), 4.40ꢀ4.80 (2H),
4.05ꢀ4.21 (1H), 2.35 (t, J = 7.2 Hz, 2H), 1.44ꢀ1.64 (6H), 1.43ꢀ1.20
(14H), 0.80ꢀ0.96 (3H). HRMS (FAB) m/z [M þ H]þ calcd for
C18H33O6, 345.2277; found, 345.2274. ESI-MS/MS analysis yielded the
following diagnostic fragments: m/z 343.2 [M ꢀ H]ꢀ, m/z 213, 209, 207,
185, 125, 109, 99 (see Supporting Information).
1
58%). H NMR (400 MHz, CDCl3): δ 5.90 (m, 2H), 5.15 (s, 1H),
3.60ꢀ4.80 (11H), 3.30 (s, 9H), 3.25 (s, 3H), 2.16ꢀ2.27 (4H),
1.80ꢀ2.00 (4H), 1.00ꢀ1.60 (42 H), 0.70ꢀ0.90 (12H). ESI-MS analysis
yielded the following diagnostic peaks: m/z 894.9 [M þ H]þ, m/z 916.8
[M þ Na]þ, m/z 823.0 [M þ HꢀMiP]þ, m/z 1788.3 [2M þ H]þ (see
Figure S4 in the Supporting Information). HRMS (FAB) m/z [M þ H]þ
calcd for C46H89NO13P, 894.6071; found, 894.6059.
1-Hexadecanoyl-2-(8-(6-(1-hydroperoxyhexyl)-3,6-dihydro-
1,2-dioxin-3-yl)octanoyl)-sn-glycero-3-phosphocholine
(13-HP-Endo-PC). 13-HP-Endo-PC-MiP (7.6 mg, 0.0085 mmol) and
pyridinium p-toluene sulfonate (PPTs, 3 mg) were dissolved into 5 mL
of chloroform/methanol (1/1) and the solution stirred under argon
for 24 h. The solvents were then removed by rotary evaporation. The
residue was dissolved into water (10 mL) and extracted with chloro-
form (3 ꢁ 10 mL) to remove the PPTs. The crude product was
further purified by HPLC using a 250 ꢁ 4.60 mm luna 5 C18(2)
column (Phenomenex, Torrance, CA). The column was eluted with
methanol/water (90/10, v/v) containing 5 mM NH4OAc at 1 mL/min.
13-HP-Endo-PC was obtained (4 mg, 0.0049 mmol, yield = 58%).
HRMS (FAB) m/z [M þ H]þ calcd for C42H81NO12P, 822.5496;
found, 822.5456. ESI-MS/MS analysis yielded the following diagnostic
fragments: m/z 822.8 [M þ H]þ, m/z 184.1 (see Supporting
Information).
13-HP-Endo was further purified by HPLC using a 250 ꢁ 4.6 mm
luna 5 μm C18(2) column (Phenomenex, Torrance, CA), with a binary
solvent (water and acetonitrile) gradient at 1 mL/min. The gradient
started with 60% acetonitrile and rose to 100% acetonitrile linearly in
30 min. The gradient was then reversed to 60% acetonitrile in 0.5 min
and then held for 4.5 min at 60% acetonitrile.
8-(2-Hydroxy-5-oxo-2,5-dihydrofuran-2-yl)octanoic Acid
(HODFO). To a magnetically stirred solution of NaH2PO4 (110 mg,
0.81 mmol) and NaClO2 (80% purity, 180 mg, 1.59 mmol) in 5 mL
t-BuOH/water (5:1) was added FOA (112 mg, 0.53 mmol) in 5 mL
t-BuOH/water (5:1) solution dropwise via a syringe. The resulting
mixture was stirred at room temperature for 1.5 h. Solvents were
removed by rotary evaporation, and the crude product was purified by
flash chromatography on silica gel eluting with methanol and chloroform
(1:10) to give the pure HODFO (102.8 mg, 0.425 mmol, yield = 80%);
1-Hexadecanoyl-2-(8-(5-oxo-2,5-dihydrofuran-2-yl)oct-
anoyl)-sn-glycero-3-phosphocholine (ODFO-PC). ODFO
(24 mg, 0.11 mmol), 1-hexadecanoyl-2-hydroxy-sn-glycero-3-phos-
phocholine (lyso-PC, 40 mg, 0.08 mmol), dicyclohexylcarbodiimide
(DCC, 100 mg, 0.48 mmol), and 4-dimethylaminopyridine (DMAP,
10 mg, 0.08 mmol) were dissolved in dry chloroform (5 mL) and the
solution stirred at room temperature for 72 h. Solvents were removed
by rotary evaporation to get the crude product, and the residue was
purified on a silica gel column (CHCl3/CH3OH/H2O = 16/9/1) to
1
Rf = 0.25 with 15% methanol in chloroform. H NMR (400 MHz,
CDCl3): δ 7.10ꢀ7.22 (1H), 6.07 (d, J = 6.4 Hz, 1H), 2.24ꢀ2.38 (2H),
1.85ꢀ2.05 (2H), 1.50ꢀ1.65 (2H), 1.20ꢀ1.40 (8H). HRMS (FAB) m/z
[M þ Na]þ calcd for C12H18O5Na, 265.1052; found, 265.1028.
8-(5-Oxo-2,5-dihydrofuran-2-yl)octanoic Acid (ODFO).
HODFO (16 mg, 0.066 mmol) was dissolved in methanol (3 mL),
and the mixture was cooled in an ice bath. NaBH4 (15 mg, excess) was
added in small portions, and the resulting mixture was stirred for 1 h at
room temperature. Water (100 μL) was then added, and the mixture was
stirred for another 1 h. Then 1 M HCl was added to acidify the solution
to pH 3, and the resulting mixture was stirred at room temperature for 30
min. The product was extracted into CHCl3 (10 mL ꢁ 3) and solvents
evaporated. Chromatography on silica gel eluting with 5% CH3OH in
CHCl3 delivered pure ODFO (12 mg, 0.053 mmol, yield = 80%); Rf =
0.25 with 10% methanol in chloroform. 1H NMR (400 MHz, CDCl3): δ
7.38 (dd, J = 5.6, 1.6 Hz, 1H), 6.05 (dd, J = 6.0, 2.4 Hz, 1H), 4.97 (m,
1H), 2.28 (t, J = 7.6 Hz, 2H), 1.65 (m, 1H), 1.50ꢀ1.63 (3H), 1.30ꢀ1.42
(2H), 1.20ꢀ1.32 (6H). 13C NMR (400 MHz, CD3OD): δ 176.5, 174.6,
1
give pure ODFO-PC (30.2 mg, 0.04 mmol, yield = 50%). H NMR
(400 MHz, CDCl3/CD3OD): δ 7.58 (dd, J = 5.6, 1.6 Hz, 1H), 6.08
(dd, J = 6.0, 2.4 Hz, 1H), 5.18 (m, 1H), 5.07 (m, 1H), 4.38 (dd, J = 12,
3.2 Hz, 1H), 4.20 (m, 2H), 4.12 (dd, J = 12, 6.4 Hz, 1H), 3.96 (t, J =
6.4 Hz, 2H), 3.57 (m, 2H), 3.20 (s, 9H), 2.32ꢀ2.20 (4H), 1.50ꢀ1.62
(4H), 1.20ꢀ1.40 (34H), 0.84(t, J = 6.8 Hz, 3H). HRMS (FAB) m/z
[M þ H]þ calcd for C36H67NO10P, 704.4502; found, 704.4503. ESI-
MS/MS analysis yielded the following diagnostic fragments: m/z
704.6 [M þ H]þ, m/z 184.1 (see Supporting Information).
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Thermal Decomposition of the Linoleate Derived Hydro-
peroxy Endoperoxide 13-HP-Endo. Small 3 dram glass vials, each
containing 13-HP-Endo (10 μg, 0.03 μmol) as a dry film, were incubated
at 37 °C for various times. After incubation, the vials were sealed under
argon and kept at ꢀ80 °C until LC-MS/MS analysis. For the decom-
position of 13-HP-Endo with vitamin E, vials containing 13-HP-Endo
(10 μg, 0.03 μmmol) and one equivalent of vitamin E as a dry film were
incubated at 37 °C for various times. After incubation, the vials were
sealed under argon and kept at ꢀ80 °C until LC-MS/MS analysis. Vials
containing 13-HP-Endo (10 μg, 0.03 μmmol) and various amounts of
vitamin E as a dry film were incubated at 37 °C for 2 h. After incubation,
the vials were sealed under argon and kept at ꢀ80 °C until LC-MS/MS
analysis. For the decomposition of 13-HP-Endo with vitamin E and
DTPA, vials containing 13-HP-Endo (10 μg, 0.03 μmmol) and various
amounts of vitamin E and 5 equivalents of DTPA as a dry film were
incubated at 37 °C for 2 h. After incubation, the vials were sealed under
argon and kept at ꢀ80 °C until LC-MS/MS analysis. Before analysis,
100 μL of internal standard solution (8-(furan-2-yl)-8-oxooctanoic acid,
1 ng/μL) in methanol was added, and 10 μL of this mixture solution was
injected into the LC/MS/MS system each time. The amounts of
HODA, ODFO, KODA, and FOA were monitored simultaneously.
9-Hydoxy-12-(methoxyimino)dodec-10-enoic Acid (HO-
DA Methoxime). To authentic HODA (50 μg) in a small vial was
added pyridine (200 μL) containing 2 wt % of methoxylamine hydro-
chloride. The mixture was kept under argon at room temperature over-
night. Pyridine was then removed under a stream of argon. The residue
was dissolved in 0.5 mL of water (pH 3) and extracted with ethyl ether
(1 mL and then 0.5 mL). Solvent was removed from the extract and the
residue analyzedvia ESI-MS/MS, producing thefollowingfragments:m/z
256.3 [M ꢀ H]ꢀ; m/z 197 (see Supporting Information).
Figure 3. Postulated fragmentation products from 13-HP-Endo.
of vitamin E were initially dissolvedinto methanol, vortexed for 1 min, and
the solvent removed under a stream of argon. Lipids were fully hydrated
by the addition of phosphate saline buffer (1 mL, 50 mM, pH 7.4) with or
without DTPA (100 μM). Following vortexing to disperse the hydrated
lipids, small unilamellar vesicles (SUVs) were generated by extrusion
through a polycarbonate filter until a clear solution was produced (about
15 times) using an Avanti Mini-Extruder Set (Avanti Polar Lipids,
Alabaster, AL). The buffer solution of vesicles was aliquoted into small
vials (100 μL in each) of a buffer solution containing 13-HP-Endo-PC
(10 μg), and the vials incubated at 37 °C for various times. After
incubation, DT-PC (100 ng) was added as internal standard, and the
mixture was immediately extracted by the Bligh and Dyer method. The
extracts were sealed under argon and stored in ꢀ80 °C until LC-MS/MS
analysis (less than 24 h).
Thermal Decomposition of Linoleate Derived Hydroper-
oxy Endoperoxide Phosphocholine Ester 13-HP-Endo-PC in
Homogeneous Solution. 13-HP-Endo-PC (10 μg), with SP-PC
(90 μg) and one equivalent of vitamin E, was dissolved in a homo-
geneous mixture of methanol (100 μL) and PBS buffer (100 μL, 50 mM,
pH 7.4). The vial was incubated at 37 °C for 4 h. After incubation, DT-
PC (100 ng) was added as internal standard, and the mixture was
immediately extracted by Bligh and Dyer method. The extract was
sealed under argon and stored in ꢀ80 °C until LC-MS/MS analysis (less
than 24 h).
13-HP-Endo (50 μg, 0.15 μmmol) and one equivalent of vitamin E
were incubated as a dry film at 37 °C for 2 h. Then pyridine (200 μL)
containing 2 wt % of methoxylamine hydrochloride was added, and the
mixture was kept under argon at room temperature overnight. Pyridine
was then removed under a stream of argon. The residue was then
dissolved in 0.5 mL of water (pH 3), and the solution was extracted with
ethyl ether (1 mL and then 0.5 mL). Solvent was then removed from the
extract, and the residue was sealed under argon and kept at ꢀ80 °C until
LC-MS/MS analysis.
Perfluorobenzyl 8-(5-oxo-2,5-dihydrofuran-2-yl)octano-
ate (ODFO-PFB Ester). To authentic ODFO (50 μg) in a small vial
was added dry acetonitrile (100 μL) containing 10 wt % of pentafluor-
obenzyl bromide and 20 wt % of N,N-diisopropylethylamine. The
resulting mixture was kept at room temperature for 2 h, and the solvent
was removed with a stream of argon. The residue was dissolved in 1 mL
of water, and the product was extracted into ethyl acetate (1 mL).
ESI-MS/MS analysis in the positive mode gave [M þ NH4]þ and
characteristic daughter fragments at m/z 209, 181, 163, 145, 135
(see Supporting Information).
13-HP-Endo (50 μg, 0.15 μmmol) and one equivalent of vitamin
E were incubated as a dry film at 37 °C for 2 h. Then dry acetonitrile
(100 μL) containing 10 wt % of pentafluorobenzyl bromide and 20 wt %
of N,N-diisopropylethylamine was added. The mixture was kept under
argon at room temperature for 2 h and solvents removed under a stream
of argon. The residue was dissolved in water (1 mL), and the solution
was extracted with ethyl acetate (1 mL). Solvent was removed from the
extract, and the residue was sealed under argon and kept at ꢀ80 °C until
LC-MS/MS analysis.
Thermal Decomposition of Linoleate Derived Hydroper-
oxy Endoperoxide Phosphocholine Ester 13-HP-Endo-PC in
a Model Membrane. Vesicles comprising 13-HP-Endo-PC and
vitamin E were prepared by extrusion by a method described previously.19
Specifically, 13-HP-Endo-PC (100 μg), with 1-stearoyl-2-palmitoyl-sn-
glycero-3-phosphocholine (SP-PC, 900 μg) as carrier, and one equivalent
’ RESULTS
Synthesis of a Linoleate-Derived Hydroperoxy Endoper-
oxide and Its Predicted Fragmentation Products. We postu-
lated that fragmentation of the hydroperoxy endoperoxide
13-HP-Endo would deliver the γ-hydroxyalkenal HODA
through fragmentation of an intermediate alkoxy radical by
β-scission in conjunction with homolysis of the dialkyl peroxide
and hydrogen atom abstraction to produce the γ-hydroxy alkenal
cis-HODA (Figure 3). The cis γ-hydroxy alkenal is expected to be
unstable. It can isomerize into the trans γ-hydroxy alkenal
HODA or be oxidized further to the lactone ODFO in the
oxidative environment. The unsaturated aldehyde HODA can
also be oxidized further, e.g., to deliver KODA (Figure 3).20,21 We
previously found that a HODA ester of 2-lysophosphatidylcholine
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Figure 4. Structurally specific synthesis of a 13-hydroperoxyend-
Figure 5. Synthesis of the butenolide ODFO.
operoxide.
MS/MS Similarity of Two Expected Products, KODA and
ODFO, from Fragmenation of 13-HP-Endo. LC-ESI/MS/MS
was chosen to monitor the reaction profile. This technique allows
simultaneous quantification of each specific product based on
selected reaction monitoring (SRM), i.e., monitoring unique
transitions between the mass-to-charge (m/z) ratio of the parent
ion ([M ꢀ H]ꢀ or [M þ H]þ) and characteristic daughter ions
for each species, as well as their characteristic LC retention time
that could be established using authentic samples prepared by
unambiguous syntheses. The technique gives specific peaks with
high signal-to-noise ratio and is suitable for quantification.
During the development of an MS/MS protocol for monitoring
the fragmentation of 13-HP-Endo, we found that ODFO has an
ESI-MS/MS spectrum similar to that of KODA. Both KODA and
ODFO have isobaric parent ions and nearly identical patterns of
daughter ions (Figure 6A and B). The slight differences between
the mass spectra of these two molecules are that KODA produces
a dominant daughter ion at m/z 109 and a weak daughter ion at
m/z 110, while ODFO gives a dominant daughter ion at m/z 110.
Possible fragments that may correspond to these daughter ions
are presented in Figure 6. Furthermore, KODA and ODFO
exhibited the same LC retention times when eluting with
methanol and water. Although the mass spectra of both mol-
ecules show peaks in the 225 f 110 and 225 f 109 channels
with reversed intensity, it is difficult to distinguish them in a
mixture (Figure 6C). We subsequently found that, with an
acetonitrile and water LC eluant system, these two compounds
are well separated and show the expected reversed intensity in
the two channels (Figure 6D). The LC-MS/MS similarity of
KODA and ODFO provides a good example that demonstrates
the importance of having authentic samples, obtained through
unambiguous chemical synthesis, to confirm the putative iden-
tities of molecules characterized by LC-MS/MS.
Vitamin E Promotes the Fragmentation the Hydroperoxy
Endoperoxide 13-HP-Endo. It was anticipated that vitamin E
(R-tocopherol), that can inhibit free radical-induced oxidation by
hydrogen atom transfer that neutralizes reactive radicals, would
prevent the oxidative destruction of HODA, e.g., to produce
ODFO and KODA. The linoleate-derived hydroperoxy endo-
peroxide 13-HP-Endo was incubated at 37 °C as a dry film in
open glass vials in the absence or in the presence of one
equivalent of vitamin E (R-tocopherol). The amounts of poten-
tial fragment products (HODA, ODFO, KODA, and FOA) were
monitored by LC-ESI/MS/MS (Figure 7). In the absence of
R-tocopherol, only a small amount of ODFO was slowly
generated during the incubation (yield <3%). The addition of
R-tocopherol greatly promotes the fragmentation of 13-HP-
Endo and increases the yields of HODA, ODFO, and FOA. A
large increase in the generation of HODA was found. The yield of
HODA increased from below 0.2% to more than 4%, a more
than 20-fold increase. The yield of ODFO also increased from
cyclizes and loses one molecule of water to give a PC ester of the
furan FOA under biomimetic reaction conditions presumably
through prior isomerization to cis-HODA.22
A structurally nonspecific synthesis of linoleate-derived hydro-
peroxy endoperoxides was accomplished previously by treating
a mixture of linoleate 9- and 13-hydroperoxyoctadecadienoate
(9-and 13-HPODE) withsinglet oxygen, generatedbygaseousO2
in the presence of a photosensitizer.18 Isolation of the pure hydro-
peroxy endoperoxides from the complex reaction product mix-
ture required tedious HPLC. However, the reaction with singlet
molecular oxygen is quite slow and requires many days of
irradiation to achieve a high conversion. This is problematic
because both 13-HPODE and 13-HP-Endo are not stable under
these conditions and decompose during prolonged reaction with
singlet molecular oxygen. Therefore, the yield is low, and much
effort is required to isolate pure 13-HP-Endo from the complex
reaction product mixture (Figure S1A, Supporting Information)
by HPLC. We designed a structurally specific synthesis of pure
13-HP-Endo (Figure 4) that proceeded in much higher yields
starting from a pure monohydroperoxide (13-HPODE) that is
available through an enzymatic peroxidation of linoleic acid.23 The
hydroperoxy group was protected by treatment with 2-methoxy-
propene and a catalytic amount of pyridinium p-toluene sulfonate
(PPTs).24 The R-methoxyisopropyl (MiP) protected hydroper-
oxy endoperoxide 13-HP-Endo-MiP was obtained after the reac-
tion of 13-HP-MiP with singlet molecular oxygen. Then PPTs-
catalyzed deprotection in methanol delivered the final product
13-HP-Endo. Introduction of the protecting group lengthens the
synthesis compared to the direct reaction of 13-HPODE with
singlet molecular oxygen. However, the extra steps are worthwhile
because the MiP protected hydroperoxides are quite stable under
the reaction conditions, and the desired product is obtained in
good yield from a relatively simple product mixture (Figure S1B,
Supporting Information). The MiP protecting group is also
valuable for the synthesis of a linoleate-derived hydroperoxy
endoperoxide phosphocholine ester (vide infra).
Authentic samples of several likely fragmentation products
9-hydroxy-12-oxo-10-dodecenoic acid (HODA), 9-keto-12-oxo-
10-dodecenoic acid (KODA), and 8-(2-furyl)octanoic acid(FOA)
were prepared as described previously.25ꢀ27 The butenolide, 8-(5-
oxo-2,5-dihydrofuran-2-yl)octanoic acid (ODFO), was prepared
from the furan FOA (Figure 5). Oxidation of FOA in the presence
of sodium chlorite in a slightly acidic aqueous solution at room
temperature generates hydroxy butenolide HODFO.28 The cyclic
forms of such hydroxy butenolide compounds are not stable and
can open to form 4-oxo-2 (Z) alkenoic acids depending on pH.28
Sodium borohydride can effectively reduce the free keto group
into a hydroxyl group and after cyclization under acidic conditions
gives the butenolide compound ODFO.29
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Figure 6. Negative ESI-MS/MS spectrum of KODA (A) and ODFO (B), and HPLC chromatogram of KODA and ODFO (1:1 mixture) eluting with
methanol/water (C) and acetonitrile/water (D). The chromatogram was monitored by LC-MS in the negative ion mode with SRM of appropriate mass
transitions as noted.
below 3% to more than 8%. Furthermore, in the presence of
R-tocopherol, the generation of these fragmentation products
was very fast. The yields of HODA and ODFO reach their
maxima in less than 2 h. The amount of HODA decreases during
further incubation due to additional oxidation and fragmenta-
tion. Compared with HODA, ODFO was relatively stable, and
the yield only slightly decreased during further incubation.
KODA is not a major product in the fragmentation of hydro-
peroxy endoperoxide 13-HP-Endo under these conditions. Only
small amounts of KODA were generated, possibly due to the
further oxidation of HODA, during the incubation (<0.2%) even
in the presence of R-tocopherol. The yield of the furan com-
pound FOA also increased with the addition of R-tocopherol and
reached its maximum after 4 h of incubation.
HODA and ODFO are two major products from the frag-
mentation of 13-HP-Endo in the presence of vitamin E. To verify
their identities, two derivatizations were applied. An authentic
sample of HODA and the fragmentation reaction product
mixture from 13-HP-Endo were both derivatized with methoxy-
lamine hydrochloride in pyridine. The methoxime derivative of
HODA ([M ꢀ H]ꢀ, m/z 256) gives a dominant daughter ion
m/z 197. For the HPLC analysis, the HODA methoxime deriva-
tive was detected by ESI-MS/MS using SRM monitoring of the
225 f 197 mass transition. Before derivatization, HODA was
detected in the reaction product mixture from 13-HP-Endo by
ESI-MS/MS using SRM monitoring of the 227 f 84 mass
transition. After derivatization, the HODA peak disappeared, and
the HODA methoxime derivative was detected (Figure S2,
Supporting Information). The generation of ODFO from 13-
HP-Endo was further confirmed by pentafluorobenzyl (PFB)
esterification. The ODFO-PFB ammonia adduct ([M þ 18]þ,
m/z 424) gives characteristic daughter ions with m/z 181 and
m/z 163. The HPLC chromatogram of ODFO-PFB was mon-
itored by ESI-MS/MS with SRM of the 424 f 181 mass
transition. Reaction with PFB-Br caused the disappearance of
ODFO. The concomitant formation of ODFO-PFB from the
putative ODFO generated by fragmentation of 13-HP-Endo was
confirmed through comparison with an authentic sample (Figure
S3, Supporting Information).
Vitamin E Stoichiometrically Induces the Decomposition
of 13-HP-Endo. To further understand the role of R-tocopherol
in the decomposition of the hydroperoxy endoperoxide, we
incubated 13-HP-Endo in the presence of various amounts of
R-tocopherol at 37 °C for 2 h as a dry film and monitored the
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Figure 7. Time courses for the generation of HODA, ODFO, KODA, and FOA during the thermal decomposition of 13-HP-Endo in the absence (open
symbols) or presence (solid symbols) of R-tocopherol (Vit E). Error bars are the deviation from average of duplicate runs within less than 24 h.
Figure 9. CML-bound redox-active metal ions may catalyze reductive
fragmentation of hydroperoxy endoperoxides to γ-hydroxyalkenals.
generate an alkoxyl radical. Such chemistry has not been reported
previously for R-tocopherol. However, it is pertinent to note that
vitamin C is reported to promote fragmentation of hydroper-
Figure 8. Formation of HODA and ODFO from 13-HP-Endo with
various amounts of R-tocopherol in the absence and presence of the
chelating agent diethene triamine pentaacetic acid (DTPA). Error bars
are the deviation from average of duplicate runs within less than 24 h.
oxides through a reductive homolysis pathway.30
However, reductive cleavage of hydroperoxides to generate
alkoxyl radicals can be induced by redox active metal ions, e.g.,
through a Fenton reaction. We previously showed that electron
transfer from R-tocopherol reduces Fe3þ to its Fe2þ that
then reductively cleaves hydroperoxides, and therefore, Fenþ
can catalyze the reductive homolysis of hydroperoxides
by R-tocopherol.31 Catalysis by redox active metal ions is
especially likely in vivo because, e.g., sugar-derived N-(carboxy-
yields of HODA and ODFO simultaneously (Figure 8). The
yields of HODA and ODFO are greatly increased by the addition
of R-tocopherol and plateau after the addition of one equivalent.
The stoichiometry is consistent with a mechanism in which
R-tocopherol transfers an electron to the hydroperoxy group to
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Figure 10. Possible redox metal ion catalyzed reductive homolysis of 13-HP-Endo by R-tocopherol.
Chart 1
methyl)lysine (CML) oxidative modifications in proteins bind
metal ions, and these protein-bound metal ions can catalyze
the oxidation of ascorbate by H2O2.32,33 Electron transfer from
(oxidation of) ascorbate or R-tocopherol would be accompanied
by electron transfer to (reduction of) metal ions. The reduced
metal ions would subsequently be reoxidized by electron transfer
to H2O2 or to lipid hydroperoxides. It is likely that, in the
presence of vitamin E, protein-bound redox active metal ions can
catalyze the fragmentation of hydroperoxy endoperoxides to
deliver toxic γ-hydroxyalkenals (Figure 9).
Figure 11. Formation of HODA and ODFO from 13-HP-Endo in the
presence of various amounts of R-tocopherol or γ-tocopherol.
larger than k1. This seems likely since the β-scission with
concomitant homolysis of a dialkyl hydroperoxide is expected
to be very favorable owing to the simultaneous formation of two
new carbonyl groups (Figure 3). This scenario is consistent with
the belief that R-tocopherol is not an efficient scavenger for
hydroxyl or alkoxyl radicals in vivo.35
Although metal ions were not added to the incubation of
hydroperoxy endoperoxide 13-HP-Endo, it is conceivable that, in
the presence of R-tocopherol, traces of redox active metal ions,
e.g., Fenþ, efficiently catalyze these reactions (Figure 10). As
proposed in Figure 10, metal ion-induced reductive cleavage of
the hydroperoxy endoperoxide would produce an alkoxyl radical
that can further fragment to a γ-alkoxyl-R,β-unsaturated alde-
hyde, which gives rise to HODA, ODFO, and FOA, or can be
trapped by hydrogen atom transfer to form the hydroxy endo-
peroxide 13-H-Endo. The competition between these alter-
natives would depend on the rate constants k1 and k2 and the
concentration of accessible hydrogen atom donors such as
R-tocopherol.34 We expected that an excess of vitamin E (up
to five equivalents) would reduce the yields of fragmentation
products because it would favor production of the hydroxy
endoperoxide. One possible explanation for the lack of a decrease
in the yields of those products is that the rate constant k2 is much
DTPA, a Metal Ion-Chelating Agent, Suppresses Vitamin
E-Induced Fragmentation of 13-HP-Endo. To test the hypoth-
esis that traces of redox active metal ions are required for vitamin
E-promoted fragmentation of 13-HP-Endo, diethylene triamine
pentaacetic acid (DTPA) was introduced into our system. It was
reported previously that after chelation, redox active metal ions
are relatively resistant to reduction and thus lose the ability to
react with R-tocopherol.36,37 If the fragmentation were metal ion
independent, the addition of DTPA would not affect the reac-
tion. We incubated a mixture of DTPA and the hydroperoxy
endoperoxide with various amounts of R-tocopherol at 37 °C for
2 h as a dry film. The addition of DTPA totally quenched the
generation of HODA and greatly suppressed the generation of
ODFO (Figure 8). This supports our hypothesis that the
fragmentation is metal ion-dependent.31 We favor this explana-
tion over the possibility that DTPA suppresses the direct
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Figure 12. Synthesis of a hydroperoxy endoperoxide phosphocholine
ester 13-HP-Endo-PC.
reduction of hydroperoxy endoperoxides by tocopherol in some
unprecedented alternative manner.
γ-Tocopherol-Promoted Decomposition of 13-HP-Endo
Causes Less Fragmentation than r-Tocopherol. The term
“vitamin E” designates a group of tocopherols and tocotrienols.
Among them, R-tocopherol has been most studied and is often
said to have the highest biological activity.38 However, recent
studies indicate that γ-tocopherol, the major form of vitamin E in
the US diet, has some unique biological activities, such as
trapping electrophilic mutagens,39 and thus may be important
to human health.38,40,41 It is also found that γ-tocopherol does
not reduce Cu(II) as rapidly as R-tocopherol, partly owing to its
lack of one methyl substituent on the chromanol headgroup
(Chart 1).35,42 Incubation of the hydroperoxy endoperoxide
13-HP-Endo with various amounts of γ-tocopherol at 37 °C
for 1.5 h as a dry film generated HODA and ODFO, but in lower
yields than with R-tocopherol (Figure 11). Presumably, the
lower reducing ability of γ-tocopherol results in less fragmenta-
tion products. This suggests the possibility that replacement of
R-tocopherol with γ-tocopherol may result in less pro-oxidant
effects in vivo.
Synthesis of Hydroperoxy Endoperoxide Phosphocholine
Ester 13-HP-Endo-PC. In vivo, polyunsaturated fatty acids
(PUFAs) are present mainly esterified, for example, in phospho-
lipids. Therefore, we prepared the hydroperoxy endoperoxide
phosphocholine ester 13-HP-Endo-PC through chemical synth-
esis (Figure 12). The 2-methoxyisopropyl (MiP) derivative of
13-HPODE, 13-HP-MiP, could be esterified to 2-lyso-PC in the
presence of dicyclohexylcarbodiimide (DCC) and 4-dimethyla-
minopyridine (DMAP).43 Then reaction with singlet molecular
oxygen, followed by removal of the MiP protecting group,
delivered the title compound 13-HP-Endo-PC. The MiP pro-
tecting group is very important for the overall success of the
synthesis because the reaction with singlet molecular oxygen
proceeds very slowly and was not complete even after 4 days of
irradiation. With the protecting group, the hydroperoxy endo-
peroxide derivative 13-HP-Endo-PC-Mip is easily separated
from the starting material, 13-HP-PC-MiP, and other oxidation
products (Figure S4, Supporting Information). 2-Methoxypro-
pene also has been used recently as the protecting group to
prepare other lipid hydroperoxides.44 The potential fragmenta-
tion product HODA-PC was prepared as previously described,25
and ODFO-PC was prepared through direct esterification
of ODFO.
Figure 13. Formation of HODA-PC and ODFO-PC from 13-HP-
Endo-PC in small unilamellar vesicles, in PBS buffer (50 mM), with one
equivalent of R-tocopherol (Vit E) in PBS buffer (50 mM), or with
one equivalent of R-tocopherol in PBS buffer (50 mM) with DTPA
(100 μM).
Fragmentation of 13-HP-Endo-PC in Unilamellar Vesicles.
To evaluate the fragmentation of hydroperoxy endoperoxide 13-
HP-Endo-PC in a model membrane under physiologically
relevant conditions, we incubated unilamellar vesicles compris-
ing 13-HP-Endo-PC (10 wt %) and 1-stearoyl-2-palmitoyl-sn-
glycero-3-phosphocholine (SP-PC) as carrier (1:9 respectively)
in pH 7.4 aqueous buffer at 37 °C for various times. Only a low
yield of ODFO-PC was generated (Figure 13), even after six
hours. When R-tocopherol is embedded in liposomes, the
chromanol head of vitamin E is close to the interface of the
aqueous phase and the bilayer membrane (Figure 14).45 Incuba-
tion of the above unilamellar vesicles containing R-tocopherol
(one equivalent), generated both HODA-PC and ODFO-PC in
greatly increased yields, reaching maxima of 7% and 9%, respec-
tively. The addition of DTPA effectively inhibited the generation
of both HODA-PC and ODFO-PC, even in the presence of
tocopherol. The effects of DTPA and tocopherol are similar to
those observed with the free acid 13-HP-Endo. In the present
study, it appears that redox active metal ions in the environment
catalyze the reductive cleavage of the hydroperoxide group
leading to fragmentation of the hydroperoxy endoperoxide
13-HP-Endo-PC under biomimetic conditions.
Membrane Environment Protects γ-Hydroxyalkenals
against Oxidation to Butenolides. The product distribution
between γ-hydroxyalkenal (HODA and HODA-PC) and their
butenolide oxidation products (ODFO and ODFO-PC) exhibits
interesting differences. Fragmentation of 13-HP-Endo in the
homogeneous environment of a dry film gave HODA and ODFO
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Figure 14. Fragmentation of 13-HP-Endo-PC in unilamellar vesicles. Octanol/water partition coefficients (log P) for R-tocopherol and the methyl
esters of the oxidized PUFAs, calculated by ChemDraw, are shown as a measure of relative hydrophobicity.
in a 1:2ꢀ3 ratio. In contrast, the fragmentation of 13-HP-Endo-
PC in a model membrane delivered HODA-PC and ODFO-PC
in about a 1:1 ratio. Apparently, a membrane environment
protects the γ-hydroxyalkenal against oxidation to a butenolide.
To further test this hypothesis, we incubated 13-HP-Endo-PC
(10 wt %), SP-PC (90 wt %), and one equivalent of R-tocopherol
in a homogeneous solution in methanol and PBS buffer (1:1) at
37 °C for 4 h. The yields of HODA-PC and ODFO-PC are
presented in Figure 15 and are compared to the yields obtained
from the corresponding reaction in a model membrane (SUV).
In homogeneous solution, the product distribution from the
fragmentation of 13-HP-Endo-PC is very similar to that obtained
from the fragmentation of 13-HP-Endo (the free acid) in a dry
film, and not like that obtained from 13-HP-Endo-PC in a model
membrane. This supports the hypothesis that the membrane
environment protects HODA-PC against further oxidation.
of Figure 3 (see above), fragmentation of the hydroperoxy
endoperoxide generates an unstable cis-γ-hydroxyalkenal, which
can isomerize into a trans-γ-hydroxyalkenal or be converted,
through further oxidation, into a butenolide. Notably, the ratio of
γ-hydroxyalkenal to butenolide will depend on the oxidative
environment. The lipid whisker model1,46,47 predicts segregation
of the γ-hydroxyalkenal from lipid hydroperoxide oxidants in the
membrane environment, and this favors the accumulation of
HODA-PC at the expense of ODFO-PC. Thus, when cellular
membranes undergo peroxidation, lipid hydroperoxides, e.g.,
13-HPODE-PC (Figure 14), remain buried in the hydrophobic core
of the membrane. However, the PUFA side chain containing the
hydroperoxide becomes more hydrophilic after further oxidation
and truncation, and moves from the interior of the lipid bilayer to
the aqueous exterior. Consequently, the membrane grows lipid
whiskers on its surface. The model predicts that the oxygenated
acyl groups of 13-HP-Endo-PC and its precursor 13-HPODE-
PC prefer to remain embedded in the lipophilic core of the
membrane bilayer (because they are relatively hydrophobic as
indicated by the octanol/water partition coefficients log P = 4.85
’ DISCUSSION
Membrane Environment Compartmentalizes Lipid Oxida-
tion Intermediates and Products. As detailed in the mechanism
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Figure 16. Possible mechanism that links elevated carboxymethyllysine
(CML) accumulation with the generation of carboxyethylpyrroles
(CEPs) in the retina of AMD eyes.
been noted previously and (2), as found in the present study, by
promoting fragmentation of oxidized lipids to generate toxic
aldehydes that can, inter alia, modify proteins by covalent
adduction. A pro-oxidant effect of vitamin E, i.e., owing to H
atom abstraction by the tocopheryl radical, is known,52 but in vivo
this pathway may be inhibited by coantioxidants, e.g., ascorbate.
It was proposed that a deficiency of coantioxidants rather than
R-tocopherol-mediated peroxidation alone may be responsible
for lipoprotein lipid peroxidation in the aortic subendothelial
space.53 Indeed, in several animal models of atherosclerosis,
administration of bisphenol, which is a kinetically inferior
classical radical scavenger, yet efficient coantioxidant that can
neutralize tocopheryl radicals, dramatically lowered aortic lipo-
protein lipid peroxidation.53
Vitamins C and E can reduce metal ions that, paradoxically,
can lead to oxidation of PUFAs. For example, a prooxidant effect
of R-tocopherol may arise from either metal-catalyzed or direct
electron transfer to lipid hydroperoxides. The resulting reductive
cleavage generates hydroxyl and/or alkoxyl radicals that can
initiate free radical chain reactions between PUFAs and oxygen.
In other words, R-tocopherol might promote the oxidation of
PUFAs by oxygen because it can cause single electron reduction
of hydroperoxides and consequently generate free radical initia-
tors of autoxidation. It was reported that R-tocopherol reduces
Cu(II) to Cu(I) and concomitantly induces autoxidation in
human lipoproteins.54ꢀ60 This prooxidant effect of vitamin E
was presumed to be “mediated by its capability to reduce Cu2þ to
Cuþ which, in turn, produces, from lipid hydroperoxides, the
highly reactive alkoxyl radicals”.55 Similarly, H2O2 was previously
shown to generate hydroxyl radicals and oxidize ascorbate ∼4
times faster in the presence of proteins from a cataractous lens
than similar proteins from a normal lens. This is presumably
because the endogenous level of Cu is significantly higher in a
cataractous lens than in a normal lens. Ascorbate oxidizing
activity at 24 h of crystallins, a structural lens protein, was
significantly higher for crystallins isolated from cataractous lenses
than from normal eyes. The metal ion chelator DTPA completely
prevented this oxidation up to 24 h of incubation. Presumably,
the oxidation of ascorbate reduces Cu(II) to Cu(I) that then
reductively cleaves H2O2 to generate hydroxyl radicals.
Figure 15. Formation of HODA-PC and ODFO-PC from 13-HP-
Endo-PC in methanol and buffer homogeneous solution and in small
unilamellar vesicles.
and 5.59), while the more polar γ-hydroxyalkenal acyl group (log
P = 1.59) of HODA-PC prefers to extend into the aqueous phase
(Figure 14). cis-HODA-PC (see Figure 14) partitions between
isomerization to the trans isomer and oxidation to ODFO-PC.
Oxidation would be disfavored in the membrane environment if
lipid hydroperoxides, e.g., 13-HP-Endo-PC and 13-HPODE-PC,
are required for the oxidation because the hydroperoxide of
13-HP-endo-PC and the γ-hydroxyalkenal acyl group of HODA-
PC are localized in different phases, i.e., membrane hydrophobic
core and aqueous phase, respectively. In contrast, the reaction of
13-HP-Endo-PC in an aqueous homogeneous environment
(methanol/PBS, 1:1) or the reaction of 13-HP-Endo in the
homogeneous environment of a dry film favors oxidation of cis-
HODA-PC to ODFO-PC or of cis-HODA to ODFO because
both cis-HODA and 13-HP-Endo or their PC esters are in the
same phase.
To understand the free-radical-induced oxidative transforma-
tions of PUFAs in vivo, it is essential to investigate their chemistry
when they are incorporated into phospholipids in membranes
because the chemistry of the free fatty acids does not recapitulate
their behavior in membranes. The structure and composition of
the membrane environment profoundly influences product
evolution. Previous studies of model membranes constructed
from pure phospholipids, available by our unambiguous synth-
eses, established that the hydroperoxydiene modifications of
PUFAs esterified into phospholipids, which are the major initial
products of free radical- or singlet-oxygen-mediated oxidation,
remain buried in the hydrophobic membrane core. With further
oxidation and truncation, oxidized acyl groups become hydro-
philic and adopt a conformation in which the oxidized acyl group
protrudes from the membrane into the aqueous phase. Hydro-
peroxydienes are sources of chain-carrying alkoxy radicals that
can either abstract doubly allylic hydrogen to generate pentadie-
nyl radical precursors of additional hydroperoxides or abstract
aldehydic hydrogen resulting in the oxidation of aldehydes to
carboxylic acids.
Possible Contribution of Hydroperoxy Endoperoxide
Fragmentation to Oxidative Injury in the Eye. The present
study shows that vitamin E can promote the fragmentation of
hydroperoxy endoperoxides, products that may be generated in
the retina from the reaction PUFAs with singlet molecular
oxygen, to give toxic aldehydes. These can, in turn, react with
proteins, leading to oxidative lipid-derived protein modifications,
such as the carboxyethylpyrrole (CEP) derivatives that trigger
angiogenesis into the retina, as well as an immune-mediated
destruction of the retina. Therefore, the use of vitamin E as a
Potential Contributions of Vitamin E and Ascorbate to
Promoting Oxidative Injury. It has been more than 80 years
since vitamin E was first discovered.48 Yet the role and impor-
tance of vitamin E in vivo is still unclear.49,50 As an antioxidant,
vitamin E can scavenge lipid peroxyl and alkoxyl radicals and thus
protect the membrane from oxidative damage.34,51 However,
vitamin E can also potentially contribute to oxidative injury: (1)
by promoting lipid peroxidation, a prooxidant activity, that has
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supplement in clinical trials as a therapeutic intervention to
prevent oxidative injury should consider not only its antioxidant
but also its prooxidant effects61,62 and its ability to promote the
generation of toxic aldehydes through fragmentation of peroxidized
lipids. Strong evidence is accumulating for a major role for lipid
oxidation and lipid-derived protein modification in the pathogenesis
of AMD. It seemed reasonable, therefore, to expect that antioxidants
would have therapeutic benefits. Tocopherol’s prooxidant effects
and/or its ability to promote fragmentation of peroxidized lipids
may help to explain why clinical trials have concluded that vitamin E
in some cases provides a little but in most cases provides no benefit
in ameliorating AMD.63ꢀ65 Even if vitamin E successfully neutra-
lizes chain carrying free radicals and free-radical-induced oxidation
of lipids, it may concomitantly increase levels of toxic aldehydes
generated by vitamin E-induced fragmentation.
The tocopherol-induced in vitro fragmentations of hydroper-
oxy endoperoxides that we described above may involve direct
electron transfer leading to reductive cleavage or a process
catalyzed by traces of redox active metal ions. Such catalysis is
especially likely in vivo because carboxymethyl lysine (CML)
modifications of proteins, which have been shown to bind redox
active metal ions,32,33 are found in eyes from individuals
with AMD,66 and levels are elevated in cataractous lenses.67
Furthermore, it is likely that, in the presence of vitamin E, these
protein-bound redox active metal ions will catalyze the reductive
fragmentation of hydroperoxy endoperoxides by tocopherols to
deliver toxic γ-hydroxyalkenals which, in turn, modify proteins,
e.g., to produce CEPs (Figure 16). Mean levels of CEP adducts
are ∼60% higher, and CML adducts are ∼54% higher in AMD
relative to normal controls.68,69 It is tempting to speculate that
the correlation between elevated CML and elevated CEP in
AMD eyes indicates a role for CMLs in promoting the generation
of CEPs. Specifically, reactions of docosahexaenoate phospholi-
pids with singlet molecular oxygen can generate hydroperoxy
endoperoxides that can be converted to CEPs through the
chemistry of detailed above.
The present study provides a basis for postulating that redox
active metal ions contribute to lipid-derived oxidative protein
modification that may be pathogenic, e.g., promoting both wet
and dry forms of AMD. It also suggests a mechanism that may
compromise the efficacy of dietary R-tocopoherol suplementa-
tion as a therapeutic modality. Testing these hypotheses will
require, inter alia, comparing levels of redox active metals
and tocopherols in eyes from individuals with AMD and from
those with no disease. In a previous study, we found that dietary
R-tocopherol supplementation offered no benefit for lower-
ing levels of oxidative protein modifications in hemodialysis
patients.62 Rather, it caused a decrease in plasma γ-tocopherol
levels, presumably by competitively inhibiting uptake of
γ-tocopherol from the diet. Thus, supplementation with R-toco-
pherol resulted in a 2-fold increase in R-tocopherol levels and a
concomitant halving of γ-tocopherol levels. The finding of the
present study that γ-tocopherol-promoted decomposition of
13-HP-Endo causes less fragmentation than R-tocopherol sug-
gests that dietary supplementation with γ-tocopoherol may be
superior to R-tocopoherol as a therapeutic modality to prevent
oxidative injury caused by the generation of toxic aldehydes.
This material is available free of charge via the Internet at http://
pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: rgs@case.edu.
Funding Sources
We are grateful for support of this work by National Institutes of
Health Grants GM021249 and EY016813.
’ ABBREVIATIONS
1O2, singlet oxygen; 9,12-diHPODE, 9,12-dihydroperoxyoctade-
ca-10,13-dienoic acid; 9-HP-Endo, 9-hydroperoxy-10,13-epidi-
oxy-11-octadecenoic acid; 9-HPODE, 9-hydroperoxy-10,12-
octadecadienoic acid; 10,13-diHPODE, 10,13-dihydroperoxyoc-
tadeca-8,11-dienoic acid; 10-HPODE, 10-hydroperoxy-8,12-octa-
decadienoic acid; 12-HPODE, 12-hydroperoxy-9,13-octade-
cadienoic acid; 13-H-Endo, 13-hydroxy-9,12-epidioxy-10-octa-
decenoic acid; 13-HP-Endo, 13-hydroperoxy-9,12-epidioxy-10-
octadecenoic acid; 13-HP-Endo-MiP, 8-(6-(1-(2-methoxy-
propan-2-ylperoxy)hexyl)-3,6-dihydro-1,2-dioxin-3-yl)octanoic
acid; 13-HP-Endo-PC, 1-hexadecanoyl-2-(8-(6-(1-hydroperoxy-
hexyl)-3,6-dihydro-1,2-dioxin-3-yl)octanoyl)-sn-glycero-3-phos-
phocholine; 13-HP-Endo-PC-MiP, 1-hexadecanoyl-2-(8-(6-(1-
(2-methoxypropan-2-ylperoxy)hexyl)-3,6-dihydro-1,2-dioxin-3-yl)-
octanoyl)-sn-glycero-3-phosphocholine; 13-HP-MiP, (9Z,11E)-
13-(2-methoxypropan-2-ylperoxy)octadeca-9,11-dienoic acid;
13-HPODE, 13-hydroperoxy-9,12-epidioxy-10-octadece-
noic acid; 13-HP-PC-MiP, 1-hexadecanoyl-2-((9Z,11E)-13-(2-
oxypropan-2-ylperoxy)octadeca-9,11-dienoyl)-sn-glycero-3-phos-
phocholine; AMD, age-related macular degeneration;
CD36, cluster of differentiation 36; CEP, carboxyethylpyrrole;
CML, N-(carboxymethyl)lysine;DCC, dicyclohexylcarbodiimide;
DMAP, 4-dimethylaminopyridine; DTPA, diethylenetriamine-
pentaacetic acid; DT-PC, 1,2-ditridecanoyl-sn-glycero-3-phos-
phatidylcholine; ESI-MS/MS, electrospray ionizationꢀtandem
mass spectrometry; FOA, 8-(furan-2-yl)octanoic acid; HNE, 4-
hydroxy-2-nonenal; HODA, 9-hydroxy-12-oxo-10(E)-dodece-
noic acid; HODA-PC, 1-hexadecanoyl-2-(9-hydroxy-12-oxo-
10(E)-dodecenoyl)-3-phosphatidylcholine; HODFO, 8-(2-hy-
droxy-5-oxo-2,5-dihydrofuran-2-yl)octanoic acid; HRMS, high
resolution mass spectrometry; KODA, 9-keto-12-oxo-10-dode-
cenoic acid; LA, linoleic acid; Lyso-PC, 1-acyl-sn-glycero-3-phos-
phocholine; SRM, selected reaction monitoring; ODFO,
8-(5-oxo-2,5-dihydrofuran-2-yl)octanoic acid; ODFO-PC, 1-hexa-
decanoyl-2-(8-(5-oxo-2,5-dihydrofuran-2-yl)octanoyl)-sn-gly-
cero-3-phosphocholine; oxPCs, oxidatively truncated phos-
phatidylcholines; oxPEs, oxidatively truncated phosphatidy-
lethanolamine; PC, phosphatidylcholine; PFB, pentafluoro-
benzyl; PPTs, pyridinium p-toluene sulfonate; PUFA, polyunsa-
turated fatty acyl; RPE, retinal pigment epithelium; SIR, selected
ion recording; SP-PC, 1-stearoyl-2-palmitoyl-sn-glycerol-3-phos-
phocholine; SUVs, small unilamellar vesicles; TPP, tetraphenyl-
porphine; Vit E, vitamin E.
’ REFERENCES
’ ASSOCIATED CONTENT
(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.
S
Supporting Information. General experimental meth-
b
ods and spectroscopic and analytical data for new compounds.
1091
dx.doi.org/10.1021/tx200093m |Chem. Res. Toxicol. 2011, 24, 1080–1093

Chemical Research in Toxicology
ARTICLE
(2) 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.
(3) 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.
hydroperoxy cyclic peroxides, dihydroperoxides, keto esters and volatile
thermal decomposition products. Lipids 18, 868–876.
(19) MacDonald, R. C., MacDonald, R. I., Menco, B. P., Takeshita,
K., Subbarao, N. K., and Hu, L. R. (1991) Small-volume extrusion
apparatus for preparation of large, unilamellar vesicles. Biochim. Biophys.
Acta 1061, 297–303.
(20) Chen, X., Zhang, W., Laird, J., Hazen, S. L., and Salomon,
R. G. (2008) Polyunsaturated phospholipids promote the oxidation
and fragmentation of gamma-hydroxyalkenals: formation and reac-
tions of oxidatively truncated ether phospholipids. J. Lipid Res. 49, 832–
846.
(4) 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.
(5) Gu, X., Meer, S. G., Miyagi, M., Rayborn, M. E., Hollyfield, J. G.,
Crabb, J. W., and Salomon, R. G. (2003) Carboxyethylpyrrole protein
adducts and autoantibodies, biomarkers for age-related macular degen-
eration. J. Biol. Chem. 278, 42027–42035.
(6) 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 neovasculariza-
tion: Implications for age-related macular degeneration. Proc. Natl. Acad.
Sci. U.S.A. 103, 13480–13484.
(21) Balamraju, Y. N., Sun, M., and Salomon, R. G. (2004) Gamma-
hydroxyalkenals are oxidatively cleaved through Michael addition of
acylperoxy radicals and fragmentation of intermediate beta-hydroxype-
resters. J. Am. Chem. Soc. 126, 11522–11528.
(22) Gao, S., Zhang, R., Greenberg, M. E., Sun, M., Chen, X.,
Levison, B. S., Salomon, R. G., and Hazen, S. L. (2006) Phospholipid
hydroxyalkenals, a subset of recently discovered endogenous CD36
ligands, spontaneously generate novel furan-containing phospholipids
lacking CD36 binding activity in vivo. J. Biol. Chem. 281, 31298–31308.
(23) Funk, M. O., Isacc, R., and Porter, N. A. (1976) Preparation and
purification of lipid hydroperoxides from arachidonic and gamma-
linolenic acids. Lipids 11, 113–117.
(7) West, X. Z., Malinin, N. L., Merkulova, A. A., Tischenko, M.,
Kerr, B. A., Borden, E. C., Podrez, E. A., Salomon, R. G., and Byzova,
T. V. (2010) Oxidative stress induces angiogenesis by activating TLR2
with novel endogenous ligands. Nature 467, 972–976.
(24) Dussault, P. H., and Woller, K. R. (1997) Singlet oxygenation/
radical rearrangement as an approach to 1,4-dioxygenated peroxides:
Asymmetric total syntheses of plakorin and enantio-chondrillin. J. Am.
Chem. Soc. 119, 3824–3825.
(8) Renganathan, K., Ebrahem, Q., Vasanji, A., Gu, X., Lu, L.,
Sears, J., Salomon, R. G., Anand-Apte, B., and Crabb, J. W. (2008)
Carboxyethylpyrrole adducts, age-related macular degeneration and
neovascularization. Adv. Exp. Med. Biol. 613, 261–267.
(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) Crabb, J. W., Miyagi, M., Gu, X., Shadrach, K., West, K. A.,
Sakaguchi, H., Kamei, M., Hasan, A., Yan, L., Rayborn, M. E., Salomon,
R. G., and Hollyfield, J. G. (2002) Drusen proteome analysis: an
approach to the etiology of age-related macular degeneration. Proc.
Natl. Acad. Sci. U.S.A. 99, 14682–14687.
(11) Gugiu, B. G., Mesaros, C. A., Sun, M., Gu, X., Crabb, J. W., and
Salomon, R. G. (2006) Identification of oxidatively truncated ethanol-
amine phospholipids in retina and their generation from polyunsatu-
rated phosphatidylethanolamines. Chem. Res. Toxicol. 19, 262–271.
(12) Rando, R. R. (1991) Membrane phospholipids as an energy
source in the operation of the visual cycle. Biochemistry 30, 595–602.
(13) Bensasson, R. V., Land, E. J., and Truscott, T. G. (1993) Excited
States and Free Radicals in Biology and Medicine, Oxford University Press,
New York.
(14) Rozanowska, M., Wessels, J., Boulton, M., Burke, J. M., Rodgers,
M. A., Truscott, T. G., and Sarna, T. (1998) Blue light-induced singlet
oxygen generation by retinal lipofuscin in non-polar media. Free Radical
Biol Med 24, 1107–1112.
(15) Zhang, W., Sun, M., and Salomon, R. G. (2006) Preparative
singlet oxygenation of linoleate provides doubly allylic dihydroperox-
ides: putative intermediates in the generation of biologically active
aldehydes in vivo. J. Org. Chem. 71, 5607–5615.
(16) Schneider, C., Tallman, K. A., Porter, N. A., and Brash,
A. R. (2001) Two distinct pathways of formation of 4-hydroxynonenal.
Mechanisms of nonenzymatic transformation of the 9- and 13-hydroper-
oxides of linoleic acid to 4-hydroxyalkenals. J. Biol. Chem. 276, 20831–
20838.
(17) Schneider, C., Boeglin, W. E., Yin, H., Ste, D. F., Hachey, D. L.,
Porter, N. A., and Brash, A. R. (2005) Synthesis of dihydroperoxides of
linoleic and linolenic acids and studies on their transformation to
4-hydroperoxynonenal. Lipids 40, 1155–1162.
(25) Deng, Y., and Salomon, R. G. (1998) Total synthesis of
γ-hydroxy-R,β-unsaturated aldehydic esters of cholesterol and 2-lyso-
phosphatidylcholine. J. Org. Chem. 63, 7789–7794.
(26) Jian, W., Lee, S. H., Arora, J. S., Silva Elipe, M. V., and Blair, I. A.
(2005) Unexpected formation of etheno-20-deoxyguanosine adducts
from 5(S)-hydroperoxyeicosatetraenoic acid: evidence for a bis-hydro-
peroxide intermediate. Chem. Res. Toxicol. 18, 599–610.
(27) Gallasch, B. A., and Spiteller, G. (2000) Synthesis of 9,12-dioxo-
10(Z)-dodecenoic acid, a new fatty acid metabolite derived from
9-hydroperoxy-10,12-octadecadienoic acid in lentil seed (Lens culinaris
Medik.). Lipids 35, 953–960.
(28) Annangudi, S. P., Sun, M., and Salomon, R. G. (2005) An efficient
synthesis of 4-oxoalkenoic acids from 2-alkylfurans. Synlett 9, 1468–1470.
(29) Teijeira, M., Suarez, P. L., Gomez, G., Teran, C., and Fall, Y.
(2005) The furan approach to oxacycles. Part 5: Synthesis of a chiral
butenolide, building block towards biologically interesting natural
products. Tetrahedron Lett. 46, 5889–5892.
(30) Lee, S. H., Oe, T., and Blair, I. A. (2001) Vitamin C-induced
decomposition of lipid hydroperoxides to endogenous genotoxins.
Science 292, 2083–2086.
(31) Gu, X., Zhang, W., and Salomon, R. G. (2007) Fe2þ catalyzes
vitamin E-induced fragmentation of hydroperoxy and hydroxy endoper-
oxides that generates gamma-hydroxy alkenals. J. Am. Chem. Soc.
129, 6088–6089.
(32) Glomb, M. A., and Monnier, V. M. (1995) Mechanism of
protein modification by glyoxal and glycolaldehyde, reactive intermedi-
ates of the Maillard reaction. J. Biol. Chem. 270, 10017–10026.
(33) Saxena, A. K., Saxena, P., Wu, X., Obrenovich, M., Weiss, M. F.,
and Monnier, V. M. (1999) Protein aging by carboxymethylation of
lysines generates sites for divalent metal and redox active copper
binding: relevance to diseases of glycoxidative stress. Biochem. Biophys.
Res. Commun. 260, 332–338.
(34) Traber, M. G., and Atkinson, J. (2007) Vitamin E, antioxidant
and nothing more. Free Radical Biol. Med. 43, 4–15.
(35) Niki, E., and Noguchi, N. (2004) Dynamics of antioxidant
action of vitamin E. Acc. Chem. Res. 37, 45–51.
(36) Burkitt, M. J., and Milne, L. (1996) Hydroxyl radical formation
from Cu(II)-trolox mixtures: insights into the pro-oxidant properties of
alpha-tocopherol. FEBS Lett. 379, 51–54.
(37) Hanna, P. M., and Mason, R. P. (1992) Direct evidence for
inhibition of free radical formation from Cu(I) and hydrogen peroxide
(18) Neff, W. E., Frankel, E. N., Selke, E., and Weisleder, D. (1983)
Photosensitized oxidation of methyl linoleate monohydroperoxides:
1092
dx.doi.org/10.1021/tx200093m |Chem. Res. Toxicol. 2011, 24, 1080–1093

Chemical Research in Toxicology
ARTICLE
by glutathione and other potential ligands using the EPR spin-trapping
technique. Arch. Biochem. Biophys. 295, 205–213.
and its dependence on the oxidative stress. Chem. Phys. Lipids 114, 81–
98.
(38) Brigelius-Flohe, R., and Traber, M. G. (1999) Vitamin E:
function and metabolism. FASEB J. 13, 1145–1155.
(59) Esterbauer, H., Gebicki, J., Puhl, H., and Jurgens, G. (1992) The
role of lipid peroxidation and antioxidants in oxidative modification of
LDL. Free Radical Biol. Med. 13, 341–390.
(60) Burkitt, M. J. (2001) A critical overview of the chemistry of
copper-dependent low density lipoprotein oxidation: roles of lipid
hydroperoxides, alpha-tocopherol, thiols, and ceruloplasmin. Arch.
Biochem. Biophys. 394, 117–135.
(61) Sesso, H. D., Buring, J. E., Christen, W. G., Kurth, T., Belanger,
C., MacFadyen, J., Bubes, V., Manson, J. E., Glynn, R. J., and Gaziano,
J. M. (2008) Vitamins E and C in the prevention of cardiovascular
disease in men: the Physicians’ Health Study II randomized controlled
trial. JAMA 300, 2123–2133.
(62) Lu, L., Erhard, P., Salomon, R. G., and Weiss, M. F. (2007)
Serum vitamin E and oxidative protein modification in hemodialysis: a
randomized clinical trial. Am. J. Kidney Dis. 50, 305–313.
(63) Jampol, L. M., and Ferris, F. L., 3rd (2001) Antioxidants and
zinc to prevent progression of age-related macular degeneration. JAMA
286, 2466–2468.
(64) Kuzniarz, M., Mitchell, P., Flood, V. M., and Wang, J. J. (2002)
Use of vitamin and zinc supplements and age-related maculopathy: the
Blue Mountains Eye Study. Ophthalmic Epidemiol. 9, 283–295.
(65) Taylor, H. R., Tikellis, G., Robman, L. D., McCarty, C. A., and
McNeil, J. J. (2002) Vitamin E supplementation and macular degenera-
tion: randomised controlled trial. BMJ 325, 11.
(66) Ishibashi, T., Murata, T., Hangai, M., Nagai, R., Horiuchi, S.,
Lopez, P. F., Hinton, D. R., and Ryan, S. J. (1998) Advanced glycation
end products in age-related macular degeneration. Arch. Ophthalmol.
116, 1629–1632.
(39) Christen, S., Woodall, A. A., Shigenaga, M. K., Southwell-Keely,
P. T., Duncan, M. W., and Ames, B. N. (1997) gamma-tocopherol traps
mutagenic electrophiles such as NO(X) and complements alpha-tocopher-
ol: physiological implications. Proc. Natl. Acad. Sci. U.S.A. 94, 3217–3222.
(40) Jiang, Q., Christen, S., Shigenaga, M. K., and Ames, B. N.
(2001) gamma-tocopherol, the major form of vitamin E in the US diet,
deserves more attention. Am. J. Clin. Nutr. 74, 714–722.
(41) Hensley, K., Benaksas, E. J., Bolli, R., Comp, P., Grammas, P.,
Hamdheydari, L., Mou, S., Pye, Q. N., Stoddard, M. F., Wallis, G.,
Williamson, K. S., West, M., Wechter, W. J., and Floyd, R. A. (2004) New
perspectives on vitamin E: gamma-tocopherol and carboxyelthylhydrox-
ychroman metabolites in biology and medicine. Free Radical Biol. Med.
36, 1–15.
(42) Yoshida, Y., Niki, E., and Noguchi, N. (2003) Comparative
study on the action of tocopherols and tocotrienols as antioxidant:
chemical and physical effects. Chem. Phys. Lipids 123, 63–75.
(43) Baba, M., Yoneda, K., Tahara, S., Iwasa, J., Keneko, T., and
Matsuo, M. (1990) A regioselective, stereoselective synthesis of a
diacylglycerophosphocholine hydroperoxide by use of lipoxygenase
and lipase. J. Chem. Soc., Chem. Commun. 1281–1282.
(44) Ibusuki, D., Nakagawa, K., Asai, A., Oikawa, S., Masuda, Y.,
Suzuki, T., and Miyazawa, T. (2008) Preparation of pure lipid hydro-
peroxides. J. Lipid Res. 49, 2668–2677.
(45) Atkinson, J., Epand, R. F., and Epand, R. M. (2008) Tocopher-
ols and tocotrienols in membranes: a critical review. Free Radical Biol.
Med. 44, 739–764.
(46) Li, X. M., Salomon, R. G., Qin, J., and Hazen, S. L. (2007)
Conformation of an endogenous ligand in a membrane bilayer for the
macrophage scavenger receptor CD36. Biochemistry 46, 5009–5017.
(47) Govindarajan, B., Junk, A., Algeciras, M., Salomon, R. G., and
Bhattacharya, S. K. (2009) Increased isolevuglandin-modified proteins
in glaucomatous astrocytes. Mol. Vision 15, 1079–1091.
(48) Evans, H. M., and Bishop, K. S. (1922) On the existence of a
hitherto unrecognized dietary factor essential for reproduction. Science
56, 650–651.
(67) Saxena, P., Saxena, A. K., Cui, X. L., Obrenovich, M., Gudipaty,
K., and Monnier, V. M. (2000) Transition metal-catalyzed oxidation of
ascorbate in human cataract extracts: possible role of advanced glycation
end products. Invest. Ophthalmol. Vis. Sci. 41, 1473–1481.
(68) Gu, J., Pauer, G. J., Yue, X., Narendra, U., Sturgill, G. M., Bena,
J., Gu, X., Peachey, N. S., Salomon, R. G., Hagstrom, S. A., and Crabb,
J. W. (2009) Assessing susceptibility to age-related macular degenera-
tion with proteomic and genomic biomarkers. Mol. Cell Proteomics
8, 1338–1349.
(49) Brigelius-Flohe, R. (2009) Vitamin E: the shrew waiting to be
tamed. Free Radical Biol. Med. 46, 543–554.
(50) Azzi, A. (2007) Molecular mechanism of alpha-tocopherol
(69) Ni, J., Yuan, X., Gu, J., Yue, X., Gu, X., Nagaraj, R. H., Crabb,
J. W., and Study Group, C. (2009) Plasma protein pentosidine and
carboxymethyllysine, biomarkers for age-related macular degeneration.
Mol. Cell Proteomics 8, 1921–1933.
action. Free Radical Biol. Med. 43, 16–21.
(51) Herrera, E., and Barbas, C. (2001) Vitamin E: action, metabo-
lism and perspectives. J. Physiol. Biochem. 57, 43–56.
(52) Bowry, V. W., and Ingold, K. U. (1999) The unexpected role of
vitamin E (R-tocopherol) in the peroxidation of human low-density
lipoprotein. Acc. Chem. Res. 32, 27–34.
(53) Upston, J. M., Terentis, A. C., and Stocker, R. (1999) Toco-
pherol-mediated peroxidation of lipoproteins: implications for vitamin E
as a potential antiatherogenic supplement. FASEB J. 13, 977–994.
(54) Yamamoto, K., and Niki, E. (1988) Interaction of alpha-
tocopherol with iron: antioxidant and prooxidant effects of alpha-
tocopherol in the oxidation of lipids in aqueous dispersions in the
presence of iron. Biochim. Biophys. Acta 958, 19–23.
(55) Maiorino, M., Zamburlini, A., Roveri, A., and Ursini, F. (1993)
Prooxidant role of vitamin E in copper induced lipid peroxidation. FEBS
Lett. 330, 174–176.
(56) Yoshida, Y., Tsuchiya, J., and Niki, E. (1994) Interaction of
alpha-tocopherol with copper and its effect on lipid peroxidation.
Biochim. Biophys. Acta 1200, 85–92.
(57) Kontush, A., Meyer, S., Finckh, B., Kohlschutter, A., and
Beisiegel, U. (1996) Alpha-tocopherol as a reductant for Cu(II) in
human lipoproteins. Triggering role in the initiation of lipoprotein
oxidation. J. Biol. Chem. 271, 11106–11112.
(58) Bittner, O., Gal, S., Pinchuk, I., Danino, D., Shinar, H., and
Lichtenberg, D. (2002) Copper-induced peroxidation of liposomal
palmitoyllinoleoylphosphatidylcholine (PLPC), effect of antioxidants
1093
dx.doi.org/10.1021/tx200093m |Chem. Res. Toxicol. 2011, 24, 1080–1093