Effect of α-tocopherol on the hemin-catalyzed decomposition of 1-palmitoyl-2-linoleoyl-3-sn-phosphatodylcholine 13-hydroperoxide in micelles and liposomes

Article abstract of DOI:10.1016/j.chemphyslip.2014.10.001

The secondary process of lipid peroxidation produces some toxic aldehydes. Since this process takes place via free radical reaction in lipophilic circumstances, α-tocopherol would suppress the formation of such aldehydes by trapping free-radical intermedi

Full text of DOI:10.1016/j.chemphyslip.2014.10.001

Chemistry and Physics of Lipids 184 (2014) 61–68
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Effect of
a
-tocopherol on the hemin-catalyzed decomposition of
1
-palmitoyl-2-linoleoyl-3-sn-phosphatodylcholine 13-hydroperoxide
in micelles and liposomes
Ryo Yamauchi *, Siori Watanabe, Ana S Martín, Satoshi Iwamoto
Department of Applied Life Science, Graduate School of Applied Biological Sciences, Gifu University, Gifu 501-1193, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 18 July 2014
Received in revised form 22 September 2014
Accepted 7 October 2014
Available online 13 October 2014
The secondary process of lipid peroxidation produces some toxic aldehydes. Since this process takes
place via free radical reaction in lipophilic circumstances,
such aldehydes by trapping free-radical intermediates. This study reports the effect of
the hemin-catalyzed decomposition of 1-palmitoyl-2-linoleoyl-3-sn-phosphatidylcholine 13-hydroper-
oxide (PLPC-OOH) in micelles and liposomes. PLPC-OOH and -tocopherol were reacted with hemin in
micelles, and the reaction products were characterized to be 1-palmitoyl-2-( -tocopheroxy-12,13-
epoxyoctadecenoyl)-3-sn-phosphatidylcholines (T-epoxyPLPC) and known compounds, 1-palmitoyl-2-
(8a-dioxy- -tocopherone)-12,13-epoxyoctadecenoyl]-3-sn-phosphatidylcholines (TOO-epoxyPLPC)
and -tocopherol dimer. The hemin-catalyzed decomposition of PLPC-OOH in micelles produced
hexanal as one of secondary aldehydic products. -Tocopherol suppressed the formation of hexanal, and
-tocopherylquinone, -tocopherol dimer, TOO-epoxyPLPC, and T-epoxyPLPC were detected during the
reaction. In liposomes, -tocopherol could partially suppress the formation of hexanal, and the main
products were TOO-epoxyPLPC and -tocopherol dimer. The results indicate that -tocopherol may
a
-tocopherol would suppress the formation of
a
-tocopherol on
a
a
Keywords:
Lipid peroxidation
[
a
a-Tocopherol
a
Phosphatidylcholine hydroperoxide
Hexanal
Hemin
a
a
a
Free radical
a
a
a
suppress the formation of hexanal by trapping the epoxyperoxyl and epoxyalkyl radicals derived from
PLPC-OOH.
ã 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
biological systems (Blair, 2001; Guéraud et al., 2010; Fritz and
Petersen, 2013). Since such aldehydes are produced from lipid
Lipid peroxidation produces lipid hydroperoxides as the
primary products. Although lipid hydroperoxides are relatively
stable at physiological temperature, they are decomposed by
transition metals to produce a wide range of secondary aldehydic
products, such as alkanals, alkenals, 4-hydroxy-2-alkenals, and
hydroperoxides via free radical reaction in hydrophobic circum-
stances, some lipid-soluble antioxidants may be expected to
suppress the formation of aldehydes by trapping free radical
intermediates.
a-Tocopherol (1,Fig. 1), the most active form of vitamin E, is
4
-oxo-2-alkenals (Esterbauer et al., 1991; Spiteller et al., 2001; Gu
known to act as a chain-breaking antioxidant against lipid
peroxidation (Kamal-Eldin and Appelqvist, 1996; Niki, 2014).
and Salomon, 2012). These aldehydic products are more stable
than hydroperoxides, and they may exert toxicological effects in
a
-Tocopherol efficiently transfers a hydrogen atom to a lipid-
peroxyl radical, giving a lipid hydroperoxide and an -tocopher-
oxyl radical. The -tocopheroxyl radical then reacts with a second
lipid-peroxyl radical to form an addition product of -tocopherol
with lipid-peroxyl radical, 8a-(lipid-dioxy)- -tocopherone
Yamauchi, 2007). The iron-catalyzed decomposition of lipid
hydroperoxides forms alkoxyl and peroxyl radicals, which react
with -tocopherol to yield some addition products (Gardner,1989;
Yamauchi et al., 1995, 2002a). Therefore, if -tocopherol traps
a
a
Abbreviations: ESI-MS, electrospray ionization mass spectrometry; HPLC, high-
performance liquid chromatography; PLPC, 1-palmitoyl-2-linoleoyl-3-sn-phospha-
tidylcholine; PLPC-OOH, 1-palmitoyl-2-[(9Z,11E)-(S)-13-hydroperoxy-9,11-octade-
a
a
(
cadienoyl]-3-sn-phosphatidylcholine;
POPC,
phosphatidylcholine; H NMR, proton nuclear magnetic resonance spectrometry;
T-epoxyPLPC, 1-palmitoyl-2-[( -tocopheroxy)-epoxyoctadecenoyl]-3-sn-phospha-
tidylcholines; TOO-epoxyPLPC, 1-palmitoyl-2-[(8a-dioxy- -tocopherone)-epox-
-tocopherylquinone; UV,
1-palmitoyl-2-oleoyl-3-sn-
1
a
a
a
a
yoctadecenoyl]-3 sn-phosphatidylcholines;
a-TQ, a
hydroperoxide-derived free radicals during the secondary oxida-
tion process, the formation of aldehydic products may be
suppressed. Raghavamenon et al. (2009) reported that some
ultraviolet.
*
Corresponding author. Tel.: +81 58 293 2930; fax: +81 58 293 2930.
E-mail addresses: yamautir@gifu-u.ac.jp (R. Yamauchi), anittasm@gmail.com
A.S. Martín), isatoshi@gifu-u.ac.jp (S. Iwamoto).
(
phenolic antioxidants, such as a-tocopherol and Trolox, did not
http://dx.doi.org/10.1016/j.chemphyslip.2014.10.001
009-3084/ã 2014 Elsevier Ireland Ltd. All rights reserved.
0

6
2
R. Yamauchi et al. / Chemistry and Physics of Lipids 184 (2014) 61–68
Fig. 1. Structures of compounds referred to in the text.
prevent the decomposition of linoleic acid hydroperoxides and
seemed even to accelerate its decomposition. However, it is
2. Materials and methods
unclear whether
a-tocopherol affects the formation of secondary
2.1. Materials
aldehydic products during the peroxidation of phospholipids in
micellar and liposomal systems.
RRR-a-Tocopherol was purchased from Sigma–Aldrich Co. (St.
Hemoproteins and hemin (or hematin) have been widely used
to study initiation and propagation steps of lipid peroxidation and
catalyze the decomposition of lipid hydroperoxides (Kim and
Sevanian, 1991; Hayashi et al., 2004; Carlsen et al., 2005). It has
been reported that decomposition of phosphatidylcholine hydro-
peroxide proceeds very rapidly by the addition of hemoproteins
and generates aldehydic products, such as 4-hydroxy-2-nonenal
Louis, MO) and purified by reversed-phase high-performance
liquid chromatography (HPLC) (Yamauchi et al.,1994).1-Palmitoyl-
2-oleoyl-3-sn-phosphatidylcholine (POPC) was purchased from
NOF Co. (Tokyo, Japan) and used with received. 1-Palmitoyl-2-
linoleoyl-3-sn-phosphatidylcholine (PLPC) was synthesized as
described previously (Yamauchi et al., 1998). PLPC-OOH was
prepared by oxidation of PLPC with soybean lipoxygenase (type 1-
B, Sigma–Aldrich Co.) in the presence of sodium deoxycholate
(Brash et al., 1987), and purified by reversed-phase HPLC using an
Inertsil Prep ODS column (20 mm ꢀ 250 mm; GL Sciences Co.,
Tokyo, Japan) with methanol: ESI-MS m/z (relative intensity, %)
(
HNE) and malondialdehyde (MDA) (Hayashi et al., 2004).
Therefore, we have studied the effect of -tocopherol on the
a
formation of aldehydic products using the hemin-catalyzed
reaction of 1-palmitoyl-2-[(9Z,11E)-(S)-13-hydroperoxy-9,11-octa-
decadienoyl]-3-sn-phosphatidylcholine (PLPC-OOH, 2) in micellar
and liposomal systems. Hexanal was measured as the secondary
product from PLPC-OOH, because this aldehyde was one of the
major aldehydes produced from linoleic acid hydroperoxides
+
+
772.2 (12, [M ꢁ OH] ), 790.2 (100, [M + H] ), and 812.2 (10,
+
1
[M + Na] ); H NMR (CDCl
(m, 38H), 1.42 (m, 1H, H -14), 1.58 (m, 4H, H
1H, H -14), 2.15 (m, 1H, H -8), 2.22 (m, 1H, H
2 and H -2), 3.30 (s, 9H, choline-(CH ), 3.74 (br m, 2H, H
(dd, J = 6.0, 6.4 Hz, 2H, H -1), 4.15 (dd, J = 6.1, 11.9 Hz, 1H, H
(br m, 2H, H -1), 4.31 (dd, J = 6.4,14.2 Hz,1H, H
11.9,1H, H -3), 5.21 (br m, 1H, H -2), 5.43 (dt, J = 10.5, 7.8 Hz, 1H, H
9), 5.59 (dd, J = 8.2, 15.1 Hz, 1H, H -12), 6.00 (t, J = 11.0 Hz, 1H, H -10),
3
)
d
0.88 (m, 6H, H
-3 and H
-8), 2.30 (m, 4H, H
p
-14 and H
l
-18), 1.25
l
p
l
-3), 1.65 (m,
l
l
l
p
-
(
Schieberle and Grosch,1981; Gu and Salomon, 2012) and from the
autoxidation of human low-density lipoprotein (Esterbauer et al.,
987). The hemin-catalyzed reaction products of PLPC-OOH with
l
3
)
3
c
-2), 3.96
-3), 4.30
g
g
1
g
l
-13), 4.37 (dd, J = 3.2,
a
a
-tocopherol were also characterized to clarify the mechanism of
-tocopherol action.
g
g
l
-
l
l

R. Yamauchi et al. / Chemistry and Physics of Lipids 184 (2014) 61–68
63
6
.50 (dd, J = 11.4, 15.1 Hz, 1H, H
where H , H , H , and H indicate protons at palmitoyl, linoleoyl,
glycerol, and choline moieties, respectively. Authentic -toco-
pherylquinone ( -TQ, 3) and -tocopherol spirodiene dimer (4)
were prepared as described previously (Yamauchi et al., 1996). A
mixture of 1-palmitoyl-2-[9-(8a-dioxy- -tocopherone)-12,13-ep-
oxy-10-octadecenoyl]-3-sn-PC (5a) and 1-palmitoyl-2-[11-(8a-
dioxy- -tocopherone)-12,13-epoxy-9-octadecenoyl]-3-sn-PC (5b)
TOO-epoxyPLPC) was prepared by the ferric acetylacetonate-
catalyzed reaction of PLPC-OOH with -tocopherol in methanol
Yamauchi et al., 2002a). Hemin (ferriprotoporphyrin chloride)
l
-11), and 11.18 (s, 1H, 13-OOH),
buffer (pH 7.4) containing 5 mM sodium deoxycholate. After pre-
ꢂ
p
l
g
c
incubated for 5 min at 37 C in a water bath, 25
m
L of 2 mM hemin
a
solution was added to the micelles. The final concentrations of
PLPC-OOH, -tocopherol, and hemin were 0.5 mM, 0.1 mM, and
10 M, respectively.
a
a
a
m
a
2.4. Hemin-catalyzed reaction of PLPC-OOH with
POPC liposomes
a-tocopherol in
a
(
a
Large unilamellar POPC liposomes containing PLPC-OOH and
a-tocopherol were prepared by the extrusion method (MacDonald
(
was purchased from Sigma–Aldrich Co., and dissolved in 0.15 M
ammonia solution before use. Hemin readily converts to hematin
et al., 1991). A mixture of POPC and PLPC-OOH without or with
-tocopherol was suspended in a 50 mM sodium phosphate buffer
a
(
2
ferriprotoporphyrin hydroxide) in aqueous solution (Carlsen et al.,
005). 2,4-Dinitrophenylhydradine (DNPH) was obtained from
(pH 7.4) containing 50 mM NaCl. The milky suspension was
transferred to a LiposoFast apparatus (Avestin, Ottawa, Canada),
extruded 21 times back and forth through a polycarbonate
membrane (pore size of 100 nm), and diluted with the same
buffer solution to obtain large unilamellar liposomes. The
Wako Pure Chemical Ind. (Osaka, Japan), and recrystallized from
acetonitrile before use. Hexanal and heptanal was obtained from
Tokyo Chemical Co. (Tokyo, Japan). Diethylenetriamine-N,N,N ,N ,
0
00
00
ꢂ
N -pentaacetic acid (DTPA) and disodium ethylenediamintetraa-
cetate (Na EDTA) were purchased from Dojindo (Kumamoto,
Japan). DTPA was dissolved in 0.1 M sodium phosphate buffer (pH
.4) before use. Other chemicals were from Wako Pure Chemical
Ind. All solvents were distilled in an all-glass still before use.
liposomes (5.9 mL) were pre-incubated for 5 min at 37 C in a
water bath, and then the reaction was started by the addition of
2
3 mM hemin solution (100
PLPC-OOH, -tocopherol, and hemin were 9 mM, 1 mM, 0.2 mM,
and 50 M, respectively.
mL). The final concentrations of POPC,
7
a
m
2.2. Apparatus
2
.5. Quantification
HPLC was performed using a PU-2089 pump or a PU-2086
pump connected to a UV-2075 UV/VIS detector (Jasco, Tokyo,
Japan). Ultraviolet (UV) spectra were measured with
PLPC-OOH was determined by reversed-phase HPLC (Yamauchi
et al., 1996). A 100- L reaction mixture was collected into an
Eppendorf micro-tube, added ethanol (0.4 mL) and 0.2 M Na EDTA
(10 L), mixed, and centrifuged 10,000 rpm for 5 min. The upper
a
m
U-2810 spectrophotometer (Hitachi Co., Tokyo, Japan). Proton
2
1
nuclear magnetic resonance spectrum ( H NMR) was recorded at
m
6
00.17 MHz with a JNM-ECA-600 FT-NMR spectrometer (JEOL, Ltd.,
layer was injected onto an Inertsil C8-3 column (4.6 mm ꢀ 150
mm) developed with 95% methanol at 1.0 mL/min, and monitored
at 235 nm.
Hexanal was determined by reversed-phase HPLC as a DNPH
derivative (Esterbauer and Zollner, 1989). The DNPH reagent was
made of 50 mg of DNPH with 10 mL of 2.4 M HCl and 90 mL of
Tokyo, Japan), using CDCl as the solvent and tetramethylsilane as
the internal standard. The H– H chemical shift correlation
technique was employed to assign H shifts and couplings.
Electrospray-ionization mass spectra (ESI-MS) were obtained on
3
1
1
1
an LCMS-QP8000
a instrument (Shimadzu Co., Kyoto, Japan).
Samples were delivered into the ion source after the separation
using a Luna C18(2) column (2 mm ꢀ 150 mm, Phenomenex,
Torrance, CA) with 10 mM ammonium acetate in ethanol/methanol
ethanol. The reaction mixture (200
tube containing 20 L of 25 mM DTPA solution and 20
heptanal in ethanol solution (25 g/mL, an internal standard).
m
L) was collected into a test
m
m
L of
m
(
7:3, v/v) at 0.2 mL/min.
Then 100
m
L of the DNPH reagent was added to the reaction
ꢂ
mixture, and incubated at 45 C for 30 min. The resulting
hydrazones were extracted with ethyl acetate, dried in vacuo,
2
.3. Hemin-catalyzed reaction of PLPC-OOH with
a-tocopherol in
micelles
and dissolved in 200 mL of ethanol for HPLC analysis. HPLC was
performed with a TSK-gel-ODS 100 Z column (4.6 mm ꢀ 150 mm;
Tohso Co., Tokyo, Japan) with 80% methanol at 1.0 mL/min. The
hydrazones were monitored by an absorbance at 365 nm.
PLPC-OOH (395 mg, 0.50 mmol) and
.25 mmol) in ethanol solution was added into a round-bottom
flask, and the solvent was removed in vacuo. To this was added
00 mL of 0.1 M sodium phosphate buffer (pH 7.4) containing 5 mM
sodium deoxycholate, and mixed vigorously with a Vortex mixer
for 1 min followed by sonication for 1 min to obtain clear micelles.
a-tocopherol (108 mg,
0
a
-Tocopherol and its reaction products were determined by
HPLC after solid-phase extraction (Yamauchi et al., 2002b). A
500- L aliquot of each reaction mixture was withdrawn, added
25 L of 25 mM DTPA solution, and extracted twice with two
5
m
m
Hemin (6.6 mg, 10
then added to the micelles, and incubated at 37 C for 30 min with
mechanical shaking (50 rpm). After the reaction, the micellar
m
mol; dissolved in 2.0 mL of 20 mM NaOH) was
volumes of hexane/2-propanol (3:2, v/v). The hexane layer was
evaporated to dryness in vacuo, dissolved in the same solvent, and
applied on a silica gel 60 NH (40–50 mm; Kanto Chem. Co., Tokyo,
2
ꢂ
solution was poured into a separatory funnel, added Na
(
(
2
EDTA
0.73 g, 1 mmol; dissolved in 5 mL of water) and methanol
400 mL), and the reaction products were extracted with chloro-
Japan) mini-column made from Pasteur pipettes. The column was
eluted with hexane/2-propanol (3:2, v/v; 4 mL) and then with
methanol (4 mL). The two fractions were evaporated in vacuo and
form (400 mL). The chloroform layer was washed with distilled
water, and the solvent was removed in vacuo to obtain brown oil
re-dissolved in 100
propanol fraction was analyzed for
a
m
L of ethanol, respectively. The hexane/2-
-tocopherol, -TQ, and
-tocopherol dimer; the methanol fraction was analyzed for the
a
a
(
ca. 600 mg). This oil was dissolved in ethanol and analyzed by
reversed-phase HPLC coupled with ESI-MS as described above. For
isolation of the reaction products, an Inertsil Prep ODS column
addition products of
radicals, respectively. The amounts of
a
-tocopherol with PLPC-OOH-derived free
-tocopherol and -TQ were
a
a
(
2 cm ꢀ 25 cm, GL Sciences, Tokyo, Japan) was developed with
determined by HPLC on an Inertsil ODS-80A column (4.6 mm
ethanol at 20 mL/min. The eluent was monitored at 210 nm.
For time-course experiment, PLPC-OOH without or with
ꢀ 150 mm) with 98% methanol at 1.0 mL/min by monitoring
elution at 285 nm. g-Tocopherol was used as the internal standard.
a
-tocopherol was dispersed in 5.0 mL of 0.1 M sodium phosphate
a-Tocopherol dimer was determined using the same column with

6
4
R. Yamauchi et al. / Chemistry and Physics of Lipids 184 (2014) 61–68
+
elution of ethanol at 1.0 mL/min and monitored at 290 nm. The
addition products of -tocopherol were quantified by HPLC using
1224.9 (35, [M + Na] ). Peak 6 was isolated by preparative
reversed-phase HPLC, and its structure was characterized as
subsequently described.
a
an Inertsil ODS-3 column (4.6 mm ꢀ 150 mm) with ethanol/
methanol (1:1, v/v) at 1.0 mL/min by monitoring elution at 210 nm.
Compound 6 was obtained as a colorless wax (114 mg yield),
and shown UV absorbance maxima at 206 (log
(shoulder, log 3.31), and 289 nm (log 3.38) in ethanol. The H
NMR spectrum of 6 was consistent with that expected for the
addition products of -tocopherol with 12,13-epoxy-PLPC radicals
(Table 1). The spectrum shows methin proton signals of
2,13-epoxy ring at 2.62–3.02, oxygen-bearing methin protons
at 3.84 (6b) and 4.02 (6a), and olefinic proton signals at
5.02–5.88 on 2-acyl moiety; methyl proton signals at
1.22, 2.06, and 2.09 and 2.10 on chroman ring of
moiety; and three methyl protons at 3.37 on choline moiety,
respectively. From these spectral data, the structure of compound 6
was determined to be a mixture of 1-palmitoyl-2-(9- -tocopher-
oxy-12,13-epoxy-10-octadecenoyl)-3-sn-phosphatidylcholine (6a)
and 1-palmitoyl-2-(11- -tocopheroxy-12,13-epoxy-9-octadece-
e 4.59), 282
1
3
. Results
e
e
3.1. Characterization of the hemin-catalyzed reaction products of
a
PLPC-OOH with
a-tocopherol in micelles
1
d
Micelles containing PLPC-OOH and
a
-tocopherol were reacted
d
d
ꢂ
at 37 C for 30 min by the addition of hemin. The reaction products
were analyzed reversed-phase HPLC coupled with ESI-MS (Fig. 2).
Peaks corresponding to the reaction products, 4–6, and other
unknown peaks appeared on the chromatogram. The identity of
each peak was confirmed by ESI-MS and by comparison of the
HPLC behavior with that of the authentic sample. Peak 4 was
d
1.21 and
a
-tocopherol
d
a
identified as the spirodiene dimer of
a
-tocopherol (Nelan and
a
Robeson, 1962): ESI-MS m/z (relative intensity, %) 857.7 (100,
noyl)-3-sn-phosphatidylcholine (6b) (T-epoxyPLPC, Fig. 1). Al-
though the magnitude of the olefinic coupling constant (J = 15.2 Hz)
for 6a was a typical of a trans olefin, we could not resolve the
olefinic configuration of 6b. The ratio of 6a and 6b was calculated to
be almost the same from the H NMR data (Table 1). The mixture of
6a and 6b could not be further separated under the present HPLC
+
+
[
M + H] ) and 879.6 (6, [M + Na] ). Peak 5 was identified as the
addition products of -tocopherol with 12,13-epoxyperoxyl
radicals derived from PLPC-OOH (TOO-epoxyPLPC) (Yamauchi
a
+
1
et al., 2002a): ESI-MS m/z 790.5 (43, [C42
H81NO10P] ), 806.5 (100,
+
+
[C
42
H81NO11P] ), and 1257.1 (57, [M + Na] ). The major peak 6 was
assumed to be the addition products of -tocopherol with
epoxyPLPC radicals: ESI-MS m/z 1202.9 (100, [M + H] ) and
a
conditions.
+
Table 1
1
H NMR chemical shifts of a mixture of 1-palmitoyl-2-(9-a-tocopheroxy-12,13-
epoxy-10-octadecenoyl)-3-sn-phosphatidylcholine (6a) and 1-palmitoyl-2-(11-
-tocopheroxy-12,13-epoxy-9-octadecenoyl)-3-sn-phosphatidylcholine (6b).
a
Chemical shift (
d
in CDCl
3
)a
Assignment
H-16c (palmitoyl), H-18 (TEODE ),
b
d
0
.84–0.89 (m , 18H)
0
0
0
0
a
H-4 a, -8 a, -12 a, -13 ( -TH)
0
0
0
a
1
1
1
1
.04–1.15 (m, 3H)
.17–2.17 (m, 64H)
.21 and 1.22 (s, 3H)
.75 (m, 1H)
.79 (m, 1H)
H-4 , -8 , -12 (
—CH
H-2a (
-TH)
2
—
a
-TH)
-TH)
-TH)
H-3 (
H-3 (
a
a
1
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
4
4
4
4
5
5
5
5
5
5
5
5
.06 (s, 6H)
.09 and 2.10
.27 and 2.29 (t, J = 8.2 Hz, 4H)
.53 (m, 1/4H)
.54 (m, 2H)
.62 (m, 1/2H)
.78 (m, 1/4H)
.89 (m, 1/4H)
.00 (m, 1/2H)
.02 (m, 1/4H)
.37 (s, 9H)
.80 (br. m, 2H)
.84 (m, 1/2H)
.91 (m, 1H)
H-5a, -8a (
(s, 3H) H-7a (
H-2 (palmitoyl), H-2 (TEODE)
H-13 (11-TEODE, 6b)
a
-TH)
a
-TH)
H-4 (a-TH)
H-13 (9-TEODE, 6a)
H-13 (11-TEODE, 6b)
H-12 (11-TEODE, 6b)
H-12 (9-TEODE, 6a)
H-12 (11-TEODE, 6b)
+
N (CH
3
)
3
(choline)
H-2 (choline)
H-11 (11-TEODE, 6b)
H-1 (glycerol)
H-1 (glycerol)
H-9 (9-TEODE, 6a)
H-3 (glycerol)
.95 (m, 1H)
.02 (m, 1/2H)
.12 (dd, J = 7.2, 12.0 Hz, 1H)
.30 (br. m, 2H)
.40 (d, J = 11.7 Hz, 1H)
.02 (dd, J = 8.2, 15.2 Hz, 1/4H)
.18 (m, 1/4H)
.19 (br. m, 1H)
.53 (m, 1/4H)
.56 (m, 1/4H)
.64 (m, 1/4H)
H-1 (choline)
H-3 (glycerol)
H-11 (9-TEODE, 6a)
H-11 (9-TEODE, 6a)
H-2 (glycerol)
H-9 (11-TEODE, 6b)
H-10 (11-TEODE, 6b)
H-10 (11-TEODE, 6b)
H-9 (11-TEODE, 6b)
H-10 (9-TEODE, 6a)
.68 (m, 1/4H)
.88 (dd, J = 8.2, 15.2 Hz, 1/2H)
a: Shifts in parts per million downfield relative to tetramethylsilane; b: multiplicity:
s, singlet; d, doublet; t, triplet; m, multiplet; br., broad; c: the numbering systems
Fig. 2. Reversed-phase HPLC and positive ESI-MS of the hemin-catalyzed reaction
used are the standard numbering systems for the fatty acid chains, the
moiety, and the glycerol moiety of sn-glycero-3-phosphocholine; d:
-tocopherol; 9-TEODE, 9-( -tocopheroxy)-12,13-epoxy-10-octadecenoyl; 11-
TEODE, 11-( -tocopheroxy)-12,13-epoxy-9-octadecenoyl.
a
-tocopherol
products of PLPC-OOH with
a-tocopherol. HPLC was done with a Luna C18(2)
a
-TH,
column (2 mm ꢀ 150 mm) that was developed with 10 mM ammonium acetate in
a
a
ethanol/methanol (7:3, v/v) at 0.20 mL/min.
a

R. Yamauchi et al. / Chemistry and Physics of Lipids 184 (2014) 61–68
65
3.2. Reaction of PLPC-OOH with a-tocopherol in micelles
Micellar solutions of PLPC-OOH without or with
were reacted with hemin (Fig. 3). The rapid decrease of PLPC-OOH
by hemin was observed in the absence of -tocopherol, and
hexanal was formed as one of the secondary oxidation product.
-Tocopherol suppressed the formation of hexanal, but it did not
affect the rate of PLPC-OOH decay. During the reaction, -tocoph-
erol disappeared rapidly and its reaction products, -TQ, -to-
a-tocopherol
a
a
a
a
a
copherol dimer, TOO-epoxyPLPC, and T-epoxyPLPC, were
observed. When micelles consisted of 0.5 mM POPC, 0.1 mM
a
-tocopherol, and 10
of the -tocopherol remained in the micelles (data not shown).
Fig. 4 summarizes the amounts of hexanal and -tocopherol
products when PLPC-OOH micelles containing different amounts
of -tocopherol were reacted with hemin for 5 min. The formation
of hexanal was almost suppressed by the tested amounts of
-tocopherol. The distribution of -tocopherol products was
depending on the added amounts of -tocopherol. The main
product at lower concentrations of -tocopherol was -TQ, while
more -tocopherol dimer and T-epoxyPLPC were observed at
higher concentrations.
mM hemin were incubated for 5 min, almost
a
a
a
a
a
a
a
a
a
Fig. 4. Formations of hexanal and
a
-tocopherol products during the hemin-
catalyzed reaction of PLPC-OOH with different concentration of
a-tocopherol in
micelles. Micelles consisted of PLPC-OOH (0.5 mM) and the indicated concentration
3.3. Reaction of PLPC-OOH with
a-tocopherol in POPC liposomes
ꢂ
of
a-tocopherol were incubated with hemin (10
m
M) at 37 C for 5 min. Each value is
expressed as the mean ꢃ S.D. of three separate experiments. Different letters in each
The hemin-catalyzed reaction of PLPC-OOH was also performed
product are significantly different by Student's t-test (p < 0.05).
in POPC liposomes (Fig. 5). PLPC-OOH in POPC liposomes was
rapidly decomposed by the addition of hemin, but only half of the
starting PLPC-OOH was consumed. The produced hexanal in POPC
Fig. 3. Hemin-catalyzed reaction of PLPC-OOH with
a-tocopherol in micelles. The
Fig. 5. Hemin-catalyzed reaction of PLPC-OOH with
a-tocopherol in POPC
reaction system consisted of PLPC-OOH (0.5 mM),
a-tocopherol (0.1 mM), and
liposomes. The reaction system consisted of POPC (9 mM), PLPC-OOH (1 mM),
hemin (10
m
M) in 0.1 M sodium phosphate buffer (pH 7.4) containing 5 mM sodium
a
-tocopherol (0.2 mM), and hemin (50
m
M) in 50 mM sodium phosphate buffer (pH
ꢂ
ꢂ
deoxycholate. The micelles were incubated at 37 C. PLPC-OOH and hexanal (O)
7.4) containing 50 mM NaCl. The liposomes were incubated at 37 C. PLPC-OOH and
without hemin, (*) with hemin, or (
4
) with hemin and
a
-tocopherol; (~)
hexanal (O) without hemin, (*) with hemin, or (
4
) with hemin and
a-tocopherol;
a-tocopherol; and its reaction products, (
5
)
a-TQ, (&)
a-tocopherol dimer, (^)
(~)
a-tocopherol; and its reaction products, (
5
)
a
-TQ, (&) -tocopherol dimer,
a
TOO-epoxyPLPC, and (^) T-epoxyPLPC, are shown. Each value is expressed as the
mean ꢃ S.D. of three separate experiments.
(^) TOO-epoxyPLPC, and (^) T-epoxyPLPC, are shown. Each value is expressed as
the mean ꢃ S.D. of three separate experiments.

6
6
R. Yamauchi et al. / Chemistry and Physics of Lipids 184 (2014) 61–68
This treatment destroys the liposomal structure to make micelles.
As a result, PLPC -OOH and -tocopherol could further react with
hemin, and additional amounts of hexanal and the reaction
products of -tocopherol were produced. Especially the marked
increase was observed in the formation of -TQ.
a
a
a
4
. Discussion
Lipid hydroperoxides are further cleaved oxidatively to yield
peroxyl radicals, or reductively to yield alkoxyl radicals by
transition metals (Gardner, 1989). Both alkoxyl and peroxyl
radicals initiate new chain reactions, while alkoxyl radicals further
cleave to yield aldehydes and other secondary oxidation products
(Grosch, 1987). Thus, the radical-trapping action of a-tocopherol
would be expected to suppress the formation of such secondary
products from lipid hydroperoxides. It has been reported that
a
-tocopherol could trap such hydroperoxide-derived free radicals
to form some addition products (Gardner et al., 1972; Kaneko and
Matsuo, 1985; Yamauchi et al., 1995, 2002a). In this study, we have
characterized such addition products on the hemin-catalyzed
reaction of PLPC-OOH with
a
-tocopherol in micellar solutions
Fig. 6. Formations of hexanal and
catalyzed reaction of PLPC-OOH with different concentration of
POPC liposomes. Liposomes consisted of POPC (9 mM), PLPC-OOH (1 mM), and the
a
-tocopherol products during the hemin-
(Fig. 2). The main products were characterized to be 6-O-
a-tocopherol in
epoxyPLPC-a-tocopherols (T-epoxyPLPC, 6), in which the 12,13-
ꢂ
epoxy carbon-centered radicals derived from PLPC-OOH directly
attacked the 6-hydroxyl group of -tocopherol. The 6-O-epox-
ylipid- -tocopherols have been reported in the iron-catalyzed
reactions of -tocopherol with linoleate 13-hydroperoxide under
anaerobic conditions (Gardner et al., 1972; Yamauchi et al., 1995).
-Tocopherol also trapped the epoxyperoxyl radicals from
PLPC-OOH to produce TOO-epoxyPLPC (5) as reported previously
Yamauchi et al., 2002a), and the self-reaction of -tocopherol
indicated concentration of
a-tocopherol were reacted with hemin (5
m
M) at 37 C
for 5 min. Each value is expressed as the mean ꢃ S.D. of three separate experiments.
a
Different letters in each product are significantly different by Student's t-test
a
(
p < 0.05).
a
a
liposomes was low compared with that in micellar systems as
indicated in Fig. 3. -Tocopherol accelerated the decomposition of
PLPC-OOH and partly suppressed the formation of hexanal. The
main products of -tocopherol were -tocopherol dimer and TOO-
epoxyPLPC with smaller formations of -TQ and T-epoxyPLPC.
Fig. 6 shows the formations of hexanal and -tocopherol products
when POPC liposomes containing PLPC-OOH and different
amounts of -tocopherol were reacted with hemin for 5 min.
The formation of hexanal was more suppressed by the high
amount of -tocopherol. The main product of -tocopherol was
TOO-epoxyPLPC, and excess addition of -tocopherol resulted in
higher production of -tocopherol dimer. Relatively small
-TQ and T-epoxyPLPC were observed in the liposomal
a
(
a
produced dimer (4).
a
a
Hemin-catalyzed reaction of PLPC-OOH with a-tocopherol (TH)
in micellar and liposomal systems would proceed with the
following process:
a
a
ꢄ
a
PLPC-OOH + Hemred ! PLPC-O + Hemox + H+
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
a
a
a
ꢄ
ꢁ
a
PLPC-OOH + Hemox ! PLPC-OO + Hem + OH
red
amounts of
systems.
a
POPC liposomes containing 1 mM PLPC-OOH and 0.5 mM
-tocopherol were reacted with 50 M of hemin for 5 min. After
ꢄ
a
m
TH + Hemox ! T + Hem
red
the incubation, an equal volume of 20 mM sodium deoxycholate in
buffer solutionwas added and further incubated for 5 min (Table 2).
ꢄ
ꢄ
Table 2
PLPC-O ! epoxyPLPC
Effect of the deoxycholate treatment after the hemin-catalyzed reaction of PLPC-
OOH with
a-tocopherol in POPC liposomes.
Treatment
Control
ꢄ
! epoxyPLPC-OOꢄ
epoxyPLPC-O + O
2
Compound
Deoxycholate
PLPC-OOH
Hexanal
167 ꢃ 7a
44 ꢃ 3b
0.7 ꢃ 0.1a
5.2 ꢃ 0.1b
31.9 ꢃ 1.9b
22.8 ꢃ 0.3b
36.9 ꢃ 1.6a
29.6 ꢃ 1.6b
2.7 ꢃ 0.2b
a
a
-Tocopherol
-TQ
64.4 ꢃ 3.2a
2.9 ꢃ 0.4a
40.3 ꢃ 0.7a
19.2 ꢃ 2.7a
1.7 ꢃ 0.2a
ꢄ
ꢄ
T + epoxyPLPC-OO ! TOO-epoxyPLPC
Dimer
TOO-epoxyPLPC
T-epoxyPLPC
Liposomes consisted of POPC (9 mM), PLPC-OOH (1 mM), and
a-tocopherol
ꢄ
ꢄ
T + epoxyPLPC ! T-epoxyPLPC
(
0.5 mM) in 50 mM sodium phosphate buffer (pH 7.4) containing 50 mM NaCl
ꢂ
were reacted with hemin (50
mM) at 37 C for 5 min. After the incubation, the same
volume of the buffer solution (control) or the buffer solution containing 20 mM
sodium deoxycholate (deoxycholate) was added, and further incubated for 5 min.
The concentration of each compound indicates the value after addition of the buffer
solution. Each value is expressed as the mean ꢃ S.D. of three separate experiments.
Different letters in a row are significantly different by Student's t-test (p < 0.05).
ꢄ
ꢄ
T + T ! T–T

R. Yamauchi et al. / Chemistry and Physics of Lipids 184 (2014) 61–68
67
a
-tocopherol were observed when the membrane structure was
destroyed by the addition of sodium deoxycholate (Table 2). This
destruction of liposomes resulted in the marked increase of -TQ,
The first step may be the reduction of PLPC-OOH by the reduced
form of hemin (Hemred) to produce the alkoxyl radical (PLPC-O )
ꢄ
a
and the oxidized form of hemin (Hemox) (Eq. (1)), or the oxidation
of PLPC-OOH by Hemox to produce the corresponding peroxyl
radical (PLPC-OO ) and Hemred (Eq. (2)) (Koga et al., 1991; Carlsen
probably due to the degradation reaction of TOO-epoxyPLPC by
hemin.
ꢄ
This study indicated that a-tocopherol could suppress the
et al., 2005). In the presence of
a
-tocopherol, Hemox is reduced by
formation of hexanal, one of secondary oxidation product, by
trapping the epoxyperoxyl and epoxyalkyl radicals derived from
ꢄ
a
-tocopherol and forms Hemred and
a-tocopheroxyl radical (T )
(
Eq. (3)) (Yamamoto and Niki,1988). Therefore, the present hemin-
PLPC-OOH. However, the inhibitory effect of
formation of was limited in POPC liposomes. Further studies will be
needed to clarify the effect of -tocopherol on the formation of
a-tocopherol on the
ꢄ
ꢄ
catalyzed reaction preferentially produced PLPC-O and T by Eqs.
(
ꢄ
1) and (3). The PLPC-O then rearranges intra-molecularly by
a
addition to the adjacent double bond and forms the carbon-
secondary aldehydic products in biological systems.
ꢄ
centered radical of 12,13-epoxyPLPC (epoxyPLPC ) (Eq. (4)) (Gard-
ꢄ
ner, 1989; Wilcox and Marnett, 1993). The epoxyPLPC couples
Conflict of Interest
with molecular oxygen at a diffusion-controlled rate and forms the
ꢄ
peroxyl radicals of 12,13-epoxyPLPC (epoxyPLPC-OO ) in the
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
presence of oxygen (Eq. (5)) (Erben-Russ et al., 1987). The
ꢄ
ꢄ
produced epoxyPLPC-OO could be trapped by T to form addition
products (TOO-epoxyPLPC, 5) (Eq. (6)) as reported previously
Transparency document
(Yamauchi et al., 2002a). In addition, we have detected another
addition product, T-epoxyPLPC (6), by the reaction between
The Transparency document associated with this article can be
found in the online version.
ꢄ
ꢄ
epoxyPLPC and T (Eq. (7)) under the
ditions in micelles (Fig. 4). The -tocopheroxyl radical also
resulted in the formation of dimer (T–T) by a bimolecular self-
reaction (Eq. (8)) under the -tocopherol-rich conditions
Figs. 4 and 6). The overall hemin-catalyzed reaction proceeded
a-tocopherol-rich con-
a
a
Acknowledgement
(
very rapidly and terminated within several minutes (Figs. 3 and 5).
Hemoproteins and hemin have been demonstrated high
reactivity with PLPC-OOH in micelles to generate aldehydic
products, such as HNE and MDA with low yields (Hayashi et al.,
This work was supported by Grants-in-Aid for Scientific
Research (Nos. 23580162 and 26450155) from Japan Society for
the Promotion of Science.
2004). In the similar reaction conditions, we detected hexanal with
relative high yield (ca. 5% of the starting PLPC-OOH after incubation
for 5 min), although HNE and MDA were not measured. Since
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