Cholesterol hydroperoxides generate singlet molecular oxygen [O 2(1Δg)]: Near-IR emission, 18O-labeled hydroperoxides, and mass spectrometry

Article abstract of DOI:10.1021/tx200079d

In mammalian membranes, cholesterol is concentrated in lipid rafts. The generation of cholesterol hydroperoxides (ChOOHs) and their decomposition products induces various types of cell damage. The decomposition of some organic hydroperoxides into peroxyl

Full text of DOI:10.1021/tx200079d

ARTICLE
pubs.acs.org/crt
Cholesterol Hydroperoxides Generate Singlet Molecular Oxygen
[O2 (1Δg)]: Near-IR Emission, 18O-Labeled Hydroperoxides,
and Mass Spectrometry
Miriam Uemi, Graziella E. Ronsein, Fernanda M. Prado, Flꢀavia D. Motta, Sayuri Miyamoto,
Marisa H. G. Medeiros, and Paolo Di Mascio*
Departamento de Bioquímica, Instituto de Química, Universidade de S~ao Paulo, CEP 05513-970, CP26077, S~ao Paulo, SP, Brazil
S Supporting Information
b
ABSTRACT: In mammalian membranes, cholesterol is con-
centrated in lipid rafts. The generation of cholesterol hydro-
peroxides (ChOOHs) and their decomposition products
induces various types of cell damage. The decomposition of
some organic hydroperoxides into peroxyl radicals is known to
be a potential source of singlet molecular oxygen [O2 (1Δg)] in
biological systems. We report herein on evidence of the
generation of O2 (1Δg) from ChOOH isomers in solution or
in liposomes containing ChOOHs, which involves a cyclic
mechanism from a linear tetraoxide intermediate originally
proposed by Russell. Characteristic light emission at 1270 nm,
corresponding to O2 (1Δg) monomolecular decay, was ob-
served for each ChOOH isomer or in liposomes containing
ChOOHs. Moreover, the presence of O2 (1Δg) was unequivocally demonstrated using the direct spectral characterization of near-
infrared light emission. Using 18O-labeled cholesterol hydroperoxide (Ch18O18OH), we observed the formation of 18O-labeled O2
(1Δg) [18O2 (1Δg)] by the chemical trapping of 18O2 (1Δg) with 9,10-diphenylanthracene (DPA) and detected the corresponding
18O-labeled DPA endoperoxide (DPA18O18O) and the 18O-labeled products of the Russell mechanism using high-performance
liquid chromatography coupled to tandem mass spectrometry. Photoemission properties and chemical trapping clearly
demonstrate that the decomposition of Ch18O18OH generates 18O2 (1Δg), which is consistent with the Russell mechanism
and points to the involvement of O2 (1Δg) in cholesterol hydroperoxide-mediated cytotoxicity.
’ INTRODUCTION
oxygen has been shown to be generated in biological
systems.21,22 As possible biological sources of O2 (1Δg), some
examples include enzymatic processes catalyzed by peroxidases
or oxygenases, reactions of hydrogen peroxide with hypochlorite
or peroxynitrite, and the thermodecomposition of dioxetanes,
endoperoxides, and type II photosensitization reactions.23ꢀ29
UV irradiation of aromatic amino acids in protein immunoglo-
bulins is responsible for the production of O2 (1Δg).30ꢀ33 The
presence of a pair of electrons with opposite spin in the highest
occupied molecular orbital confers dienophilic properties to the
O2 (1Δg), which explains its substantial reactivity toward
electron-rich organic molecules, particularly with those exhibit-
ing conjugated double bonds.19,20 The reactions of O2 (1Δg)
with unsaturated fatty acids, proteins, and DNA have been
extensively studied since this activated oxygen species can
induce various types of cell damage related to aging, cancer,
and other cytotoxic effects.34
Cholesterol is a neutral lipid found in all mammalian mem-
branes and is especially concentrated in lipid rafts. Lipid rafts are
subdomains of the plasma membrane that contain high con-
centrations of cholesterol and glycosphingolipids.1 Cholesterol,
as an unsaturated lipid, is also susceptible to oxidation in the
presence of reactive oxygen species (ROS), giving rise to
mutagenic and cytotoxic products.2,3 Cholesterol hydroperox-
ides (ChOOHs) are the first oxidation products formed during
cholesterol photooxidation4ꢀ9 and lipid peroxidation.10,11 The
generation of ChOOH and its decomposition products in lipid
rafts can affect the binding of signaling molecules, receptors, and
proteins.12,13 These molecules can be targets of ROS produced
through ChOOH decomposition, leading to the loss of function
or altered signaling pathways. Thus, the identification of
ChOOHs provides important insights into the role ROS plays
in biological damage.14ꢀ18
Singlet molecular oxygen [O2 (1Δg)] in its first excited state,
denoted as O2 (1Δg), exhibits a substantial reactivity toward
electron-rich organic molecules, leading to the formation of
allylic hydroperoxides, dioxetanes, or endoperoxides.19,20 Singlet
Received: February 18, 2011
Published: April 21, 2011
r
2011 American Chemical Society
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Scheme 1. Chemical Trapping of 16O2 (1Δg) and 18O-Labeled
O2 (1Δg) by DPA To Form the Stable Corresponding
Anthracene Endoperoxide DPAxOxO (x = 16 or 18)
column (ThermoQuest) (Supporting Information, Figure S1). The
mobile phase was acetonitrile, water, and methanol (90:8:2,
v/v/v), and compounds were eluted in isocratic mode at flow rate of
3.6 mL/min. The UV detector was set at 210 nm.7 The quantification of
each isolated ChOOH was done using the iodometric method described
by Girotti et al.43 and Thomas et al.44
Synthesis and Purification of 18O-Labeled Cholesterol
Hydroperoxide (Ch18O18OHs). Photooxidation of cholesterol
(51.7 mM dissolved in chloroform) in an 18O2-saturated atmosphere
was performed in the presence of methylene blue (0.2 mM in
methanol).41 Irradiation was carried out in an ice bath for 3 h using
light from a tungsten lamp (500 W). The oxygen contained in the system
was removed by successive freezing and thawing under vacuum. This
procedure was repeated at least five times to ensure complete removal of
16O2. Thereafter, the whole system was connected to an 18O2 gas
cylinder under 0.5 atm. Cholesterol-oxidized products generated by
photosensitization, namely, 7R-OOH, 5R-OOH, 6R-OOH, 6β-OOH,
and 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (ChAld),
were purified by TLC and HPLC according to Ronsein et al.41 HPLC
coupled to dopant-assisted atmospheric pressure photoionization
(APPI) tandem mass spectrometry of ChOOH and ChAld was
performed in an Agilent HPLC system (1200 series, Waldbronn,
Germany) connected to a 4000 Q Trap mass spectrometer with an
APPI source (Applied Biosystems, Foster City, CA) (Supporting
Information, Figure S2).41
It is well-known that lipid hydroperoxides can generate singlet
molecular oxygen through the self-reaction of peroxyl radicals via
the Russell mechanism.35ꢀ38 To study the possibility of ChOOHs
generating O2 (1Δg), photooxidation of cholesterol was per-
formed, and ChOOHs 3β-hydroxicholest-5-ene-7R-hydroper-
oxide (7R-OOH), 3β-hydroxycholest-4-ene-6β-hydroperoxide
(6β-OOH), and 3β-5R-cholest-6-ene-5-hydroperoxide (5R-OOH)
were isolated and purified by high-performance liquid chroma-
tography (HPLC).39ꢀ41 O2 (1Δg) was measured using chemi-
luminescence, which showed that ChOOHs are able to produce
O2 (1Δg) in the presence of cerium ions (Ce4þ). Moreover, the
reaction of ChOOHs with Ce4þ produced an alcohol and a
ketone, two products formed via the Russell mechanism, which
were detected by high-pressure liquid chromatography coupled
to tandem mass spectrometry (HPLC/MS/MS). Additionally,
18O-labeled O2 (1Δg) [18O2 (1Δg)] was also observed by
chemical trapping with 9,10-diphenylanthracene (DPA) in
a two phase CHCl3/D2O, generating the corresponding
18O-labeled DPA endoperoxide (DPA18O18O) (Scheme 1),
detectable by HPLC/MS/MS.
Monomol Light Emission Measurements of O2 (1Δg).
Monomol light emission of O2 (1Δg) at 1270 nm was monitored during
the reaction of ChOOH mixture or each isolated ChOOH and Ce4þ
ions. For this assay, the Ce4þ solution (final concentration, 3.1 mM,
prepared in D2O) was infused into a quartz cuvette containing a
ChOOH (final concentration, 9.4 mM in ethanol) at a flow rate of
0.6 mL/min.
For the acquisition of monomol light emission spectrum of O2 (1Δg),
Ce4þ ions (final concentration, 6 mM in D2O) was infused at flow rate of
0.6 mL/min into a mixture of ChOOH (final concentration, 5.4 mM).
As a reference, the spectrum of O2 (1Δg) produced in the reaction of
H2O2 and HOCl and DMNO2 was also acquired. For H2O2 and HOCl
reaction, HOCl was infused into H2O2 solution (final concentration,
0.28 and 1 M, respectively) at a flow rate of 1.4 mL/min and for
DMNO2, a 60 mM concentration of the endoperoxide in methanol was
incubated at 40 °C.
HPLC/MS/MS Analysis of Products Generated in the Reac-
tion of ChOOH and Ce4þ Ions. The products generated in the
reaction of ChOOHs (7R-OOH, 5R-OOH, and 6β-OOH) and Ce4þ
ions were analyzed by a Shimadzu HPLC system (Tokyo, Japan) coupled
to a triple-quadrupole mass spectrometer (Quattro II, Micromass,
Altricham, United Kingdom) (HPLC/MS/MS) (Supporting Informa-
tion, Figures S3 and S4).
For HPLC/MS/MS analysis, 30 μL of the reaction mixture was
injected into a Shimadzu C18 column (250 mm ꢁ 4.6 mm, 5 μm
particle size) and eluted in isocratic mode with acetonitrile and
1% ammonium formate (91:9, v/v) as the solvent. The flow rate
was 1.5 mL/min. The column oven and UV detector were set at 25 °C
and 210 nm, respectively. The analysis in the mass spectrometer was
performed using positive atmospheric pressure chemical ionization
source (APCIþ). The mass spectrometer parameters were as follows:
source temperature, 150 °C; APCI probe temperature, 400 °C;
sample cone voltage, 25 V; extractor cone voltage, 5 V; corona
potential, 3.5 kV; and collision energy, 15 eV. The flow rates of drying
and nebulizing gases were 400 and 30 L/h, respectively. The HPLC/
MS/MS detection of alcohol (ChOH) and ketone (ChKeto) was
performed by SRM (selected reaction monitoring) mode by monitor-
ing the mass transition of m/z 385 f 383 for ChOH and m/z 401 f
383 for ChKeto.
’ MATERIAL AND METHODS
Materials. Cholesterol (cholest-5-en-3β-ol) was obtained from
Sigma (St. Louis, MO). Silica gel 60 (230ꢀ400 mesh), ammonium
cerium(IV) nitrate, dimyristoylphosphatidylcholine (DMPC), DPA,
9,10-dibromoanthracene (DBA), and sodium azide were purchased from
Aldrich (Steinheim, Germany). The 18O2 gas cylinder (99% 18O) was
from Isotec-Sigma (St. Louis, MO). Deuterium oxide (D2O) was
acquired from Cambridge Isotope Laboratories (Rio de Janeiro, Brazil).
Silica gel 60 F254 plates, methylene blue, and solvents of HPLC grade
were acquired from Merck (Rio de Janeiro, Brazil). The water used in the
experiments was treated with the Nanopure Water System (Barnstead,
Dubuque, IA). The 1,4-dimethylnaphthalene endoperoxide (DMNO2)
was synthesized as described by Di Mascio et al.42
Synthesis and Purification of ChOOHs. ChOOHs (7R-OOH,
5R-OOH, and 6β-OOH) were synthesized by photooxidation of choles-
terol using methylene blue as a sensitizer. Two hundred milligrams of
cholesterol was dissolved in 20 mL of chloroform containing
10 μM methylene blue, prepared in methanol. The solution under
continuous stirring and oxygen-saturated atmosphere was cooled at
4 °C and irradiated using two tungsten lamps (500 W) for 2.5 h. The
ChOOHs were purified by silica gel 60 column (230ꢀ400 mesh). The
column was equilibrated with hexane, and then, a gradient of hexane and
ethyl ether was used. The ChOOHs were analyzed by thin-layer chro-
matography (TLC) using Merck 0.25 mm coated silica gel 60 F254 plates.
The TLC plate was eluted with ethyl acetate and isooctane (1:1, v/v).
The ChOOH isomers were purified using a Shimadzu HPLC system
(Tokyo, Japan), and a 250 mm ꢁ 10 mm (particle size, 5 μm) ODS
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temperature of 300 °C, sample cone voltage of 15 V, extractor cone
voltage of 4 V, corona potential of 3.5 kV, and collision energy of 15 eV.
The flow rates of drying and nebulizing gases were 400 and 30 L/h,
respectively.
A mixture of Ch18O18OHs (final concentration, 20 mM in
chloroform) was reacted with DPA (final concentration, 60 mM in
chloroform) and Ce4þ ions (final concentration, 20 mM in D2O). The
reaction was protected from light and maintained at 37 °C during 15 min
under stirring (1300 rpm). After that, 20 μL of reaction was dried with
nitrogen gas, and the residue was dissolved in 50 μL of acetone. From
this solution, 40 μL was mixed with 40 μL of acetonitrile. Finally, an
aliquot of 70 μL was injected into the HPLC/MS/MS system.
Preparation of Liposomes Containing ChOOH. Liposomes
of defined size (100 nm) were prepared by an extrusion technique.45
Briefly, a solution containing ChOOH mixture (2 mM, final con-
centration) and DMPC (2 mM, final concentration) in ethanol was
dried under nitrogen atmosphere and by vacuum. Then, 1 mL of D2O
was added. The solution was mixed vigorously for 1 min and extruded
21 times through a 100 nm polycarbonate membrane filter using the
Liposofast kit (Avestin Inc., Ontario, Canada).
Figure 1. Monomol light emission of O2 (1Δg) at 1270 nm generated in
reactions containing a mixture of ChOOHs (ChOOHs) or the isolated
isomers (5R-, 6β-, and 7R-OOH). Arrows indicate the injection of
Ce4þ solution.
’ RESULTS
The chemiluminescence studies provided important infor-
mation about the excited species generated during the reac-
tion. This method is used for the detection and character-
ization of the radioactive monomol transition of O2 (1Δg) to
Chemical Trapping of O2 (1Δg) and HPLC Analysis of the
Reaction ChOOH/Ce4þ/DPA. Evidence of O2 (1Δg) generation in
the reaction ChOOH and Ce4þ was obtained by chemical trapping of O2
(1Δg) with DPA, being the corresponding endoperoxide [9,10-diphe-
nylanthracene endoperoxide (DPAO2)] detected by HPLC
(Supporting Information, Figure S5). For this assay, 50 μL of each
isolated ChOOH (final concentration, 25 mM in chloroform) and 50 μL
of DPA (final concentration, 60 mM in chloroform) were reacted in a
glass tube that was protected from light. Following, 100 μL of Ce4þ ions
was added (final concentration, 25 mM in D2O), and the reaction was
incubated at 37 °C during 1 h with string (1300 rpm). After incubation,
20 μL of the organic phase was removed and dried with nitrogen gas, and
200 μL of acetone was added to the residue. An aliquot of 50 μL was
separated and mixed with 50 μL of NaN3 [final concentration, 20 mM in
H2O/acetonitrile (10:90, (v/v)] and 25 μL of DBA (final concentration,
0.2 mM in acetonitrile).
its ground state (1Δg f Σgꢀ) in the near-infrared region
3
(NIR) at 1270 nm (eq 1).37,38,46
1
3
O2ð ΔgÞ f O2ð ΣgꢀÞ þ hv ðλ ¼ 1270 nmÞ
ð1Þ
Monomol light emission of O2 (1Δg) was measured for the
mixture of ChOOHs and each isolated ChOOH, using a special
photon counting apparatus developed in our laboratory.37,38
For the assay, Ce4þ (final concentration, 3.1 mM in D2O) was
infused into a quartz cuvette containing a mixture of ChOOHs or
the isolated isomers (5R-, 6β-, and 7R-OOH) (final concentra-
tion, 9.4 mM in ethanol) at a flow rate of 0.6 mL/min. The
injection of Ce4þ into the solution produced an intense mono-
mol light emission signal, generated by the monomolecular decay
of O2 (1Δg) (Figure 1).
DPAO2 (30 μL of incubation) was analyzed through a Shimadzu C18
column (250 mm ꢁ 4.6 mm, 5 μm particle size), using 1% ammonium
formate (solvent A) and acetonitrile (solvent B) as the mobile phase
using the following linear gradient: 75ꢀ100% B in 15 min, 100% B for
10 min, 100ꢀ75% B in 5 min, and 75% until 40 min. The flow rate and
UV detector were set at 1 mL/min and 210 nm, respectively.
In addition, the monomol light emission spectrum of O2
(1Δg) produced in the reaction containing the ChOOH mixture
and Ce4þ was also confirmed by recording the spectrum of
the light emitted in the NIR (Figure 2A). For comparative
purposes, the spectrum of the O2 (1Δg) generated by the
reaction of H2O2 with HOCl47ꢀ50 (eq 2, Figure 2B) and by
DMNO2 thermodecomposition (eq 3, Figure 2C) was also
acquired (eq 2).
Chemical Trappingof18O2 (1Δg) and HPLC/MS/MS Analysis
of the Reaction Ch18O18OHs/Ce4þ/DPA. 18O-Labeled O2 (1Δg)
can be produced in the reaction of Ch18O18OHs and Ce4þ. The
generation of 18O2 (1Δg) was confirmed by chemical trapping of
18O2 (1Δg) using DPA. DPA18O18O and DPA18O16O were detected
by HPLC/MS/MS in the SRM mode (m/z 367 f 330 for DPA18O18O
and m/z 365 f 330 for DPA18O16O). Besides 18O-labeled DPA
endoperoxides, it was possible to observe the formation of unlabeled
DPA endoperoxide (DPA16O16O, m/z 363 f 330).
1
H2O2 þ OClꢀ f H2O þ HOCl þ O2ð ΔgÞ ð100%Þ ð2Þ
The HPLC/MS/MS analysis were performed using the same system
described for ChOOH analysis. For analytical purposes a Phenomenex
Gemini C-18 column (250 mm ꢁ 4.6 mm i.d., 5 μm particle size) was
used, and the UV detector was set to 210 nm. The samples were analyzed
using 0.1% formic acid as solvent A and acetonitrile as solvent B, with a
flow rate of 0.8 mL/min. The linear gradient was 75ꢀ100% B for 10 min,
100% B for 10 min, 100ꢀ75% B during 2 min, and 75% B until 30 min.
The flux of 0.25 mL/min was directed to the mass spectrometer from
10 to 15 min, using a FCV-12AH Shimadzu valve. Mass spectrometry
parameters were set at source temperature of 150 °C, APCI probe
All spectra showed an emission band with a maximum
intensity at 1270 nm, characteristic of O2 (1Δg) monomolecular
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Figure 2. NIR monomol light emission spectrum of O2 (1Δg) obtained from the reactions in the (A) ChOOHs/Ce4þ/D2O, (B) H2O2/HOCl/H2O
systems, and in the thermodecomposition of DMNO2.
Scheme 2. Russell Mechanism for the Self-Reaction of Cholesterol Peroxyl Radical (7Ch18O18O• or 5Ch18O18O•), Generating
18O2 (1Δg), 5-Ch18OH, 7-Ch18OH, and 7-Ch18O Keto
decay and, thus, clearly demonstrating that O2 (1Δg) was formed
in the reaction of ChOOHs with Ce4þ. Further evidence that the
light emitted in the reaction corresponds to O2 (1Δg) was
obtained by testing the effect of nondeuterated versus deuterated
solvent and by testing the effect of sodium azide, a known
O2 (1Δg) quencher.51ꢀ53 The intensity of the light emitted in
the reaction conducted in buffer prepared in D2O was higher
than in the reaction conducted in buffer containing H2O. This
observation is consistent with the fact that the lifetime of
O2 (1Δg) is about 10 times longer in D2O than in H2O. The
quenching effect of azide on the chemiluminescent reaction was
also observed.
peroxyl radicals react via a cyclic mechanism35 involving a
acyclic tetraoxide intermediate that decomposes to generate
an alcohol, a ketone, and molecular oxygen (Scheme 2).
This reaction may generate either O2 (1Δg) or an electro-
nically excited ketone.35,36 In fact, it was shown that the genera-
tion of O2 (1Δg) is favored over the electronically excited
ketone.54
To investigate the mechanism involved in the generation of
O2 (1Δg) by ChOOH, the products formed in the reaction of a
2 mM ChOOH isomer (7R-, 6β-, and 5R-OOH) with 2 mM
Ce4þ ions in D2O at 37 °C with 5 min of vigorous mixing were
analyzed by HPLC/MS/MS using the APCI source in the
positive mode. The UV detector connected to the system
was set at 210 nm. The HPLC/MS/MS analyses were per-
formed in the SRM mode by monitoring the loss of one or
two water molecules from the ketone (m/z 401 f 383, peak at
In previous studies, we demonstrated that the generation
of O2 (1Δg) using linoleic acid hydroperoxide in the presence
of metal ions, peroxynitrite, and HOCl involves the Russell
mechanism.28,37,38 In this mechanism, primary or secondary
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Figure 3. HPLC/MS/MS detection of the products generated in the reaction of 7R-OOH and Ce4þ. UV chromatogram at 210 nm of 7R-OOH (A)
and the reaction of 7R-OOH with Ce4þ (B). SRM chromatograms of the mass transition: m/z 401 f 383 (C) and m/z 385 f 367 (D).
23.3 min, Figure 3C) and the alcohol (m/z 385 f 367, peak
at 20.7 min, Figure 3D), respectively.
The peak at 26.0 min (Figure 4D) can be attributed to
7β-ketonecholestan, generated by a dissociative mechanism
from 7R-OOH to 7β-OOH, as previously described by
Beckwith et al.55
The results obtained from the reaction of 7R-OOH and
Ce4þ are shown in Figure 3. The UV chromatogram at 210 nm
shows two peaks, one corresponds to the alcohol 7-ChOH and
the other to the ketone 7-ChKeto (Figure 3B), beyond the peak
of 7R-OOH (Figure 3A) (see the Supporting Information,
Figure S6).
The generation of O2 (1Δg) was also confirmed through the
detection of 18O2 (1Δg) formed in the reaction of 18O-labeled
ChOOHs (Ch18O18OHs; final concentration, 20 mM in
ethanol) with Ce4þ (final concentration, 20 mM in D2O).
18O-labeled O2 (1Δg) was chemically trapped with DPA (final
concentration, 60 mM in chloroform), and the corresponding
18O-labeled DPA endoperoxide (Scheme 1) was detected by
HPLC/MS/MS (Figure 5). The formation of DPAO2 was
observed through monitoring the mass transition of unlabeled
DPA endoperoxide (DPA16O16O, m/z 363 f 330, Figure 5A)
and the mass transition of DPA18O18O (m/z 367 f 330,
Figure 5B and (DPA18O16O, m/z 365 f 330, Figure 5C).
The fragment ion spectrum at m/z 363 (DPA16O16O,
Figure 5A inset), m/z 367 (DPA18O18O, Figure 5B inset),
and m/z 365 (DPA18O16O, Figure 5C inset) shows an intense
fragment ion at m/z 330 that corresponds to the lost of
molecular oxygen from the DPAxOxO (Scheme 1) molecule.
The DPA endoperoxide with one 18O isotopically labeled
oxygen atom (DPA18O16O, m/z 365 f 330) was also detected
by HPLC/MS/MS (Figure 5C). Detection of DPA18O18O
(m/z 367 f 330, Figure 5B) clearly demonstrated the genera-
tion of 18O2 (1Δg) that occurs by the self-reaction of two
18O-labeled peroxyl radicals of ChOOH (Ch18O18O•)
(Scheme 2). The formation of 16O2 (1Δg) and detection of
The results obtained from the reaction of isomer 6β-OOH
and Ce4þ also showed the formation of the corresponding
ketone and alcohol products (see the Supporting Information,
Figures S3 and S4), the two characteristic products of the Russell
mechanism. Yet, analysis of the 5R-OOH/Ce4þ reaction
showed a different pattern of products (Figure 4). Before the
reaction, the UV chromatogram (Figure 4A) showed one major
peak, corresponding to the 5R-OOH at 21.8 min and another
minor peak with the same retention time for 7R-OOH at
19.5 min (Figure 3A). After the reaction, four peaks were
detected (Figure 4B) and the SRM analysis demonstrated that
two of these had the same retention time as did the 7R-OOH
decomposition products (7-ChOH at m/z 385 f 367,
Figure 4C, and 7-ChKeto at and m/z 401 f 383, Figure 4D).
The peak at 22.0 min (Figure 4C) corresponds to a 5R-OOH
decomposition product, 5R-hydroxycholest-6-ene (5-ChOH at
m/z 385 f 367). In the chromatogram in Figure 4D, the peak at
21.8 min corresponds to the residual 5R-OOH, which has the
same SRM mass transition of 7-ChKeto (m/z 401 f 383) and
thus represents the loss of two water molecules from 5R-OOH.
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Figure 4. HPLC/MS/MS detection of the products generated in the reaction of 5R-OOH and Ce4þ. UV chromatogram at 210 nm of 5R-OOH
(A) and reaction of 5R-OOH with Ce4þ (B). SRM chromatogram of the mass transition: m/z 385 f 367 (C) and m/z 401 f 383 (D).
DPA16O16O could be (i) related to a reaction between two
unlabeled peroxyl radicals (Ch16O16O•) generated from resi-
dual unlabeled ChOOHs (12.5%)37 or derived from the re-
placement of 18O2 from the peroxyl moiety of Ch18O18O• to
16O2 dissolved in the reaction system,56 (ii) due to an energy
transfer mechanism from 18O2 (1Δg) to 16O2 in the funda-
mental ground state (3Σgꢀ),57,58 or (iii) due to the reaction of
two peroxyl radicals containing only one labeled oxygen
(Ch18O16O•), which can be formed by oxygen exchange reac-
tions with water or molecular oxygen.37 The detection of
DPA18O16O is probably due to the reaction of labeled and
unlabeled peroxyl radicals.
radical may react with another labeled 7-Ch18O18O• radical,
yielding 18O2 (1Δg), 18O-labeled 5R-hydroxycholestan-6-ene
(5-Ch18OH), and 7-Ch18OKeto (Scheme 2B).
To investigate the biological relevance of these data, we
incorporated pure ChOOH as a mixture of ChOOHs into a
unilamellar liposome. The liposome was prepared using an
extrusion method.45 For the liposome, 2 mM ChOOHs were
incorporated into 2 mM DMPC in D2O. Chemiluminescence
assays were performed using the same instrument as mentioned
above. For the reaction, Ce4þ (final concentration, 8.3 mM in
ethanol) was injected into a 1.5 mL of liposome solution
containing ChOOHs and DMPC in D2O (final concentration,
2 mM) (Figure 6). In this system, O2 (1Δg) generation is
detected by an intense monomol light emission of O2 (1Δg) at
1270 nm (Figure 6A), and sodium azide (NaN3; final concen-
tration, 2 mM) (Figure 6B), a well-known quencher of
O2 (1Δg), is used to suppress emitted light. Our results show,
for the first time, that ChOOHs contained in liposomes yield
O2 (1Δg).
On the basis of these observations and the fact that the
presence of hydrogen-R (Scheme 2) is known to be critical for
the generation of O2 (1Δg) by the Russell mechanism, we
propose the mechanism of O2 (1Δg) generation by 7R-OOH
and 5R-OOH. The mechanism involved in the generation
of 18O2 (1Δg) by the isomer 18O-labeled 7R-OOH
(7R-18O18OH) depends on the formation of the 18O-labeled
7-cholesterol peroxyl (7-Ch18O18O•) radical, which can react
with another 7-Ch18O18O• radical to generate a tetraoxide
intermediate (Scheme 2A). This tetraoxide intermediate
can decompose into 18O2 (1Δg), 18O-labeled alcohol
(7-Ch18OH), and 18O-labeled ketone (7-Ch18OKeto)
(Scheme 2).
In conclusion, the results provide direct evidence of O2 (1Δg)
generation in the self-reaction of two cholesterol peroxyl radicals
via the Russell mechanism, in which two peroxyl radicals
combine via a cyclic mechanism, generating O2 (1Δg), an alcohol,
and a ketone (Scheme 2). Moreover, the detection of the
alcohols 7-ChOH, 5-ChOH, and the ketone 7-ChKeto as the
decomposition products formed in the reaction is strong evi-
dence for the Russell mechanism. In addition, the detection of O2
(1Δg) produced in the ChOOHs/DMPC system indicated the
importance of ChOOH as biological source of O2 (1Δg) in
causing cell damage.
In the case of 18O-labeled 5R-OOH (5R-18O18OH), this isomer
can suffer sigmatropic rearrangement into the isomer
7R-18O18OH,55 which can be oxidized to the 7-Ch18O18O• radical
and also generate 18O2 (1Δg) via the Russell mechanism. Addi-
tionally, the 18O-labeled 5-cholesterol peroxyl (5-Ch18O18O•)
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Figure 5. HPLC/MS/MS analyses of unlabeled (DPA16O16O) and labeled DPA endoperoxide (DPA18O18O and DPA18O16O) formed in the reaction
of 18O-labeled ChOOHs with Ce4þ. Mass transition of DPA16O16O, m/z 363 f 330 (A), DPA18O18O, m/z 367 f 330 (B), and DPA18O16O,
m/z 365 f 330 (C). Inset: Fragment ion spectrum of DPA16O16O at m/z 363 (A), DPA18O18O at m/z 367 (B), and DPA18O16O at m/z 365 (C).
with Ce4þ; HPLC/MS/MS detection of the products gener-
ated in the reaction of 6β-OOH and Ce4þ; HPLC chromato-
gram of DPAO2 produced in the reaction of 6β-OOH/Ce4þ
and mass spectra of ketone and alcohol products formed in the
reaction of 7R-OOH/Ce4þ. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: (þ55)11-3091-8498. Fax: (þ55)11-3815-5579. E-mail:
pdmascio@iq.usp.br.
Funding Sources
This work was supported by the following Brazilian research
funding institutions: FAPESP (Fundac-~ao de Amparo ꢁa Pesquisa
do Estado de S~ao Paulo), CNPq (Conselho Nacional para o
Desenvolvimento Científico e Tecnolꢀogico), Instituto do Mil^enio:
Redoxoma, INCT de Processos Redox em Biomedicina—
Figure 6. NIR monomol light emission of O2 (1Δg) at 1270 nm
generated by ChOOHs incorporated in DMPC unilamellar liposomes.
Arrows indicate the injection of Ce4þ into (A) ChOOHs/DMPC (1:1)
Redoxoma and Prꢀo-reitoria de Pesquisa da USP. G.E.R. and
F.D.M. are recipients of FAPESP fellowship.
and (B) ChOOHs/DMPC (1:1) and 2 mM of NaN3.
’ ASSOCIATED CONTENT
’ ABBREVIATIONS
ChOOH, cholesterol hydroperoxides; Ch18O18OH, 18O-labeled cho-
S
Supporting Information. HPLC chromatogram of
b
lesterol hydroperoxides; O2 (1Δg), singlet molecular oxygen; 18O2
7R-, 5R-, and 6β-OOH; SRM chromatogram and enhanced
product ion mass spectra obtained in the positive APPI mode
(1Δg), 18O-labeled singlet molecular oxygen; DPA, 9,10-di-
phenylanthracene; DPAO2, 9,10-diphenylanthracene endoper-
for cholesterol-oxidized products; comparative HPLC chro-
matograms of 7R-, 5R-, or 6β-OOH before and after reaction
oxide; DPA18O18O, 18O-labeled DPA endoperoxide; DBA,
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9,10-dibromoanthracene; ROS, reactive oxygen species; 7R-OOH,
3β-hydroxicholest-5-ene-7R-hydroperoxide; 6β-OOH, 3β-hy-
droxycholest-4-ene-6β-hydroperoxide; 5R-OOH, 3β-5R-chol-
est-6-ene-5-hydroperoxide; DMNO2, 1,4-dimethylnaphthalene
endoperoxide; ChAld, 3β-hydroxy-5β-hydroxy-B-norcholes-
tane-6β-carboxaldehyde; DMPC, dimyristoylphosphatidylcho-
line; TLC, thin-layer chromatography; HPLC/MS/MS, HPLC-
tandem mass spectrometry; APCI, atmospheric pressure chemi-
cal ionization; SRM, selected reaction monitoring; ChOH,
cholesterol alcohol; ChKeto, cholesterol ketone; D2O, deute-
rium oxide; NIR, near-infrared region.
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