Formation of 4-hydroxynonenal from cardiolipin oxidation: Intramolecular peroxyl radical addition and decomposition

Article abstract of DOI:10.1016/j.freeradbiomed.2010.10.709

We report herein that oxidation of a mitochondria-specific phospholipid tetralinoleoyl cardiolipin (L₄CL) by cytochrome c and H ₂O₂ leads to the formation of 4-hydroxy-2-nonenal (4-HNE) via a novel chemical mechanism that involves cross-chain peroxyl radical addition and decomposition. As one of the most bioactive lipid electrophiles, 4-HNE possesses diverse biological activities ranging from modulation of multiple signal transduction pathways to the induction of intrinsic apoptosis. However, where and how 4-HNE is formed in vivo are much less understood. Recently a novel chemical mechanism has been proposed that involves intermolecular dimerization of fatty acids by peroxyl bond formation; but the biological relevance of this mechanism is unknown because a majority of the fatty acids are esterified in phospholipids in the cellular membrane. We hypothesize that oxidation of cardiolipins, especially L₄CL, may lead to the formation of 4-HNE via this novel mechanism. We employed L₄CL and dilinoleoylphosphatidylcholine (DLPC) as model compounds to test this hypothesis. Indeed, in experiments designed to assess the intramolecular mechanism, more 4-HNE is formed from L₄CL and DLPC oxidation than 1-palmitoyl-2-linoleoylphosphatydylcholine. The key products and intermediates that are consistent with this proposed mechanism of 4-HNE formation have been identified using liquid chromatography-mass spectrometry. Identical products from cardiolipin oxidation were identified in vivo in rat liver tissue after carbon tetrachloride treatment. Our studies provide the first evidence in vitro and in vivo for the formation 4-HNE from cardiolipin oxidation via cross-chain peroxyl radical addition and decomposition, which may have implications in apoptosis and other biological activities of 4-HNE.

Full text of DOI:10.1016/j.freeradbiomed.2010.10.709

Free Radical Biology & Medicine 50 (2011) 166–178
Contents lists available at ScienceDirect
Free Radical Biology & Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Original Contribution
Formation of 4-hydroxynonenal from cardiolipin oxidation: Intramolecular peroxyl
radical addition and decomposition
Wei Liu a, Ned A. Porter a, Claus Schneider b, Alan R. Brash b, Huiyong Yin a,b,c,
⁎
a
Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA
b
c
Department of Medicine, Vanderbilt University, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein that oxidation of a mitochondria-specific phospholipid tetralinoleoyl cardiolipin (L4CL) by
cytochrome c and H2O2 leads to the formation of 4-hydroxy-2-nonenal (4-HNE) via a novel chemical mechanism
that involves cross-chain peroxyl radical addition and decomposition. As one of the most bioactive lipid
electrophiles, 4-HNE possesses diverse biological activities ranging from modulation of multiple signal
transduction pathways to the induction of intrinsic apoptosis. However, where and how 4-HNE is formed in
vivo are much less understood. Recently a novel chemical mechanism has been proposed that involves
intermolecular dimerization of fatty acids by peroxyl bond formation; but the biological relevance of this
mechanism is unknown because a majority of the fatty acids are esterified in phospholipids in the cellular
membrane. We hypothesize that oxidation of cardiolipins, especially L4CL, may lead to the formation of 4-HNE via
this novel mechanism. We employed L4CL and dilinoleoylphosphatidylcholine (DLPC) as model compounds to
test this hypothesis. Indeed, in experiments designed to assess the intramolecular mechanism, more 4-HNE is
formed from L4CL and DLPC oxidation than 1-palmitoyl-2-linoleoylphosphatydylcholine. The key products and
intermediates that are consistent with this proposed mechanism of 4-HNE formation have been identified using
liquid chromatography–mass spectrometry. Identical products from cardiolipin oxidation were identified in vivo
in rat liver tissue after carbon tetrachloride treatment. Our studies provide the first evidence in vitro and in vivo
for the formation 4-HNE from cardiolipin oxidation via cross-chain peroxyl radical addition and decomposition,
which may have implications in apoptosis and other biological activities of 4-HNE.
Received 13 August 2010
Revised 25 September 2010
Accepted 25 October 2010
Available online 1 November 2010
Keywords:
4-Hydroxy-2-nonenal
Cardiolipin
Cytochrome c
LC–MS
Lipid peroxidation
Free radicals
Mitochondria
Apoptosis
© 2010 Elsevier Inc. All rights reserved.
Free radical-induced oxidation products of polyunsaturated fatty
acids (PUFAs) have been implicated as a major upstream component in
signal transduction cascades in many cellular functions including
apoptosis, proliferation, inflammatory responses, stimulating adhesion
molecules, and chemoattractant production [1–3]. Fatty acid hydroper-
oxides are the primary products in enzymatic and nonenzymatic
oxidation of fatty acids. These lipid hydroperoxides not only have potent
biological activities related to human physiology and pathophysiology,
but also can serve as precursors to form other highly oxidized lipid
oxidation products or decompose to generate an array of reactive lipid
electrophiles [4]. Over the past 2 decades, 4-hydroxy-2-nonenal
(4-HNE) has become one of the most studied reactive lipid electrophiles
[5–9]. As a highly reactive α,β-unsaturated aldehyde, 4-HNE exhibits a
variety of biological activities including inhibition of protein and DNA
synthesis and inactivation of enzymes. 4-HNE can also form protein
adducts with amino acid residues such as cysteine, histidine, arginine,
and lysine because of its strong electrophilic character [10–12].
Moreover, the presence of these protein adducts can serve as
biomarkers for lipid peroxidation and oxidative stress [5]. 4-HNE can
also trigger multiple signaling events in a physiological context [8].
In contrast to the biology of 4-HNE, the mechanisms for its
formation are much less understood [13–15]. Several mechanisms
have been proposed that may account for the formation of 4-HNE,
which include free radical-induced decomposition of lipid hydro-
peroxides such as 13-hydroperoxyoctadecadienoic acid
(13-HPODE) or 15-hydroxyeicosatetraenoic acid (15-HETE)
Abbreviations: ALDH, aldehyde dehydrogenase; C-0, a water-soluble azo initiator;
CID, collision-induced dissociation; CL, cardiolipin; DLPC, 1,2-dilinoleoyl-sn-glycero-3-
phosphocholine; DNPH, 2,6-dinitrophenylhydrazine; ESI, electrospray ionization; 4-HNE,
4-hydroxy-2-nonenal; 4-HPNE, 4-hydroperoxy-2-nonenal; HPLC, high-performance
liquid chromatography; HODE, hydroxyoctadecadienoic acid; HPODE, hydroperoxyocta-
decadienoic acid; HETE, hydroxyeicosatetraenoic acid; IsoP, isoprostane; L4CL,
tetralinoleoyl cardiolipin; MeOAMVN, 2,2′-azobis-(4-methoxy-2,4-dimethylvalero-
nitrile); MS, mass spectrometry; O4CL, tetraoleoyl cardiolipin; 4-ONE, 4-oxo-2-
nonenal; PLPC, 1-palmitoyl-2-linoleoylphosphatidylcholine; POPC, 1-palmitoyl-2-
oleoylphosphatidylcholine; PC, phosphatidylcholine; PHGPX, phospholipid hydro-
peroxide glutathione peroxidase; PPh3, triphenylphosphine; PUFA, polyunsaturated
fatty acid; ROS, reactive oxygen species; SRM, selective reaction monitoring; UPLC,
ultrapressure liquid chromatography.
⁎
Corresponding author. Division of Clinical Pharmacology, Departments of Medicine,
Pharmacology, Chemistry, Vanderbilt University, Nashville, TN 37232, USA. Fax: +1 615 322
3669.
E-mail address: huiyong.yin@vanderbilt.edu (H. Yin).
0891-5849/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.freeradbiomed.2010.10.709

W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
167
[16,17]. Ferrous iron (Fe2+)- or vitamin C-induced decomposition of
lipid hydroperoxides has also been proposed [18,19]. Recently, an
alternative mechanism was suggested, which involves an intermo-
lecular cross-linking of a peroxyl radical [13,17]. This mechanism has
precedence in styrene–oxygen polymerization and depolymerization
[20]. There is also evidence to support the decomposition of these cross-
linked peroxides of linoleic acid as initiators of free radical lipid
oxidation and formation of reactive lipid aldehydes in vitro [21–25]. It
should be noted that all these studies were carried out using linoleic acid
or methyl ester as models and thus the biological relevance of this
mechanism remains unclear because a majority of linoleic acids are
esterified on phospholipids in cellular membranes.
typically the distribution of linoleate mitochondrial CL is around 85–90%
[26–28]. Recently, cardiolipin oxidation has attracted much attention
because it is involved in regulation of programmed cell death (apoptosis)
initiated in mitochondria [29]. During apoptosis, CL interacts with
cytochrome c to form a peroxidase complex that catalyzes CL oxidation,
and accumulating evidence indicates that the oxidation products of CL
play a critical role in the mitochondrial stage of the execution of the cell
death program [30,31].
We hypothesize that L4CL is the best model compound with
biological relevance to test the idea of interchain peroxyl radical
addition and decomposition to form 4-HNE. In this case, the addition of a
peroxyl radical on one linoleic acid to another fatty acid side chain
occurs intramolecularly in L4CL instead of intermolecularly, as in the
model systems that have been studied previously. Moreover, the
intramolecular addition of a peroxyl radical in L4CL is favored kinetically
Cardiolipin (CL) is a unique class of phospholipids containing four fatty
acyl side chains and three glycerol moieties (Fig. 1A). In most mammalian
tissues the predominant form of CL is tetralinoleoyl CL (L4CL), and
O
-
O
A
C5H11
O
O
P O
O
H
C5H11
O
O
HO
O
O
P
O
O
C5H11
C5H11
O
O
H
-
O
O
(a) Tetralinoleoyl Cardiolipin (L4CL)
O
O
P
O
-
O
C5H11
C5H11
O
O
O
H
O
N
(b) di-linoleoyl-phosphatidylcholine (DLPC)
O
P
O
O
O
-
O
O
C5H11
O
H
N+
O
(c) 1-palmitoyl-2-linoleoyl-phosphatydylcholine (PLPC)
B
13
R1
R2
R2
O
R1
H
H
HOO
R1
R2
1b, 13-HPODE
HOO
O
9
R2
1a, Linoleic acid (C18:2)
R1
R1
R2
OOH
1c, 9-HPODE
R1=-C5H11; R2= - (CH2)7COOH
Dimerization
R2
R1
R2
O
R1
O
O
HOO
R1
HOO
HOO
R2
O
O
R1
HOO
R2
R1
1j
R2
1d
1e
-H2O
C5H11
4
reduction
4
C5H11
HO
1h, 4-HNE
O
C5H11
4
O
O
HOO
O
1g, 4-HPNE
1i, 4-ONE
+
O
7COOH
1f, 9-oxononanoic acid,
Fig. 1. Chemical structures of linoleoylphospholipids and proposed chemical mechanism for 4-HNE formation. (A) Chemical structures of L4CL, DLPC, and PLPC. (B) Formation of
4-HNE from dimeric peroxides of two linoleic acids under free radical conditions. Note. Other isomers of 1e can be formed during the process, but only the structures that are relevant
to the proposed mechanism are illustrated for simplicity.

168
W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
over the intermolecular addition. Because of the complexity of the
peroxidation of L4CL, we first use DLPC as a simplified model to test this
idea. A number of mass spectrometry (MS)-based techniques have been
employed to study the formation of 4-HNE from free radical oxidation of
1,2-dilinoleoyl-3-phosphatidylcholine (DLPC) and L4CL; decomposition
products and their postulated dimeric precursor have also been
identified using state-of-the-art MS methods. Furthermore, we identi-
fied thekey products with epoxyalcohol ononesidechain and truncated
aldehyde on the adjacent side chains in vivo in rat liver tissue after
treatment with carbon tetrachloride. Our data are consistent with the
concept that intramolecular cross-chain peroxyl radical addition and
decomposition may contribute significantly to the enhanced peroxida-
tion of CL and 4-HNE formation. These findings link CL oxidation and
4-HNE formation to mitochondrial dysfunction, apoptosis, and other
biological activities in mitochondria caused by oxidative stress and free
radical lipid peroxidation.
(PBS; 50 mM, pH 7.4) with 100 μM DTPA was added to make the
above concentrations for the phospholipids. After the lipid mixture
was vortexed and sonicated for 10 min under nitrogen in water,
0.06 mM C-0 in PBS was added to the mixture immediately to initiate
oxidation. The reaction mixture was incubated at 37 °C under air.
Oxidation of L4CL with cytochrome c and hydrogen peroxide
Oxidation of CL by cytochrome c and H2O2 was carried out based
on a published protocol [29]. CL and POPC stored in chloroform were
added to a glass vial and the solvent was removed by a flow of
nitrogen. PBS (50 mM, pH 7.4) with 100 μM DTPA was added and the
lipid mixture was vortexed and sonicated for 10 min under nitrogen
in water. Then to the lipid mixture were added cytochrome c and
H2O2 at 37 °C under air. H2O2 was added four times (every 15 min)
during the incubation period. The final reaction mixture contained
250 μM CL, 250 μM POPC, 20 μM cytochrome c, and 100 μM H2O2.
Oxidation of PLPC and DLPC using the Cyt c/H2O2 system was carried
out in the presence of O4CL.
Experimental procedures
Reagents
Synthesis of phospholipid hydroperoxides
Phospholipids, 1-palmitoyl-2-oleoyl-3-phosphatidylcholine (POPC),
1-palmitoyl-2-linoleoyl-3-phosphatidylcholine (PLPC), DLPC, and tetra-
oleoyl cardiolipin (O4CL) were purchased from Avanti Polar Lipids
(Alabaster, AL, USA) and used without further purification. Bovine heart
cardiolipin was purchased as a chloroform solution and purified by HPLC
as described below. Bovine heart cytochrome c, diethylenetriaminepen-
taacetic acid (DTPA), dinitrophenylhydrazine (DNPH), and soybean
lipoxygenase were purchased from Sigma–Aldrich Chemical Co. (Mil-
waukee, WI, USA). HPLC-quality solvents, such as methanol, water,
2-propanol, and acetonitrile, were purchased from either Fisher Chemical
(Phillipsburg, NJ, USA) or EM Science (Gibbstown, NJ, USA). The free
radical azo initiator 2,2′-azobis-(4-methoxy-2,4-dimethylvaleronitrile)
(MeOAMVN) and C-0 (VA-044) were generously donated by Wako
Chemicals USA Inc. (Richmond, VA, USA). 4-HNE, 4-oxo-2-nonenal
(4-ONE), 4-hydroperoxy-2-nonenal (4-HPNE), and 4-HNE-d3 were
purchased from Cayman Chemicals (Ann Arbor, MI, USA) and used
without further purification. All animal procedures were performed in
accordance with institutional guidelines after approval from the Animal
Care and Use Committee of Vanderbilt University.
A previously published protocol was used to synthesize PLPC and
DLPC hydroperoxides [32]. Soybean lipoxygenase (20 mg) was
dissolved in 30 ml of borate buffer that contained 10 mM deoxycho-
late at pH 9.0. PLPC (25 mg) or DLPC (20 mg) was taken up in 3 ml of
the same buffer and added to the enzyme solution. The reaction was
stirred at room temperature for 30–60 min. The reaction was
monitored by measuring UV absorbance at 234 nm. The reaction
mixtures were extracted with chloroform/methanol (2/1, v/v) three
times. The combined organic phase was evaporated and reconstituted
with methanol. The hydroperoxide was further purified by reverse-
phase HPLC using a Discovery C18 HPLC column (Supelco, Bellefonte,
PA, USA; 25 cm×21.2 mm, 5 μm) equilibrated with methanol/water
(90/10) at a flow rate of 15 ml/min. The detection was monitored by
the absorbance at 234 nm.
Mass spectrometry
Oxidized phospholipids were separated online by UPLC using a
Waters Acquity UPLC system (Waters Corp.) with a Phenomenex Luna
3 μ C8 column (150×4.6 mm) at a flow rate of 1.0 ml/min and a flow
splitter, which sent approximately 20% of the mobile phase to the
mass spectrometer. LC separation was performed using the same
gradient that was used to purify L4CL from bovine heart cardiolipin.
Mass spectrometry analysis was carried out on a Finnigan LTQ iontrap
mass spectrometer or Thermo Quantum Ultra triple-quadrupole mass
spectrometer. The electrospray ionization (ESI) source was fitted with
a stainless steel capillary (100 μm inner diameter). The mass
spectrometer was operated in the negative ion mode for structural
identification and quantitative analysis was carried out using selective
reaction monitoring (SRM) in positive mode for some oxidized PC.
Analysis of cardiolipins was performed in the negative ion mode.
Nitrogen was used as the sheath gas at 38 psi. The capillary
temperature was 350 °C. The spray voltage was 4.5 kV, and the tube
lens voltage was 100 V. Spectra were displayed by averaging scans
across chromatographic peaks. Data acquisition and analysis were
performed using Xcaliber software, version 2.0 (San Jose, CA, USA).
Purification of L4CL from bovine heart cardiolipin
Bovine heart cardiolipin contains about 80% L4CL. HPLC was carried
out on a Waters Model 600E pump with a Waters 996 photodiode
array detector. Millenium32 chromatography software (Waters Corp.,
Milford, MA, USA) was used to control the array detector and to collect
and process data. Purification of L4CL was performed on a Phenomenex
(Torrance, CA, USA) Luna C8 column (3 μm, 4.6×150 mm) with a flow
rate of 1.0 ml/min. Mobile phase A contained MeOH/CH3CN/NH4Ac
(2 mM) 60/20/20 (v/v/v) and mobile phase B was methanol. The
gradient started with 100% A and increased to 100% B in 15 min and
was held for 5 min before it returned to 100% A. L4CL eluted from 18 to
20 min under these conditions. The purity of L4CL was analyzed by
LC–MS described below. The stock solution of the purified L4CL was
stored at −80 °C under argon.
Oxidation of PLPC, DLPC, and L4CL in liposomes with free radical azo
initiator C-0
Analysis of 4-HNE, 4-HPNE, and 4-ONE by LC–MS
PLPC, DLPC, L4CL, and POPC were dissolved in chloroform as stock
solutions. Appropriate volumes of the stock solution were added into
separate glass vials to make final solutions of 0.6, 0.3, and 0.15 mM
PLPC, DLPC, and L4CL, respectively. A constant concentration of
14.4 mM POPC was added to each vial before the chloroform solutions
were evaporated by a stream of nitrogen. Phosphate-buffered saline
Analysis of 4-HNE, 4-HPNE, and 4-ONE was carried out using a
modified literature method [33]. Briefly, phospholipid oxidation
mixtures were extracted by CHCl3/MeOH (2/1, v/v) and 4-HNE-d3 was
added as the internal standard. After the solvent was removed by a flow
of nitrogen, 0.5 ml of saturated DNPH solution containing 1 N HCl was

W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
169
added to the residue and stored in the dark at room temperature for 2 h.
DNPH derivatives were extracted twice from the mixture with CH2Cl2
and evaporated to dryness under a stream of nitrogen. The sample was
reconstituted with MeOH/water (3/1, v/v). LC–MS/MS was conducted
using a Thermo Finnigan TSQ Quantum Ultra equipped with a Finnigan
Surveyor Autosampler Plus. Reverse-phase HPLC was performed using a
Phenomenex Luna C18 column (250×4.6 mm, 5 μm) with an isocratic
elution of MeOH/water (3/1, v/v) at a flow rate of 1 ml/min. The MS was
operated in the negative ion mode using ESI in the SRM mode. MS
parameters were optimized for 4-HNE–DNPH derivative and were as
follows: auxiliary gas pressure was set at 45 psi, sheath gas pressure was
50 psi, utilizing nitrogen for both. The vaporizer temperature was set at
400 °C. Collision-induced dissociation (CID) for the 4-HNE–DNPH,
4-ONE–DNPH, and 4-HPNE–DNPH was optimized at 25, 24, and 24 eV,
respectively, under 1.0 mTorr of argon. Data acquisition and analysis
were performed using Xcaliber software, version 2.0.
comparable for the two substrates. That is, the mole fraction of PLPC
used in these test reactions was twice that of DLPC used in parallel
experiments.
More 4-HNE is formed from the oxidation of DLPC and L4CL than that
of PLPC
Based on the mechanism illustrated in Fig. 1B, more 4-HNE is
expected to be formed from DLPC and L4CL than from PLPC in the
oxidation of liposomes containing equivalent mole fractions of
linoleate ester. We developed an LC–MS method to simultaneously
quantify the levels of 4-HNE, 4-HPNE, and 4-ONE from the oxidation
of different phospholipids. As shown in Supplementary Fig. S1, SRM
experiments were carried out on the derivatives of 4-ONE, 4-HPNE,
and 4-HNE after derivatization with DNPH. A deuterated internal
standard of 4-HNE was used to quantify these compounds. As shown
in Fig. 2, significantly higher levels of 4-HNE are formed in the
oxidation of DLPC and L4CL than in that of PLPC using a free radical
initiator. Similar results are obtained for the formation of 4-HPNE and
4-ONE (data not shown). It is noteworthy that when the oxidation
was carried out with either an azo free radical initiator or a
cytochrome c/H2O2 system, similar results were observed, i.e., the
amounts of 4-HNE, L4CLNDLPCNPLPC. These data suggest that
phospholipids containing two or more linoleate fatty acids in the
same molecule generate more 4-HNE and related compounds under
free radical conditions, consistent with the mechanism proposed in
Fig. 1B.
Identification of cardiolipin oxidation products that are consistent with
this novel pathway in vivo
After intragastric administration of CCl4 (2 mg/kg) in corn oil to
Sprague–Dawley rats for 2 h, the animals were anesthetized with
pentobarbital (60 mg/kg) intraperitoneally and sacrificed, and the
livers were harvested. About 1 g of liver tissue was homogenized in a
mixture of chloroform and methanol (2/1) with triphenylphosphine
and BHT: Butylated hydroxytoluene. The organic phase was evapo-
rated and reconstituted in methanol for LC–MS analysis.
Statistical analysis
DLPC dihydroperoxide is a valid model to identify the key products and
precursors for 4-HNE formation
Data are presented as the means SEM. Statistical analysis was
performed using analysis of variance followed by Student's t test.
Differences were considered significant when pb0.05.
In addition to the formation of 4-HNE and related compounds,
epoxyalcohol or epoxyhydroperoxides are predicted products from
the decomposition of the dimeric intermediates [17]. We propose that
DLPC dihydroperoxides 3a (Fig. 3) can be used as a simplified model
to test this mechanism. As shown in Fig. 3A, dihydroperoxides DLPC
3a form a cross-linked peroxide 3b, which readily decomposes to
form two alkoxyl radicals. The alkoxyl radical on sn-1 cyclizes to form
epoxyhydroperoxide or hydroxide 3c, whereas the alkoxyl radical on
sn-2 undergoes β-cleavage to form an aldehyde on C9 and 4-HPNE. It
should be noted that other isomers can be formed during the process,
but only the structures that are relevant to the proposed mechanism
are illustrated for simplicity.
Results
Proposed mechanism of 4-HNE formation: peroxyl radical addition to
conjugated dienes and peroxide decomposition
Despite the substantial efforts to study the biological activities of
4-HNE, there is a lack of understanding of the mechanisms of its
formation in vivo. Evidence provided here suggests that free radical-
induced 4-HNE formation from PUFAs may be due to peroxyl radical
addition to a conjugated diene followed by the decomposition of the
cross-linked peroxides due to the instability of these intermediates
[17]. As shown in Fig. 1B, linoleic acid 1a is one of the major fatty acids
that serve as the precursor for 4-HNE. 13-HPODE 1b and 9-HPODE 1c
are the two primary oxidation products formed under free radical
conditions. In this mechanism, a peroxyl radical from one of the
hydroperoxides adds to the conjugated double bond of another
hydroperoxide to form compound 1e after addition of O2 and
hydrogen atom abstraction. This dimeric hydroperoxide has limited
stability and is proposed to decompose to give 9-oxononanoic acid 1f
and 4-HPNE 1g; the latter can be reduced to form 4-HNE 1h or
dehydrated to generate 4-ONE 1i. The epoxide hydroperoxide 1j is
also formed on the other fatty acid in this mechanism. It is of some
interest that the linoleic acid-containing L4CL may be the best source
for the 4-HNE formation by this mechanism because this CL contains
four linoleic acids in the same molecule. The peroxyl radical addition
reaction is therefore intramolecular in this instance because it occurs
between pendant chains of the cardiolipin.
Fig. 2. Levels of 4-HNE from free radical-induced oxidation of PLPC, DLPC, and L4CL. The
reaction was carried out with 0.15, 0.3, and 0.6 mM L4CL, DLPC, and PLPC, respectively,
in 14.4 mM POPC. Analysis of 4-HNE was performed by LC–MS after derivatization with
DNPH. **pb0.01, ***p b 0.001. The phospholipids were oxidized using a free radical
initiator but similar results were observed when the oxidation was carried out using
cytochrome c and H2O2.
To simplify a model study, we first compared oxidation of DLPC, in
which peroxyl radical addition could occur intramolecularly with PLPC,
a compound that requires that addition occur intermolecularly. These
studies were carried out in liposomes diluted with an inert phospholipid
such that the concentrations of linoleate in the liposomes were

170
W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
A
3b
3a
3d
3c
B
C
1.0
DLPC-OOH
PLPC-OOH
0.8
0.6
0.4
0.2
0.0
**
*
0
2
4
6
8
Time (hr)
Fig. 3. Formation of 4-HNE from phospholipid hydroperoxides. (A) Pathway for 4-HNE formation from oxidation of DLPC hydroperoxides via two cross-linked side chains.
(B) Chemical structure of 13-HPODE–PLPC. (C) Time course of 4-HNE formation from decomposition of DLPC and PLPC hydroperoxides. *pb0.05, **p b 0.01, relative amounts of
4-HNE from DLPC vs PLPC. The phospholipid hydroperoxides were synthesized by soybean lipoxygenase and further oxidation was carried out using a free radical initiator.
To show that DLPC dihydroperoxide 3a can be used as a valid
model to test this mechanism, we synthesized DLPC dihydroperoxide
3a and PLPC hydroperoxide 3e by the use of soybean lipoxygenase.
These two precursor hydroperoxides were diluted in an unoxidizable
phospholipid, POPC (2% DLPC dihydroperoxide and 4% PLPC hydro-
peroxides, respectively) and decomposed as a thin film in the
presence of an azo free radical initiator, MeOAMVN. Time-dependent
formation of 4-HNE was observed and significantly more 4-HNE was
generated from decomposition of DLPC dihydroperoxide 3a than was
formed from PLPC hydroperoxide 3e (Fig. 3C). Similar results were
obtained for 4-HPNE and 4-ONE (Supplementary Fig. S2). Even
though the hydrogen atoms in OOH are more easily abstracted than
those at the bisallylic positions of unoxidized phospholipids, the data
shown in Fig. 3C suggest that DLPC-dihydroperoxide 3a offers a valid
model for study of the proposed mechanism when the levels of 4-HNE
from these two hydroperoxides are compared.
limited structural information can be obtained in this way because the
fragmentation from CID leads only to a prominent ion of the choline
head group with m/z 184. Thus, the acetate (Ac−) adduct of
compound 3c (m/z 764) was subjected to CID in the negative ion
mode. The results are illustrated in Fig. 4. MS2 of m/z 764 gives a
fragment of m/z 704 that represents the loss of acetic acid, whereas a
fragment of m/z 690 corresponds to the further loss of a methyl group
on the choline head group [34,35]. A characteristic fragment of m/z
311 is consistent with the carboxylate of the epoxyalcohol side chain.
Dehydration of m/z 311 gives rise to m/z 293. MS3 of m/z 311 is shown
in Fig. 4B. The fragments with m/z 171 and 139 are indicative of the
epoxyalcohol moiety in compound 3c. Taken together, these data
support the notion that decomposition of DLPC dihydroperoxide 3a
generates the key product epoxyalcohol 3c, a conclusion that is
consistent with the formation of 4-HNE through the dimerization
mechanism.
Identification of the critical decomposition products epoxyalcohol and
epoxyhydroperoxide 3c from DLPC dihydroperoxide
Identification of the intact cross-linked peroxides 3b
To identify the cross-linked peroxide precursors 3b, we fraction-
ated the reaction mixture derived from oxidation of DLPC dihydro-
peroxide by HPLC. The separated fractions were evaporated and
allowed to stand as a thin film for 2 h to determine whether they
The reaction mixture of DLPC dihydroperoxide taken at the 7-h
time point was treated with PPh3 and analyzed by LC–iontrap MS. PC
species are readily ionized in the positive ion mode MS but only

W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
171
2
(A) MS : m/z 764
m/z 619 (-CH3COOH-Me)
OH
O
690.4
704.3
C5H11
100
m/z 311
O
Ac-
O
O
O
O
P
-
O
N+
O
O
3c, m/z 764 (704 + Ac-)
O
50
619.3
293.3
311.2
0
200
300
400
500
600
700
m/z
3
(B) MS : m/z 764 to 311
m/z 139
O
-H, m/z 171
O
C5H11
O-
171.3
OH
3, m/z 764 to 311
100
50
0
MS
311.2
293.3
275.2
209.2
139.2
100
140
180
220
260
300
340
m/z
Fig. 4. Mass spectra of epoxyalcohol 3c from decomposition of DLPC hydroperoxides analyzed by LC–iontrap MS. (A) MS2 of acetate adduct of compound 3c, m/z 764. (B) MS3 of m/z
764 to 311. The oxidation mixture used was from the experiment described for Fig. 3.
contained 4-HNE precursors. Six fractions were collected and the
levels of 4-HNE and epoxyalcohol 3c were analyzed before and after
the 2-h incubation. The results are summarized in Supplementary
Fig. S3. Two fractions had elevated levels of 4-HNE and 3c after further
decomposition, which suggested that cross-linked peroxides 3b
eluted in these fractions. Structural elucidation of this compound
was carried out by LC–ESI–iontrap MS experiments. The analyses are
summarized in Fig. 5 and the proposed fragmentation pathways are
shown in Fig. 6. Multistage CID experiments were employed to
characterize the proposed structure 3b in the negative mode of
MS. MS2 was carried out on the acetate adduct of 3b with m/z 936 and
further MS3 experiments were performed on the two major fragments
of m/z 862 and 791. Based on the MS data, the intact cross-linked
peroxide 3b was unambiguously identified by the LC–MS technique.
Formation of 4-HNE from L4CL via a similar mechanism that involves
decomposition of intramolecular cross-linked peroxide
Using DLPC as a model compound, we have provided evidence that
4-HNE and other reactive aldehydes are derived from the novel
chemical mechanism, and we show here that L4CL is the precursor for
these reactive aldehydes in vivo with biological relevance.
The proposed key products consistent with the peroxyl radical
addition mechanism are epoxyalcohol on one acyl side chain and an
aldehyde at C9 on the other side chain. The putative structure as
shown in Fig. 8 has m/z 1371 and it lacks a conjugated diene structure
as shown in Supplementary Fig. S5. Cardiolipin is an anionic
phospholipid that is readily detected in the negative ion mode of
MS. The two oxidized side chains can be on the same (8a) or different
(8b) glycerol backbones. As shown in the MS2 of m/z 1371.6 (Fig. 8A),
in addition to the common fragments of m/z 1217 and 1091 for these
different regioisomers, fragments with m/z 619 and 695 are consistent
with structure 8a and m/z 727 and 587 are indicative of the structure
8b. The assignment of these two critical fragments m/z 619 and 695
was further supported by respective MS3 experiments. MS3 on m/z
1371.6 to 619 generated characteristic fragments with m/z 311, 307,
447, and 293 (Fig. 8B). A simpler fragmentation pattern of MS3 on m/z
1371.6 to 695 was obtained as shown in Fig. 8C. Fragments with m/z
279, 415, and 433 were consistent with the two intact linoleoyl side
chains on the same glycerol backbone. The presence of the key
epoxyalcohol in the structure was evident in MS4 of m/z 1371.6 to
619.3 to 311.2 (Fig. 8D). In addition to the fragments of m/z 293 and
275, two fragments with m/z 171 and 211 were consistent with the
presence of an epoxyalcohol moiety in this compound. MSn experi-
ments were carried out for ions m/z 727 and 587 and the data are
consistent with the proposed structure of 8b (data not shown).
We also attempted to identify the cross-linked peroxide pre-
cursors that would lead to 4-HNE. The stabilized precursor by PPh3
reduction had an alcohol moiety on one side chain and three hydroxyl
groups on the neighboring acyl chain. The LC–MS data of the reduced
oxidation mixture are summarized in Fig. 9. As shown in Fig. 9A, the
Derivatization of the intact cross-linked peroxides 3b and characterization
of the resulting products
Because of the limited stability of the cross-linked peroxides,
characterization of these precursors proved to be difficult. Reaction of
the peroxides 3b with PPh3 led to compounds that are more stable and
can be characterized by MS methods. Reduction of the cross-linked
peroxide 3b leads to compound 7a as shown in Fig. 7. Successful
conversion of 3b to 7a was confirmed by MS analysis and the data
were summarized in Supplementary Fig. S4.
Characterization of compound 7a was performed by iontrap MS in
the negative ion mode and the results are shown in Fig. 7. MS2 of the
acetate adduct of compound 7a with m/z 906 (Fig. 7B) produced two
major fragments: a peak with m/z 832 was consistent with the loss of
acetic acid and a methyl group on the choline head group; the
fragment of m/z 761 represented the loss of choline head group. MS3
on m/z 832 clearly indicated the two acyl chains with m/z 329 and m/z
295 (Fig. 7C). The unambiguous structural support of these two
oxidized acyl chains was obtained in the MS4 spectra (Fig. 7D). Taking
these data together, the chemical structure of compound 7a was
unambiguously confirmed by the iontrap MS.

172
W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
Identification of the key products of cardiolipin oxidation via the novel
mechanism in vivo
Carbon tetrachloride-induced liver injury is a well-established
animal model for in vivo free radical generation and oxidative stress
[36]. Elevated levels of lipid peroxidation products, such as F2-IsoP's,
malondialdehyde (MDA), thiobarbituric acid-reactive substances
(TBARS), and 4-HNE, have been detected in this model. We performed
experiments to identify the key products of cardiolipin oxidation in
vivo in rat liver tissue after carbon tetrachloride treatment. The results
are shown in Fig. 10. We identified characteristic fragments for
compound 8a in CID experiments on m/z 1371.8 (singly charged ions)
or m/z 685.6 (doubly charged ion) from in vitro oxidation of L4CL and
extracts of rat liver tissue (data not shown). A peak at 16.2 min in
Figs. 10A and B demonstrates the formation of this key product in
vitro and in vivo. It is noted that unoxidized L4CL and the major
oxidation product with addition of one oxygen atom were also
detected. These data indicate that the novel mechanism for 4-HNE
also operates in vivo and it may play an important role in 4-HNE
formation in vivo.
Discussion
Free radical-induced oxidation of PUFAs has been extensively
studied for more than half a century, and 4-HNE was first discovered
in vivo almost 3 decades ago [37]. Investigation of the toxicological
and physiological actions of this reactive lipid electrophile has been
extensive, but the mechanism of its formation is much less
understood. A novel mechanism for the formation of 4-HNE by
intermolecular peroxyl radical addition and decomposition was
recently proposed [13,17]. We provide evidence here that this
mechanism may play an important role for the oxidation of the
mitochondria-specific phospholipid cardiolipin, especially L4CL. For-
mation of 4-HNE and other reactive aldehydes from this process may
have significant implications in apoptosis and other functions of
cardiolipins in mitochondria.
Substantial efforts have been made to understand the mechanism
of 4-HNE formation in vivo since the discovery of 4-HNE [14,15].
PUFAs containing ω-6 chains, such as linoleic acid (C18:2) and
arachidonic acid (C20:4), are precursors for 4-HNE and much
attention has been paid to the decomposition of lipid hydroperoxides
to generate 4-HNE and related lipid electrophiles. We have utilized
different lipid hydroperoxides as precursors to study the formation of
4-HNE. Our previous studies using 15-HETE and 15-HPETE identified
epoxyalcohol and epoxyhydroperoxide derivatives as major products
during the formation of 4-HNE from these precursors [17]. These
results prompted us to propose a novel mechanism of 4-HNE
formation via intermolecular addition of a peroxyl radical to form a
dimeric peroxide, followed by decomposition of this unstable
precursor (Fig. 1B). There is precedence for the dimerization of
linoleic acid during the free radical oxidation in vitro and these
resulting peroxides can be used as initiators for further free radical
processes [21]. Successful separation and characterization of several
dimeric lipid hydroperoxides were carried out and their decomposi-
tion gave rise to a number of reactive aldehydes. These studies also
discovered the instability of these peroxide dimers [22–25,38].
However, the biological relevance of the intermolecular cross-
reaction mechanism is unknown because a majority of the fatty
acids are esterified on phospholipids in cellular membranes and the
linoleate acyl chain is often separated by saturated acyl side chains.
The data we present here suggest that CL is one of the major sources of
4-HNE because it has four linoleate acyl chains in the same molecule
and the formation of these important peroxyl dimers occurs
intramolecularly. Thus, it is this unique chemical structure of CL that
is responsible for the enhanced oxidation of CL (Supplementary Fig.
S1B) and the formation of 4-HNE under free radical conditions (Fig. 2).
Fig. 5. Mass spectra of intact cross-linked peroxide 3b analyzed by LC–iontrap MS.
(A) MS2 of acetate adduct of compound 3b, m/z 936. (B) MS3 of m/z 936 to 862. (C) MS3
of m/z 936 to 791. The oxidation mixture used was from the experiment described for
Fig. 3.
two oxidized side chains can be on the same (9a) or different (9b)
glycerol backbones. MS2 of m/z 1514 showed two characteristic
fragments of m/z 761 and 695 for 9a and m/z 711 and 745 are derived
from 9b. Further structural characterization was performed by MS3
and MS4. MS3 on m/z 1514 to 761 generated m/z 465 and 295. In the
same way, fragments with m/z 431 and 329 were consistent with the
presence of three hydroxyls on the other side chain. MS3 on m/z 1514
to 695 demonstrated that the compound had two intact linoleate side
chains on the same glycerol moiety because of the fragments with m/z
279, 415, and 433. MS4 on m/z 1514 to 761 to 329 clearly showed the
proposed structure due to the presence of the characteristic fragment
m/z 229. MS4 on m/z 1514 to 761 to 295 indicated that there was a
mixture of regioisomeric hydroxide on one of the side chains: m/z 171
resulted from 9-HODEs whereas m/z 195 was indicative of 13-HODE.
Similar data were obtained for structure 9b. Therefore, the precursor
with cross-chain peroxides is unambiguously identified based on MS
data.
Taken together, these data indicate that the oxidation of L4CL
generates 4-HNE through a mechanism similar to that of DLPC based
on the presence of the key products as well as the putative cross-
linked peroxide precursors.

W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
173
Fig. 6. Fragmentation pathways of multistage CID experiments for the intact cross-chain peroxide 3b. (A) MS2 of m/z 936: m/z 876 represents the loss of acetic acid, whereas
fragments with m/z 862 and 791 are further loss of the methyl group on the choline head group (6a) and complete loss of the head group (6b), respectively. (B) MS3 of m/z 862: m/z
774 (6c) is derived from the cleavage of the tail end of the fatty acid, whereas a peak with m/z 568 (6d) is consistent with a fragment having one of the side chain and head group
attached; m/z 343 (6e) and m/z 311 (6f) are consistent with the cleavage of the cross-linked peroxyl bond. (C) MS3 on m/z 936 to 791: m/z 755 may be due to the loss of two
molecules of water from 6b; m/z 479 (6 h) is derived from the loss of one of the side chains when the phosphate anion cyclizes and removes one of the side chains followed by
dehydration; m/z 329 and 293 are indicative of the proposed side chains. The oxidation mixture used was from the experiment described for Fig. 3.
It is noted that intramolecular peroxyl radical addition to a conjugated
diene is favored kinetically over the intermolecular addition. Similar
chemistry has been reported in vitro in other lipids that have two or
more linoleate side chains in the same molecule. For example, kinetic
studies showed that triglycerides containing two or more linoleate
side chains underwent enhanced autoxidation because of the arm-to-
arm hydrogen atom abstraction under free radical conditions [39,40].
We also detected the characteristic product compound 8a that is
postulated to be formed via this novel mechanism in vivo in rat liver
tissue after carbon tetrachloride treatment. As a well-established
rodent model for in vivo oxidative stress, CCl4 treatment induces free
radical generation in the liver that leads to massive lipid peroxidation,
protein modification, and DNA damage [36]. Recent studies also
demonstrate that CCl4 treatment inhibits hepatic mitochondrial
aldehyde dehydrogenases through JNK-mediated phosphorylation
and further exacerbates the liver damage by 4-HNE and other reactive
lipid aldehydes [41].
phospholipids generates an array of oxidation products and most of
them are unstable peroxides. It is extremely difficult to isolate these
oxidation products and characterize them by the conventional
techniques such as NMR. MS coupled with LC separation has
advantages in providing the molecular weight and structural
information for the proposed compounds. Iontrap MS has been
widely employed for this purpose because of its multistage CID
capability [45–47]. In the negative ion mode, the chemical structures
that are consistent with the proposed mechanism were unambigu-
ously identified using consecutive CID experiments. On the other
hand, the iontrap technique has limitations in efficient trapping of the
ions with lower m/z ratios. We also employed triple-quadruple MS to
conform the formation of these characteristic smaller fragments that
did not show in MS2 of iontrap instruments (data now shown). These
two complementary techniques enable us to identify the key products
that are consistent with our proposed mechanisms from a complex
oxidation mixture of phospholipids.
The MS techniques that we employed here have proved indis-
pensible for characterizing the proposed oxidation products of
phospholipids [42–44]. Free radical oxidation of PUFA-containing
The mechanism presented here links 4-HNE formation to CL
oxidation in mitochondria where a substantial amount of ROS, such as
superoxide anion and hydrogen peroxides, is generated. Lipid

174
W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
(A)
O
O
C5H11
C5H11
PPh3
6
6
O
O
O
HO
O
HO
HO
O
C5H11
HOO
O
C5H11
HO
O
-O P O
O
O
P
6
6
O
-O
O
O
HOO
O
N
N
3b
7a
2
(B) MS : m/z 906
761.3
100
832.4
50
0
775.3
846.4
200
300
400
500
600
700
800
900
m/z
O
C5H11
Ac-
O
HO
O
m/z 832 (M-CH3COOH-CH3)-
N+
HO
HO
C5H11
O
O-
O
HO
P
O
O
-
7a, m/z 906 (846 + Ac )
+H, m/z 761
O
3
(C) MS : m/z 906 to 832
C5H11
m/z 295
HO
O
m/z 554 (M-R1C=C=O)-
HO
HO
m/z 832
295.2
100
C5H11
HO
O
O
P
329.3
O-
O
O
O
m/z 329
N
50
554.3
277.2
761.3
0
250
350
450
550
650
750
850
m/z
(D) MS4: m/z 906 to 832 to 295
4
MS : m/z 906 to 832 to 329
171.0
295.3
100
100
50
0
195.1
171.0
112.8
211.3
50
229.1
293.4
0
150
250
150
250
m/z
m/z
+H, m/z 195
(CH2)7COO-
+H, m/z 229
HO
(CH2)7COO-
-H, m/z 171
C5H11
C5H11
HO
HO
HO
m/z 295
m/z 329
Fig. 7. Characterization of the reduced cross-chain peroxides 7a by LC–iontrap MS. (A) Reduction of compound 3b by PPh3 leads to a more stable compound 7a. (B) MS2 of acetate adduct of
compound 7a, m/z 906. (C) MS3 of m/z 906 to 832. (D) MS4 of m/z 906 to 832 to 295; MS4 of m/z 906 to 832 to 329. In addition to the characteristic fragments of m/z 229 and 171, m/z 293
represents loss of two molecules of water from m/z 329 and m/z 211 is from dehydration of m/z 229. The oxidation mixture used was from the experiment described for Fig. 3.

W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
175
(A) MS2 m/z 1371.6
619
100
80
695
60
40
20
0
415.2
447.2
465.2
755.2
727
831.3
861.4
587
1217.5
675.42
1091.3
700
800
900
1000
1100
1200
1300
O
-
m/z 1217.5, (M-R C=C=O)
1
O
O
OH
OH
+H, m/z 727
C H
O
O
O
5
11
O
O
P
O
O
O
P
O +H, m/z 619
O
H
O
H
O
O
C H
5
11
C H
5
11
OH
OH
O
HO
HO
O
m/z 1091.5
O
O
P
O
P
O
O
O
O
O
C H
O O
5
11
H
-
C H
O
O
H
O
5
11
-
C H
+H, m/z 587
O
5
11
+H, m/z 695
O
O
m/z 1371.6, 8b
m/z 1371.6, 8a
m/z
3
(B) MS m/z 1371.6 to 619
-
m/z 465, (M-R C=C=O)
1
-
447
m/z 447 (M-R COOH)
100
80
1
O
OH
P
311
O
O
O
O
O
H
-
O
O
C H
60
-
5
11
m/z 307 (M-R COOH)
2
OH
O
465
40
307
293
m/z 311
m/z 619
20
0
325.2
391.3
431.2
200
250
300
350
400
450
500
550
600
650
m/z
3
(C) MS m/z 1371.6 to 695.3
279
m/z 279
O
100
-
m/z 416, (M-R COOH)
3
415.2
433
O
80
60
40
20
0
C H
O
O
P OH
5
11
H
-
C H
O
O
5
11
-
m/z 433 (M-R C=C=O)
4
O
695.92
200
250
300
350
400
450
500
550
600
650
700
m/z
4
(D) MS m/z 1371.6 to 695.3 to 311.2
-H, m/z 211 (-H O)
2
171
-H, m/z 171
O
100
-
O
C H
275.2
5
11
80
60
40
20
0
OH
O
m/z 311
211.2
293.01
249.38
312.60
100
120
140
160
180
200
220
240
m/z
260
280
300
320
340
360
380
400
Fig. 8. Characterization of the key product of epoxyalcohol from L4CL oxidation by LC-iontrap MS. (A) MS2 of m/z 1371.6. The two oxidized side chains can be on the same glycerol
backbone (8a) or different glycerol backbones (8b). In addition to the common fragments such as m/z 1217 and 1091, fragments m/z 619 and 695 are characteristic of 8a and m/z 587
and 727 are from 8b. (B) MS3 of m/z 1371.6 to 619. (C) MS3 of m/z 1371.6 to 695. (D) MS4 of m/z 1371.6 to 619 to 311. L4CL was oxidized using cytochrome c and H2O2 as described in
detail under Experimental procedures.

176
W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
2
(A) MS m/z 1514
761
745
100
695
700
50
711
831.39
800
465.19
897.46
0
500
600
900
1000
1100
1200
1300
1400
m/z
OH
OH
OH
O
O
O
C H
OH
OH
P O
O
5
11
C H
O
O
+H, m/z 761
O
O
5
11
+H, m/z 745
O
O
P
OH
O
H
H
C H
C H
5
O
O
O
5
11
11
OH
OH
OH
OH
HO
HO
O
O
O
O
P
O
P
O
C H
O
5
11
O
C H
5
11
O
O
O
H
-
-
C H
H
O
C H
O
5
11
5
11
+H, m/z 695
+H, m/z 711
O
m/z 1514.0, 9a
O
m/z 1514.0, 9b
m/z 295
O
-
3
m/z 465 (M-R COOH)
1
(B) MS m/z 1514 to 761
100
OH
C H
5 11
OH
465
O
OH
OH
O
P
O
O
H
-
C H
5
O
11
-
m/z 431 (M-R COOH)
329
50
0
295
2
483
431
400
m/z 329
O
OH
200
250
300
350
450
500
550
600
650
700
750
m/z
3
m/z 279
O
(C) MS m/z 1514 to 695
100
-
m/z 415 (M-R COOH)
3
279
O
C H
5
C H
OH
P
O
O
O
11
-
H
O
5
11
-
415
433
m/z 433 (M-R C=C=O)
50
0
4
O
200
250
300
350
400
450
500
550
600
650
700
750
m/z
+H, m/z 229
4
(D) MS m/z 1514 to 695 to 329
100
OH
-
C H
229
O
5
11
OH
OH
O
50
183.32
211.08
274.00
331.54
162.57
118.86
307.24
266.76
129.79 139.14
254.93
290.29
197.46
200
244.18
0
100
4
120
140
160
180
220
240
260
280
300
320
m/z
OH
O
(E) MS m/z 1514 to 695 to 295
C H
5
11
-
O
171
295
100
195
-H, m/z 171
245.04 258.91
240 260
163.59
50
113.22
179.30
180
214.35
143.32 153.00
201.00
0
100
120
140
160
200
220
280
300
m/z
Fig. 9. Characterization of reduced cross-chain peroxide derived from oxidation of L4CL by LC–iontrap MS. (A) MS2 of m/z 1514. The two oxidized side chains can be on the same
glycerol backbone (9a) or different glycerol backbones (9b). Fragments m/z 761 and 695 are characteristic of 9a and m/z 711 and 745 are from 9b. (B) MS3 of m/z 1514 to 761. The
fragments of m/z 465, 431, 295, and 329 indicate the presence of a hydroxyl group on one side chain and three hydroxyl groups on a neighboring side chain. (C) MS3 of m/z 1514 to
695. The fragments with m/z 279, 415, and 433 suggest two intact linoleoyl side chains. (D) MS4 of m/z 1514 to 761 to 329. (E) MS4 of m/z 1514 to 761 to 295. m/z 171 and 195 are
characteristic of 9- and 13-HODE, respectively. L4CL was oxidized using cytochrome c and H2O2 as described in detail under Experimental procedures.
peroxidation and subsequent formation of 4-HNE in mitochondria
have been suggested to play an important role in mitochondrial
dysfunction [48]. Paradies et al. showed a direct correlation between
lipid peroxidation, decreased CL, and decreased cytochrome c oxidase
activity when they exposed rat heart mitochondria to t-butyl
hydroperoxides [49]. Sen et al. oxidized rat brain mitochondria
using iron–ascorbate and found that increased lipid peroxidation as
measured by MDA/TBARS correlated with decreased levels of
cardiolipin [50]. A seminal work carried out by Kagan et al.
demonstrated that oxidation of CL by the peroxidase function of
cytochrome c due to its association with CL was required to trigger
intrinsic apoptotic pathways [29,31]. Using a state-of-the-art oxida-
tive lipidomics approach they showed that CLs in mitochondria were
preferentially oxidized in the presence of other highly oxidizable
phospholipids, such as arachidonic acid, eicosapentaenoic acid, and
docosahexaenoic acid containing PC and phosphatidylethanolamine.
Previous studies also established that hydroperoxides of CL were
important in the induction of apoptotic events [51–53]. Our studies
suggest that 4-HNE formation from these CL–OOHs may also play a
significant role in these events because 4-HNE has been shown to
induce apoptosis [8,54].
This novel mechanism for 4-HNE formation from CL oxidation in
mitochondria may potentially have some physiological and pathophys-
iological significance. Generation of 4-HNE in vivo has been implicated
in cardiovascular diseases, such as atherosclerosis [55], and neurode-
generative diseases including Alzheimer disease and Parkinson disease
[5,56–60]. L4CL is the major CL species in the mitochondria of most
mammalian tissues and it constitutes more than 70% in the heart [61].

W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
177
3.4x105
activation of caspase-3, and apoptosis caused by 2-deoxyglucose,
whereas cells overexpressing nonmitochondrial PHGPX were prone
to apoptosis [51,66]. Furthermore, liver apoptosis and cytochrome c
release were suppressed in transgenic mouse overexpression of this
enzyme when mice were treated with diquat to induce apoptosis.
Levels of cardiolipin oxidation products in these transgenic animals
were also suppressed compared to the wild-type animals [67].
Reduction of mitochondrial hydrogen peroxide by overexpressing
mitochondria-specific peroxiredoxin 3 led to reduced levels of
F2-IsoP's, a widely accepted standard for oxidative stress in vivo
[68], and 4-HNE [69]. Last, some antioxidants may also have inhibitory
effects on the formation of these reactive lipid aldehydes if they can be
incorporated into the cytochrome c/CL peroxidase complex and
compete with the intramolecular hydrogen atom abstraction or
addition to the double bonds.
Alternative approaches to mitigating the detrimental effects of
4-HNE and related compounds may involve stimulation of the
deactivation mechanism for these compounds [70]. Aldehyde
dehydrogenase-2 (ALDH2) is a mitochondria-specific enzyme that
is involved in oxidation of 4-HNE to its acid analogue. A recent study
demonstrated that ischemic damage to the heart was significantly
reduced by activation of ALDH2 using a small-molecule agonist,
Alda-1 [71,72]. Moreover, increasing evidence suggested that
mitochondrial detoxification pathways for 4-HNE play a significant
role in delay of the development of Alzheimer disease and other
neurodegenerative diseases [62].
A
16 2
100
60
.
SRM: m/z 685.6 to 279
20
100
19.5
1.3x107
60
SRM: m/z 723.5 to 279
SRM: m/z 731.6 to 279
20
100
17.8
7.2x106
60
20
0
10
12
14
16
18
20
22
24
Time (min)
1.1x104
B 100
16.6
SRM: m/z 685.6 to 279
60
In summary, we have provided evidence that 4-HNE and related
reactive lipid electrophiles can be generated through a novel chemical
mechanism of cardiolipin oxidation under free radical conditions in
vitro and in vivo. This mechanism links 4-HNE generation to
cardiolipin oxidation in mitochondria where abundant ROS are
generated. Biological studies are under way to investigate the role
of CL oxidation and 4-HNE formation in mitochondrial function.
20
100
19.8
7.3x105
SRM: m/z 723.5 to 279
SRM: m/z 731.6 to 279
60
20
100
18.1
Acknowledgment
8.0x104
60
20
0
This work is supported by NIH Grants GM15431, DK48831, and
ES13125.
Appendix A. Supplementary data
10
12
14
16
18
20
22
24
Time (min)
Supplementary data to this article can be found online at
doi:10.1016/j.freeradbiomed.2010.10.709.
Fig. 10. Identification of the key oxidation product (compound 8a) of cardiolipin from
the novel mechanism in vivo. (A) In vitro oxidation of L4CL by cytochrome c/H2O2.
(B) Rat liver extracts after CCl4 treatment. SRM transitions: m/z 685.6 to 279, compound
8a; L4CL, m/z 723.5 to 279; primary oxidation product with the addition of one oxygen,
m/z 731.6 to 279. The experiments were performed on a triple-quadruple MS in the
negative ion mode coupled to reverse-phase LC separation. The arrows indicate
compound 8a. See Experimental procedures for details.
References
[1] Niki, E. Lipid peroxidation: physiological levels and dual biological effects. Free
Radic. Biol. Med. 47:469–484; 2009.
[2] Berliner, J. A.; Leitinger, N.; Tsimikas, S. The role of oxidized phospholipids in
atherosclerosis. J. Lipid Res. 50:S207–212; 2009.
[3] Gardner, H. W. Oxygen radical chemistry of polyunsaturated fatty acids. Free
Radic. Biol. Med. 7:65–86; 1989.
[4] Yin, H.; Porter, N. A. New insights regarding the autoxidation of polyunsaturated
fatty acids. Antioxid. Redox Signaling 7:170–184; 2005.
Thus formation of 4-HNE via this novel mechanism is probably
important. 4-HNE alters multiple essential functions of brain mitochon-
dria, which plays a pivotal role in initiation and progression of
neurodegenerative diseases [62]. 4-HNE also induces mitochondrial
dysfunction and aberrant axonal outgrowth in adult sensory neurons
that mimics features of diabetic neuropathy [63].
[5] Uchida, K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog.
Lipid Res. 42:318–343; 2003.
[6] Esterbauer, H.; Schaur, R. J.; Zollner, H. Chemistry and biochemistry of 4-
hydroxynonenal, malonaldehyde and related aldehydes. Free Radic. Biol. Med. 11:
81–128; 1991.
[7] Esterbauer, H.; Zollner, H.; Schauer, R. J. Aldehydes formed by lipid peroxidation:
mechanisms of formation, occurrence, and determination. In: Vigo-Pelfrey, C.
(Ed.), Membrane Lipid Oxidation. CRC Press, Boca Raton, FL,pp. 239–268; 1991.
[8] West, J. D.; Marnett, L. J. Endogenous reactive intermediates as modulators of cell
signaling and cell death. Chem. Res. Toxicol. 19:173–194; 2006.
[9] Rudolph, T. K.; Freeman, B. A. Transduction of redox signaling by electrophile–
protein reactions. Sci. Signal. 2 (re7); 2009.
[10] Carini, M.; Aldini, G.; Facino, R. M. Mass spectrometry for detection of 4-hydroxy-
trans-2-nonenal (HNE) adducts with peptides and proteins. Mass Spectrom. Rev.
23:281–305; 2004.
Modulation of the formation of 4-HNE and other lipid electrophiles
from CL oxidation in mitochondria may lead to novel approaches to
control the cytotoxic activities of these compounds. A number of
strategies have been explored to modulate the oxidation of CL in
mitochondria [64]. The peroxidase activity of cytochrome c and CL can
be activated by hydrogen peroxide or lipid hydroperoxides [65]. Thus
disruption of this interaction by scavenging H2O2 or reducing lipid
hydroperoxides may lead to the inhibition of apoptosis. Overexpres-
sion of phospholipid hydroperoxide glutathione peroxidase (PHGPX)
in mitochondria of RBL2H3 cells prevented the release of Cyt c,
[11] Doorn, J. A.; Hurley, T. D.; Petersen, D. R. Inhibition of human mitochondrial
aldehyde dehydrogenase by 4-hydroxynon-2-enal and 4-oxonon-2-enal. Chem.
Res. Toxicol. 19:102–110; 2006.

178
W. Liu et al. / Free Radical Biology & Medicine 50 (2011) 166–178
[12] Petersen, D. R.; Doorn, J. A. Reactions of 4-hydroxynonenal with proteins and
cellular targets. Free Radic. Biol. Med. 37:937–945; 2004.
[42] Yin, H.; Cox, B. E.; Liu, W.; Porter, N. A.; Morrow, J. D.; Milne, G. L. Identification of
intact oxidation products of glycerophospholipids in vitro and in vivo using
negative ion electrospray iontrap mass spectrometry. J. Mass Spectrom. 44:
672–680; 2009.
[13] Schneider, C.; Porter, N. A.; Brash, A. R. Routes to 4-hydroxynonenal: fundamental
issuesinthemechanismsof lipid peroxidation. J. Biol. Chem. 283:15539–15543; 2008.
[14] Pryor, W. A.; Porter, N. A. Suggested mechanisms for the production of 4-hydroxy-
2-nonenal from the autoxidation of polyunsaturated fatty acids. Free Radic. Biol.
Med. 8:541–543; 1990.
[15] Schneider, C.; Tallman, K. A.; Porter, N. A.; Brash, A. R. Two distinct pathways of
formation of 4-hydroxynonenal: mechanisms of nonenzymatic transformation of
the 9- and 13-hydroperoxides of linoleic acid to 4-hydroxyalkenals. J. Biol. Chem.
276:20831–20838; 2001.
[16] Schneider, C.; Boeglin, W. E.; Yin, H.; Donald, F. Stec.; Porter, D. L. H. A.; Brash, A. R.
Synthesis of dihydroperoxides of linoleic and linolenic acids and studies on their
transformation to 4-hydroperoxynonenal. Lipids 40:1155–1162; 2005.
[17] Schneider, C.; Boeglin, W. E.; Yin, H.; Porter, N. A.; Brash, A. R. Intermolecular
peroxyl radical reactions during autoxidation of hydroxy and hydroperoxy
arachidonic acids generate a novel series of epoxidized products. Chem. Res.
Toxicol. 21:895–903; 2008.
[43] Pulfer, M.; Murphy, R. C. Electrospray mass spectrometry of phospholipids. Mass
Spectrom. Rev. 22:332–364; 2003.
[44] Domingues, M. R. M.; Reis, A.; Domingues, P. Mass spectrometry analysis of
oxidized phospholipids. Chem. Phys. Lipids 156:1–12; 2008.
[45] Tyurin, V. A.; Tyurina, Y. Y.; Kochanek, P. M.; Hamilton, R.; DeKosky, S. T.;
Greenberger, J. S.; Bayir, H.; Kagan, V. E.; Roya Khosravi-Far, Z. Z. R. A. L.; Mauro, P.
Oxidative lipidomics of programmed cell death. Meth. Enzymol. 442:375–393; 2008.
[46] Tyurina, Y. Y.; Tyurin, V. A.; Epperly, M. W.; Greenberger, J. S.; Kagan, V. E.
Oxidative lipidomics of γ-irradiation-induced intestinal injury. Free Radic. Biol.
Med. 44:299–314; 2008.
[47] Tyurina, Y. Y.; Tyurin, V. A.; Kapralova, V. I.; Amoscato, A. A.; Epperly, M. W.;
Greenberger, J. S.; Kagan, V. E. Mass-spectrometric characterization of phospho-
lipids and their hydroperoxide derivatives in vivo: effects of total body irradiation.
Meth. Mol. Biol. 580:153–183; 2009.
[18] Lee, S. H.; Oe, T.; Blair, I. A. Vitamin C-induced decomposition of lipid
hydroperoxides to endogenous genotoxins. Science 292:2083–2086; 2001.
[19] Gu, X.; Zhang, W.; Salomon, R. G. Fe2+ catalyzes vitamin E-induced fragmentation
of hydroperoxy and hydroxy endoperoxides that generates gamma-hydroxy
alkenals. J. Am. Chem. Soc. 129:6088–6089; 2007.
[48] Roede, J. R.; Jones, D. P. Reactive species and mitochondrial dysfunction:
mechanistic significance of 4-hydroxynonenal. Environ. Mol. Mutagen. 51:
380–390; 2010.
[49] Paradies, G.; Ruggiero, F. M.; Petrosillo, G.; Quagliariello, E. Peroxidative damage
to cardiac mitochondria: cytochrome oxidase and cardiolipin alterations. FEBS
Lett. 424:155–158; 1998.
[20] Mayo, F. R.; Miller, A. A. Oxidation of unsaturated compounds. II. Reactions of
styrene peroxide 1. J. Am. Chem. Soc. 78:1023–1034; 1956.
[21] Morita, M.; Tokita, M. The real radical generator other than main-product
hydroperoxide in lipid autoxidation. Lipids 41:91–95; 2006.
[50] Sen, T.; Sen, N.; Tripathi, G.; Chatterjee, U.; Chakrabarti, S. Lipid peroxidation
associated cardiolipin loss and membrane depolarization in rat brain mitochon-
dria. Neurochem. Int. 49:20–27; 2006.
[22] Miyashita, K.; Hara, N.; Fujimoto, K.; Kaneda, T. Formation of dimers during the initial
stage of autoxidation in methyl linoleate. Agric. Biol. Chem. 46:751–755; 1982.
[23] Miyashita, K.; Hara, N.; Fujimoto, K.; Kaneda, T. Structural studies of polar dimers
in autoxidized methyl linoleate during the initial stages of autoxidation. Agric.
Biol. Chem. 48:2511–2515; 1984.
[24] Miyashita, K.; Hara, N.; Fujimoto, K.; Kaneda, T. Decomposition products of dimers
arising from secondary oxidation of methyl linoleate hydroperoxides. Agric. Biol.
Chem. 49:2633–2640; 1985.
[51] Nakagawa, Y. Initiation of apoptotic signal by the peroxidation of cardiolipin of
mitochondria. Ann. NY Acad. Sci. 1011:177–184; 2004.
[52] Orrenius, S.; Zhivotovsky, B. Cardiolipin oxidation sets cytochrome c free. Nat.
Chem. Biol. 1:188–189; 2005.
[53] Ott, M.; Zhivotovsky, B.; Orrenius, S. Role of cardiolipin in cytochrome c release
from mitochondria. Cell Death Differ. 14:1243–1247; 2007.
[54] Ji, C.; Amarnath, V.; Pietenpol, J. A.; Marnett, L. J. 4-Hydroxynonenal induces
apoptosis via caspase-3 activation and cytochrome c release. Chem. Res. Toxicol.
14:1090–1096; 2001.
[25] Miyashita, K.; Hara, N.; Fujimoto, K.; Kaneda, T. Dimers formed in oxygenated
methyl linoleate hydroperoxides. Lipids 20:578–587; 1985.
[55] Uchida, K. Role of reactive aldehyde in cardiovascular diseases. Free Radic. Biol.
[26] Hauff, K. D.; Hatch, G. M. Cardiolipin metabolism and Barth syndrome. Prog. Lipid
Res. 45:91–101; 2006.
[27] Lesnefsky, E. J.; Hoppel, C. L. Cardiolipin as an oxidative target in cardiac
mitochondria in the aged rat. Biochim. Biophys. Acta 1777:1020–1027; 2008.
[28] Schlame, M.; Ren, M.; Xu, Y.; Greenberg, M. L.; Haller, I. Molecular symmetry in
mitochondrial cardiolipins. Chem. Phys. Lipids 138:38–49; 2005.
[29] Kagan, V. E.; Tyurin, V. A.; Jiang, J.; Tyurina, Y. Y.; Ritov, V. B.; Amoscato, A. A.;
Osipov, A. N.; Belikova, N. A.; Kapralov, A. A.; Kini, V.; Vlasova, I. I.; Zhao, Q.; Zou,
M.; Di, P.; Svistunenko, D. A.; Kurnikov, I. V.; Borisenko, G. G. Cytochrome c acts as
a cardiolipin oxygenase required for release of proapoptotic factors. Nat. Chem.
Biol. 1:223–232; 2005.
[30] Gonzalvez, F.; Gottlieb, E. Cardiolipin: setting the beat of apoptosis. Apoptosis 12:
877–885; 2007.
[31] Kagan, V. E.; Bayir, H. A.; Belikova, N. A.; Kapralov, O.; Tyurina, Y. Y.; Tyurin, V. A.;
Jiang, J.; Stoyanovsky, D. A.; Wipf, P.; Kochanek, P. M.; Greenberger, J. S.; Pitt, B.;
Shvedova, A. A.; Borisenko, G. Cytochrome c/cardiolipin relations in mitochon-
dria: a kiss of death. Free Radic. Biol. Med. 46:1439–1453; 2009.
[32] Milne, G. L.; Seal, J. R.; Havrilla, C. M.; Wijtmans, M.; Porter, N. A. Identification and
analysis of products formed from phospholipids in the free radical oxidation of
human low density lipoproteins. J. Lipid Res. 46:307–319; 2005.
Med. 28:1685–1696; 2000.
[56] Uchida, K. Future of toxicology—lipid peroxidation in the future: from biomarker
to etiology. Chem. Res. Toxicol. 20:3–5; 2007.
[57] Liu, Q.; Raina, A. K.; Smith, M. A.; Sayre, L. M.; Perry, G. Hydroxynonenal, toxic
carbonyls, and Alzheimer disease. Mol. Aspects Med. 24:305–313; 2003.
[58] Lin, D.; Lee, H. -g.; Liu, Q.; Perry, G.; Smith, M. A.; Sayre, L. M. 4-Oxo-2-nonenal is
both more neurotoxic and more protein reactive than 4-hydroxy-2-nonenal.
Chem. Res. Toxicol. 18:1219–1231; 2005.
[59] Sayre, L. M.; Perry, G.; Smith, M. A. Oxidative stress and neurotoxicity. Chem. Res.
Toxicol. 21:172–188; 2008.
[60] Sayre, L. M.; Smith, M. A.; Perry, G. Chemistry and biochemistry of oxidative stress
in neurodegenerative disease. Curr. Med. Chem. 8:721–738; 2001.
[61] Wang, H. -Y. J.; Jackson, S. N.; Woods, A. S. Direct MALDI-MS analysis of cardiolipin
from rat organs sections. J. Am. Soc. Mass Spectrom. 18:567–577; 2007.
[62] Picklo, S. M. J.; Montine, T. J. Mitochondrial effects of lipid-derived neurotoxins. J.
Alzheimers Dis. 12:185–193; 2007.
[63] Akude, E.; Zherebitskaya, E.; Roy Chowdhury, S.; Girling, K.; Fernyhough, P. 4-Hydroxy-
2-nonenal induces mitochondrial dysfunction and aberrant axonal outgrowth in adult
sensory neurons that mimics features of diabetic neuropathy. Neurotox. Res. 17:28–38;
2010.
[33] Uchida, T.; Gotoh, N.; Wada, S. Method for analysis of 4-hydroxy-2-(E)-nonenal
[64] Hoye, A. T.; Davoren, J. E.; Wipf, P.; Fink, M. P.; Kagan, V. E. Targeting mitochondria.
Acc. Chem. Res. 41:87–97; 2008.
with solid-phase microextraction. Lipids 37:621–626; 2002.
[34] Harrison, K. A.; Davies, S. S.; Marathe, G. K.; McIntyre, T.; Prescott, S.; Reddy, K. M.;
Falck, J. R.; Murphy, R. C. Analysis of oxidized glycerophosphocholine lipids using
electrospray ionization mass spectrometry and microderivatization techniques. J.
Mass Spectrom. 35:224–236; 2000.
[35] Pulfer, M.; Murphy, R. C. Electrospray mass spectrometry of phospholipids. Mass
Spectrom. Rev. 22:332–364; 2003.
[36] Kadiiska, M. B.; Gladen, B. C.; Baird, D. D.; Germolec, D.; Graham, L. B.; Parker, C. E.;
Nyska, A.; Wachsman, J. T.; Ames, B. N.; Basu, S.; Brot, N.; FitzGerald, G. A.; Floyd, R. A.;
George, M.; Heinecke, J. W.; Hatch, G. E.; Hensley, K.; Lawson, J. A.; Marnett, L. J.;
Morrow, J. D.; Murray, D. M.; Plastaras II, J.; L.J. R.; Rokach, J.; Shigenaga, M. K.; Sohal,
R. S.; Sun, J.; Tice, R. R.; Thiel, D. H. V.; Wellner, D.; Walter, P. B.; Tome, K. B.;
Mason, R. P.; Barrett, J. C. Biomarkers of Oxidative Stress Study II: are oxidation
products of lipids, proteins, and DNA markers of CCl4 poisoning? Free Radic. Biol.
Med. 38:698–710; 2005.
[65] Belikova, N. A.; Tyurina, Y. Y.; Borisenko, G.; Tyurin, V.; Samhan Arias, A. K.;
Yanamala, N.; Furtmuller, P. G.; Klein-Seetharaman, J.; Obinger, C.; Kagan, V. E.
Heterolytic reduction of fatty acid hydroperoxides by cytochrome c/cardiolipin
complexes: antioxidant function in mitochondria. J. Am. Chem. Soc. 131:
11288–11289; 2009.
[66] Nomura, K.; Imai, H.; Koumura, T.; Kobayashi, T.; Nakagawa, Y. Mitochondrial
phospholipid hydroperoxide glutathione peroxidase inhibits the release of
cytochrome c from mitochondria by suppressing the peroxidation of cardiolipin
in hypoglycaemia-induced apoptosis. Biochem. J. 351:183–193; 2000.
[67] Liang, H.; Ran, Q.; Jang, Y. C.; Holstein, D.; Lechleiter, J.; McDonald-Marsh, T.;
Musatov, A.; Song, W.; Van Remmen, H.; Richardson, A. Glutathione peroxidase 4
differentially regulates the release of apoptogenic proteins from mitochondria.
Free Radic. Biol. Med. 47:312–320; 2009.
[68] Milne, G. L.; Yin, H.; Morrow, J. D. Human biochemistry of the isoprostane
[37] Benedetti, A.; Comporti, M.; Esterbauer, H. Identification of 4-hydroxynonenal as a
cytotoxic product originating from the peroxidation of liver microsomal lipids.
Biochim. Biophys. Acta 620:281–296; 1980.
[38] Miyashita, K.; Hara, N.; Fujimoto, K.; Kaneda, T. Structure of dimers produced from
methyl linoleate during the initial stages of autoxidation. Agric. Biol. Chem. 46:
2293–2297; 1982.
pathway. J. Biol. Chem. 283:15533–15537; 2008.
[69] Chen, L.; Na, R.; Gu, M.; Salmon, A. B.; Liu, Y.; Liang, H.; Qi, W.; Remmen, H. V.;
Richardson, A.; Ran, Q. Reduction of mitochondrial H2O2 by overexpressing
peroxiredoxin 3 improves glucose tolerance in mice. Aging Cell 7:866–878; 2008.
[70] Brichac, J.; Ho, K. K.; Honzatko, A.; Wang, R.; Lu, X.; Weiner, H.; Picklo, M. J.
Enantioselective oxidation of trans-4-hydroxy-2-nonenal is aldehyde dehydro-
genase isozyme and Mg2+ dependent. Chem. Res. Toxicol. 20:887–895; 2007.
[71] Chen, C. -H.; Budas, G. R.; Churchill, E. N.; Disatnik, M. -H.; Hurley, T. D.; Mochly-Rosen,
D. Activation of aldehyde dehydrogenase-2 reduces ischemic damage to the heart.
Science 321:1493–1495; 2008.
[39] Cosgrove, J.; Church, D.; Pryor, W. The kinetics of the autoxidation of
polyunsaturated fatty acids. Lipids 22:299–304; 1987.
[40] Bowry, V. W. Arm-to-arm autoxidation in a triglyceride: remote group reaction
kinetics. J. Org. Chem. 59:2250–2252; 1994.
[41] Moon, K. H.; Lee, Y. M.; Song, B. J. Inhibition of hepatic mitochondrial aldehyde
dehydrogenase by carbon tetrachloride through JNK-mediated phosphorylation.
Free Radic. Biol. Med. 48:391–398; 2010.
[72] Hill, B. G.; Awe, S. O.; Vladykovskaya, E.; Ahmed, Y.; Liu, S. -Q.; Bhatnagar, A.;
Srivastava, S. Myocardial ischaemiainhibits mitochondrialmetabolism of 4-hydroxy-
trans-2-nonenal. Biochem. J. 417:513–524; 2009.