Novel carbonyl and nitrile products from reactive chlorinating species attack of lysosphingolipid

Article abstract of DOI:10.1016/j.chemphyslip.2006.10.006

Lysosphingolipids are important lipid signaling molecules that are associated predominantly with high density lipoproteins (HDL) in human plasma. Further, HDL has been shown to be a target for the reactive chlorinating species (RCS) produced by myeloperox

Full text of DOI:10.1016/j.chemphyslip.2006.10.006

Chemistry and Physics of Lipids 145 (2007) 72–84
Novel carbonyl and nitrile products from reactive chlorinating
species attack of lysosphingolipid
Viral V. Brahmbhatta, Fong-Fu Hsuc, Jeff L.-F. Kaob,
Erin C. Franka, David A. Forda,∗
Department of Biochemistry and Molecular Biology, St. Louis University Medical Center,
a
1402 South Grand Blvd., St. Louis, MO 63104, United States
Department of Internal Medicine, Washington University, St. Louis, MO 63110, United States
b
c
Department of Chemistry, Washington University, St. Louis, MO 63110, United States
Received 30 August 2006; received in revised form 26 October 2006; accepted 26 October 2006
Available online 16 November 2006
Abstract
Lysosphingolipids are important lipid signaling molecules that are associated predominantly with high density lipoproteins
(HDL) in human plasma. Further, HDL has been shown to be a target for the reactive chlorinating species (RCS) produced by
myeloperoxidase (MPO). Accordingly, RCS attack of lysosphingolipids was characterized in these studies. It was shown that
RCS attack of sphingosylphosphorylcholine results in the formation of 2-hexadecenal and 1-cyano methano phosphocholine. The
structures were identified and confirmed predominantly using mass spectrometric analyses. Further, it was demonstrated that
RCS attack of another bioactive lysosphingolipid sphingosine 1-phosphate also results in the formation of 2-hexadecenal from its
sphingosine base. Using a synthetically prepared, deuterated 2-hexadecenal internal standard, it was determined that 2-hexadecenal
quickly accumulated in HDL treated with MPO/RCS generating system. Thus, the present studies characterize the formation of a
novel group of lipid products generated following RCS attack of lysosphingolipids.
© 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Myeloperoxidase; Reactive chlorinating species; Lysosphingolipids; Mass spectrometry; 2-Hexadecenal; 1-Cyano methano phospho-
choline
1. Introduction
base (OCl)-, from hydrogen peroxide and chloride ion
(Hampton et al., 1998; Harrison and Schultz, 1976).
Hypochlorous acid, is a strong oxidizing agent and can
react with primary amines to generate chloramines (R-
NHCl), which retain their oxidizing potential (Lampert
and Weiss, 1983; Weiss et al., 1983). In vivo, the primary
role of MPO is bactericidal. In fact, individuals with
MPO deficiency show decreased capacity of microbial
killing by their neutrophils (Cech et al., 1979; Kitahara
et al., 1981; Lehrer and Cline, 1969). However, RCS
also attack the host tissue (Weiss, 1989) and several
studies have established a role of MPO in heart disease.
For example, MPO polymorphisms have been shown
Myeloperoxidase (MPO),
a
heme-containing
enzyme, is present in the ␣ granules of neutrophils and
is released into phagolysosomes (Hampton et al., 1998;
Jiang et al., 1997) as well as the extracellular space
by activated phagocytes (Edwards et al., 1987). MPO
catalyzes formation of the reactive chlorinating species
(RCS), hypochlorous acid (HOCl) and its conjugate
∗
Corresponding author. Tel.: +1 314 977 9264;
fax: +1 314 977 9205.
E-mail address: fordda@slu.edu (D.A. Ford).
0009-3084/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2006.10.006

V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
73
to affect coronary artery disease (Asselbergs et al.,
2004; Nikpoor et al., 2001) and expression of human
MPO has been shown to promote atherosclerosis in
mice (McMillen et al., 2005). Still, the role of MPO in
cardiovascular disease remains to be fully elucidated.
MPO-derived RCS attack a wide variety of molecules
including nucleic acids, proteins and lipids. Previous
studies have established plasmalogens as important tar-
gets of MPO-derived RCS. MPO-derived RCS attack
on plasmalogens generates 2-chlorohexadecanal (Albert
et al., 2001), which is increased in atherosclerotic tis-
sue (Thukkani et al., 2003) and can inhibit endothelial
nitric oxide synthase activity (Marsche et al., 2004).
MPO-derived RCS also attack the unsaturated –C C–
bonds within aliphatic chains of phospholipids and
lipoprotein-associated cholesterol which leads to the
production of chlorohydrins (Winterbourn et al., 1992)
and dichlorinated products (Hazen et al., 1996). Fur-
thermore, RCS attack the primary amine group of
phosphatidylethanolamine to produce chloramines (Carr
et al., 1998).
Lysosphingolipids such as d-erythro sphingosylphos-
phorylcholine (SPC) and sphingosine 1-phosphate (S1P)
as well as sphingosine, have emerged as a new
class of signaling molecules (Alewijnse et al., 2004).
These lipids associate predominantly with high-density
lipoproteins (HDL) in the plasma (Nofer et al., 2001).
HDL-associated lysosphingolipids have been suggested
to mediate, in part, the atheroprotective effects of HDL
such as nitric oxide dependent vasorelaxation (Nofer et
al., 2004), decrease of E-selectin expression on human
endothelial cells (Nofer et al., 2003) and decrease of
endothelial cell apoptosis (Kimura et al., 2001; Nofer
et al., 2001). Other atheroprotective effects of lysosph-
ingolipids include inhibition of thrombin generation
(Deguchi et al., 2004) and inhibition of platelet func-
tion (Altmann et al., 2003). Additionally, it is known
that MPO derived RCS targets HDL and affects choles-
terol efflux (Bergt et al., 2004). Accordingly, the present
study tested the hypothesis that MPO-derived RCS can
attack HDL-associated lysosphingolipids. The results
here demonstrate that MPO-derived RCS attack SPC to
generate 2-hexadecenal and 1-cyano methano phospho-
choline. Moreover, we demonstrate that 2-hexadecenal
is increased when MPO-derived RCS react with HDL.
phorylcholine, d-erythro sphingosylphosphorylcholine
(SPC), sphingosine and sphingosine 1-phosphate
(S1P) were purchased from Matreya, Inc.
Sodium(meta)periodate was purchased from Fluka.
[13,13,14,14,14-d5]-tetradecanoic acid was purchased
from C/D/N isotopes. All other reagents were purchased
from Aldrich, Sigma or Fisher.
2.2. Analysis of reaction products from RCS
treatment of sphingolipids
Incubations were performed in chlorine-demand-
free and chloride-free reaction vessels (Weil and
Morris, 1949). One hundred micrograms of lipid
or 200 ␮g of protein (for HDL) was incubated in
1 ml of 20 mM phosphate-buffer comprised of 0.1 mM
diethylenetriamine-pentaacetic acid (pH 4.0–7.0) in the
presence or absence of indicated amounts of MPO
(1–2 U/ml), hydrogen peroxide (H2O2) (1 mM) or NaCl
(100 mM) for 15 min at 37 ◦C. Synthetic lipids were
sonicated in buffer, prior to initiating reactions. Reac-
tions were terminated by the addition of methanol. The
reaction products were extracted into chloroform by the
method of Bligh and Dyer (Bligh and Dyer, 1959).
Thin-layer chromatography (TLC) analysis was done
˚
on silica gel 60 A plates (Whatman) utilizing a mobile
phase comprised of petroleum ether/ethyl ether/acetic
acid (90/10/1, v/v/v) for neutral lipid separation. To sep-
arate polar lipids, chloroform/acetone/methanol/acetic
acid/water (6/8/2/2/1, v/v/v/v/v) was used as mobile
phase and the TLC plate was run twice in the same
direction after drying in between runs. In both cases,
the reaction products were visualized by charring con-
centrated sulfuric acid-treated plates.
2.3. Preparation of 2-hexadecenal
Sphingosinewasreactedwithsodium(meta)periodate
to generate 2-hexadecenal (Baumann et al., 1969).
2-Hexadecenal was purified by high-pressure liquid
chromatography (HPLC) using a solid-phase silica col-
umn (250 mm × 4.6 mm, 5 ␮m; Beckman). An isocratic
gradient of hexane/isopropanol (100/0.5, v/v) was uti-
lized at a flow rate of 2 ml/min. UV monitoring was set
at 206 nm for detection of 2-hexadecenal.
2. Experimental procedures
2.4. Synthesis of 2-[d5]-hexadecenal
Synthesis of 2-[15,15,16,16,16-d5]-hexadecenal
(2-[d5]-hexadecenal) was achieved by the following
scheme: (1) [13,13,14,14,14-d5]-tetradecanoic acid
was converted to [13,13,14,14,14-d5]-tetradecanol
2.1. Materials
Human myeloperoxidase (MPO), catalase and HDL
were purchased from Calbiochem. Sphinganylphos-

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V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
using sodium bis(2-methoxyethoxy)aluminum hydride,
which was further converted to [13,13,14,14,14-d5]-
tetradecanal using oxalyl chloride-activated dimethyl
sulfoxide as described earlier (Mancuso et al., 1978);
(2) [13,13,14,14,14-d5]-tetradecanal was converted to
2-[15,15,16,16,16-d5]-hexadecenoic acid by addition
of propanedioic acid in the presence of pyridine and
piperidine as described by Hino et al (Tohru Hino et
al., 1986); (3) cis and trans forms of 2-[15,15,16,16,16-
d5]-hexadecenoic acid were separated by HPLC using
reverse-phase solid-phase (Econosil C18 10 ␮m,
250 mm × 10 mm) with gradient elution using water
(pH 2 using phosphoric acid) and acetonitrile as mobile
phase A and B, respectively. Following injection of
the reaction mixture, the column was eluted with 50%
A and 50% B for 15 min, followed by linear gradient
from 50% A to 100% B over 15 min. Following the
gradient the column was further eluted with 100% B for
90 min. UV was set at 192 nm. Trans 2-[15,15,16,16,16-
d5]-hexadecenoic acid elutes at ∼34 min; (4) Trans
2-[15,15,16,16,16-d5]-hexadecenoic acid was converted
to 2-[15,15,16,16,16-d5]-hexadecenol using sodium
bis(2-methoxyethoxy)aluminum hydride (Thukkani
et al., 2002); (5) 2-[15,15,16,16,16-d5]-hexadecenol
was oxidized to 2-[d5]-hexadecenal using pyridinium
chlorochromate and extracted from silica after TLC
purification in neutral lipid solvent system as described
above; (6) 2-[d5]-hexadecenal purity was confirmed by
GC–MS of its pentafluorobenzyl (PFB) oxime deriva-
tive (see below) and quantified by gas chromatography
with flame ionization detection of its acid methanolysis
derivatives using arachidic acid as an internal standard.
rate of 25 ◦C/min to 300 ◦C. The oven temperature was
held at 300 ◦C for an additional 4 min. All spectra were
acquired on a Hewlett Packard 5973 mass spectrometer.
The source temperature was set at 230 ◦C. The elec-
tron energy was 69.9 eV and the emission current was
34.6 ␮A.
For PFB hydroxylamine-derivatized products, either
TLC-purified reaction products or crude reaction prod-
uct extracts were derivatized using PFB hydroxylamine
and analyzed as previously described (Thukkani et al.,
2002). Briefly, extracts were dried under nitrogen and
resuspended in 300 ␮l of ethanol. Three hundred micro-
liters of PFB hydroxylamine solution (6 mg/ml) was
added, vortexed for 5 min and allowed to incubate for
25 min at room temperature. Following the addition of
1.2 ml of water, the reaction products were sequen-
tially extracted into 2 ml of cyclohexane: diethyl ether
(4:1, v/v), dried under a stream of nitrogen and resus-
pended in 50–100 ␮l of petroleum ether for GC–MS
analysis. GC–MS analysis was performed in the nega-
tive ion chemical ionization mode (NICI) with methane
as the reagent gas. The instruments and the separat-
ing column used were the same as described above.
The source temperature was set at 150 ◦C. The elec-
tron energy was 193.3 eV and the emission current was
49.4 ␮A. The injector and transfer lines were maintained
at 250 ◦C and 280 ◦C, respectively. The GC oven was
maintained at 150 ◦C for 3.5 min, increased at the rate of
25 ◦C/minto310 ◦Candheldat310 ◦Cforanother5 min.
Quantification of 2-hexadecenal was performed utilizing
selected ion monitoring (SIM) by comparing the inte-
grated area corresponding to m/z = 413 to that produced
fromthedeuteratedinternalstandard2-[d5]-hexadecenal
at m/z = 418.
For methanolic HCl-derivatized products, crude reac-
tion product extracts or TLC-purified reaction products
were resuspended in 1 ml of 1N methanolic HCl and
incubated at 90 ◦C for 90 min under nitrogen. The reac-
tion was allowed to cool for 10 min before addition
of sodium carbonate (∼200 mg). For GC–MS analysis,
1 ml of water and 2 ml of petroleum ether was added
to extract the derivatized reaction products into organic
phase. The organic phase was evaporated under nitro-
gen and the products were resuspended in 50 ␮l of
petroleum ether. The derivatized products were sepa-
rated using DB-1 column and analyzed with GC–MS
EI ionization as described earlier. Alternatively, for
electrospray ionization-tandem mass spectrometry (ESI-
MS/MS) analysis, 5 ml of methanol was added after
derivatization and sodium carbonate addition. This
mixture was centrifuged at 1000 rpm for 2 min. The
supernatant was then analyzed by ESI-MS/MS.
2.5. Gas chromatography-mass spectrometry
(GC–MS) analysis of reaction products
TLC-purified reaction products were extracted from
silica into chloroform by a modified Bligh and Dyer
technique. TLC-purified reaction products were ana-
lyzed either directly, or following derivatization using
methanolic HCl or PFB hydroxylamine. For GC–MS
electron impact (EI) ionization analysis, reaction prod-
ucts in chloroform were dried under nitrogen and
resuspended in 50–100 ␮l ethyl acetate. One microliter
was injected using a Hewlett Packard 7683 series injec-
tor. The products were separated on a DB-1 column
(12.0 m, 0.2 mm inner diameter, 0.33 ␮m methyl sili-
cone film coating; Cobert, St. Louis, MO) utilizing a
Hewlett Packard 6890 gas chromatograph. The injector
temperature was set to 250 ◦C and transfer lines were
maintained at 280 ◦C. The GC oven was maintained at
125 ◦C for 2 min and temperature was increased at a

V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
75
2.6. ESI-MS/MS
mixture, the column was eluted with 50% solvent A and
50% solvent B for 2 min, the gradient was increased to
100% B over the next 3.6 min and held at 100% B for
the next 2.4 min, the gradient was again brought to 50%
solvent A and 50% solvent B over the next 0.1 min and
held for the next 6.9 min. The 223 → 136.9 transition
reaction (for 1-cyano methano phosphocholine) and the
465 → 184.1 transition reaction (for SPC) were mon-
itored in positive ion mode at 28 eV collision energy.
Argon gas pressure was set at 1.5 mTorr.
The crude reaction product extracts were dried
under nitrogen and resuspended either in methanol or
methanol/water/acetic acid (50/50/1, v/v/v). These were
then injected into the electrospray ionization interface in
the direct infusion mode (TSQ quantum ultra, Thermo
Electron Corporation) at a flow rate of 3 ␮l/min. Tan-
dem mass spectrometry (MS/MS) was performed with
collision energies ∼22 eV. Typically, spectra were aver-
aged 3–5 min and processed utilizing Xcalibur software
(Finnigan). Ions were monitored in positive ion mode.
In some cases, ESI-MS/MS was performed on TSQ
7000 (Finnigan). Skimmer collision induced dissocia-
tion (CID)–MS/MS was performed at 25 eV.
2.8. H-D exchange
Crude reaction products generated by MPO treat-
ment were extracted by Bligh and Dyer technique
as mentioned earlier. Chloroform was evaporated and
the products resuspended in deuterated methanol with
0.1 mM ammonium deuteroxide and analyzed by ESI-
MS/MS.
2.7. HPLC-ESI-MS/MS of 1-cyano methano
phosphocholine and SPC
Crude reaction product extracts collected as described
earlier were dried under nitrogen and resuspended in
10 ml methanol. Ten microliters was injected on a solid-
phase reverse-phase Discovery HS C18 column (5 ␮m,
150 mm × 2.1 mm). The products were eluted using sol-
vent A: water/methanol/acetic acid (69/30/1, v/v/v) with
5 mM ammonium acetate and solvent B: methanol/acetic
acid (99/1, v/v) with 5 mM ammonium acetate as
described by Sullards et al for SPC analysis (Sullards
and Merrill, 2001). Following injection of the reaction
2.9. Proton NMR spectrometry
Crude reaction products from multiple reactions were
extracted in hexane, pooled together and purified by
HPLC to separate 2-hexadecenal, as above. Proton NMR
spectrometry and analysis were performed at ambient
temperature in CDCl3 using Varian Unity 300 spectrom-
eter (7.05 T). 1H shifts were referenced to solvent peak at
7.24 ppm. The magnetic field strength used was 14.1 T.
Fig. 1. TLC analysis of MPO-treated SPC. SPC (100 ␮g) was incubated for 15 min at 37 ◦C in the presence or absence of each of the MPO-derived
RCS generating reagents including, MPO (1U), heat-inactivated (ꢀ) MPO, H2O2 (1 mM), NaCl (100 mM) and 20 mM phosphate buffer at pH 4–7
as indicated. Reactions were terminated by the addition of methanol and products were sequentially extracted into chloroform and separated by
TLC using either petroleum ether/ethyl ether/acetic acid (90/10/1, v/v/v) (A and B) or chloroform/acetone/methanol/acetic acid/water (6/8/2/2/1,
v/v/v/v/v) as mobile phase (C). The reaction products were visualized by sulfuric acid treatment and charring.

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3. Results
MPO is thought to have important roles in the patho-
physiology of atherosclerosis. The biological effects of
MPOaremediatedbyRCS. Invasculardiseaseitislikely
that MPO-derived RCS attack lipoprotein lipids and pro-
teins (Bergt et al., 2004; Marsche et al., 2004). Since
lysosphingolipids are biologically active lipids present
in HDL (Nofer et al., 2001), initial experiments were
performed to determine the stability of SPC in pres-
ence of RCS. TLC analysis revealed that in presence
of the complete MPO/RCS generating system (MPO,
H2O2 and Cl−) SPC is degraded (Fig. 1C, lane 2) result-
ing in the production of a neutral lipid (Fig. 1A, lane
2). This neutral lipid was not observed in the absence
of MPO, H2O2 or sodium chloride from the complete
MPO/RCS generating system. Additionally, this neutral
product was not produced when heat inactivated MPO
was used (Fig. 1A, lane 4). Since acidic conditions are
optimal for MPO-derived RCS generation at the concen-
tration of H2O2 and sodium chloride used (Hollenberg et
al., 1974; Zgliczynski et al., 1977), we characterized the
effectofpHonthegenerationoftheneutrallipidproduct.
It is observed that the neutral lipid product was present at
pH 4 and 5 but absent at pH 6 and 7 (Fig. 1B). This is most
likely due to the decrease in generation of MPO-derived
RCS at neutral pH (Hollenberg et al., 1974; Zgliczynski
et al., 1977). SPC incubation with sodium hypochlorite
also results in the production of this neutral lipid product
(data not shown).
Fig. 2. Electron impact GC–MS analysis of MPO-treated SPC. SPC
(100 ␮g) was incubated with MPO-derived RCS generating reagents,
which includes MPO (1U), H2O2 (1 mM) and NaCl (100 mM) in
20 mM phosphate buffer at pH 4. The reaction products were extracted
into chloroform. The neutral lipid product was purified by TLC and
subsequently analyzed by GC–MS using electron impact (EI) ioniza-
tion as described under Section 2. The EI spectra of the neutral lipid
product is depicted.
et al., 1999). The presence of the carbonyl group in the
molecule was further confirmed by acid methanolysis
that results in the formation of a dimethyl acetal from
TLC-purified material. The dimethyl acetal derivative
was analyzed by GC–MS with electron impact ioniza-
tion. A peak at 6.5 min that gives a molecular ion and
fragmentation pattern corresponding to a dimethyl acetal
of 2-hexadecenal was observed (Fig. 4).
The TLC-purified neutral lipid product generated by
RCS-attack of SPC was analyzed by GC–MS using elec-
tron impact ionization. Fig. 2 shows the mass spectra
of the TLC-purified product having a retention time
of 5.6 min. The molecular ion at m/z 238 and its frag-
mentation pattern is depicted in Scheme 1, showing
the McLafferty rearrangement ion as the base peak at
m/z 70. The molecular ion and fragmentation pattern
is consistent with a 16 carbon unsaturated aldehyde,
2-hexadecenal, generated from the degradation of the
sphingosine base of SPC.
To further confirm the structure and presence of
the carbonyl group, TLC-purified products were deriva-
tized using PFB hydroxylamine (Hsu et al., 1999). The
PFB derivatives were analyzed by GC–MS in NICI
mode. Fig. 3 shows the mass spectrum and fragmen-
tation scheme of PFB-oxime of 2-hexadecenal having
a retention time of 8.24 min. The spectrum shows the
structurally informative ions at m/z 433 ([M]•¯), m/z 413
([M − HF]•¯), m/z 383 ([M − HF − NO] •¯) and m/z 252
([M − 181]•¯), along with the ions at m/z 178 and m/z 196
that arise from the dissociation of the PFB residue (Hsu
Scheme 1. Fragmentation scheme for 2-hexadecenal.

V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
77
Fig. 3. PFB hydroxylamine derivatization and GC–MS analysis of
MPO-treated SPC. SPC (100 ␮g) was incubated with MPO-derived
RCS generating reagents, which includes MPO (1U), H2O2 (1 mM)
and NaCl (100 mM) in 20 mM phosphate buffer at pH 4. The reactions
were terminated with methanol. Reaction products were sequentially
extracted into chloroform and derivatized with pentafluorobenzyl
hydroxylamine and analyzed by GC–MS in negative ion chemical
ionization (NICI) mode. The mass spectra for the PFB oxime of 2-
hexadecenal is shown along with the fragmentation scheme (inset).
To further confirm the position of the double bond
of 2-hexadecenal, proton NMR was performed. The
2-hexadecenal generated by MPO-derived RCS treat-
ment of SPC was HPLC purified for NMR analysis.
Semi-synthetic 2-trans-hexadecenal was used as refer-
ence (Baumann et al., 1969). The NMR spectrum of
HPLC purified 2-hexadecenal derived from SPC and
MPO-derived RCS reaction was identical to that of the
Fig. 5. NMRanalysisof2-hexadecenal. Semi-synthetic2-hexadecenal
(A) and the putative 2-hexadecenal product from MPO-derived RCS
targeting of SPC (B) were prepared and subjected to proton NMR as
described in Section 2. Peak assignments are shown in inset in Fig. 4A.
reference (Fig. 5). This confirmed that MPO-derived
RCS generate 2-hexadecenal from SPC.
Having identified the neutral lipid product as
2-hexadecenal, we confirmed the involvement of MPO-
derived RCS in reactions including inhibitors of MPO
production of RCS. SPC was incubated with MPO-
derived RCS generating system including MPO, H2O2
andsodiumchlorideinphosphatebuffer(pH4.0). Before
starting the reactions, MPO was pre-incubated either
with aminotriazole or sodium azide, while catalase was
pre-incubated with hydrogen peroxide. The reactions
were initiated by addition of MPO and allowed to incu-
bate for 15 min at 37 ◦C. At the end of the incubation
period the reaction products were extracted and analyzed
by TLC. As observed in Fig. 6 the presence of aminotri-
azole, sodium azide and catalase inhibited the formation
of 2-hexadecenal by MPO-derived RCS.
Fig. 4. Methanolic HCl derivatization and GC–MS analysis of MPO-
treated SPC. SPC (100 ␮g) was incubated with MPO-derived RCS
generating reagents, which includes MPO (1U), H2O2 (1 mM) and
NaCl (100 mM) in 20 mM phosphate buffer at pH 4. The reaction
products were extracted into chloroform and purified by TLC. The
TLC-purified neutral lipid product was subjected to acid methanolysis
and the derivatization product was analyzed by GC–MS using electron
impact ionization mode. The fragmentation spectra and fragmentation
scheme (inset) are depicted.
In order to test whether other bioactive lysosphin-
golipids, sharing the common sphingosine base, can
be attacked by MPO-derived RCS to generate 2-
hexadecenal, we incubated S1P with MPO-derived RCS

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V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
of PFB oxime of 2-hexadecenal in presence of the MPO-
derived RCS generating system (Fig. 7, trace a). In the
absence of either H2O2 or MPO from the reaction mix-
ture these peaks are also absent (Fig. 7, trace b and c).
Further, the identities of these peaks as PFB oximes
of 2-hexadecenal have been confirmed by mass spec-
trometry. Thus, it is believed that 2-hexadecenal can be
generated from MPO-derived RCS attack on lysosphin-
golipids containing a sphingosine base.
We have thus far confirmed the identity of 2-
hexadecenal utilizing TLC, GC–MS analysis, GC–MS
analysis of PFB oxime derivative and dimethyl acetal
derivative of 2-hexadecenal and by NMR studies. We
have also confirmed the involvement of MPO using heat-
treated MPO and MPO inhibitors. Furthermore, it has
been shown that MPO-derived RCS attack both SPC and
S1P to generate 2-hexadecenal.
Fig. 6. MPO-dependence for 2-hexadecenal generation. SPC (100 ␮g)
was incubated with MPO/RCS generating reagents, which includes
MPO (1U), H2O2 (1 mM) and NaCl (100 mM) in 20 mM phosphate
buffer at pH 4. 3-Amino triazole and sodium azide were preincubated
with MPO for 5 min and catalase was preincubated with H2O2 prior
to addition of MPO to the reaction mixture as described in Section
2. Reaction products were sequentially extracted into chloroform fol-
lowed byTLCseparationusingamobilephaseofpetroleumether/ethyl
ether/acetic acid (90/10/1, v/v/v). The products were visualized by
sulfuric acid treatment and charring.
To identify the by-product of SPC and MPO-derived
RCS reaction generating 2-hexadecenal, we performed
ESI-MS/MS analysis by direct infusion of the crude
reaction product extracts derived from SPC and MPO-
derived RCS reaction. In the positive ion mode, two
ions at m/z 223 and 245 were indicated as probable
candidates. The fragmentation spectra of these ions are
depicted in Fig. 8A and B. The presence of a peak at m/z
23 in the product-ion spectra of m/z 245 identified the ion
at m/z 245 as a sodiated adduct ion of m/z 223 (Fig. 8B).
The product-ion spectra of [M + H]+ ion at m/z 223 and of
the[M + Na]+ ionatm/z245showthebasepeakfragment
ion at m/z 86, corresponding to an ethylene-trimethyl
ammonium ion (Scheme 2). Additionally, fragment ions
at m/z 164 and 186 arising from loss of the trimethy-
lamine residue (59 Da), from [M + H]+ and [M + Na]+,
respectively, are observed. These fragment ions are char-
acteristic of the phosphocholine head group (Ayanoglu
et al., 1984). To further reveal the structure, we also
obtained the product-ion spectra of the ion at m/z 164
generated by skimmer CID (Fig. 8C). The spectra con-
tains ions at m/z 137 probably arising from a loss of
HCN residue and at m/z 107 arising from the cleavage
of the P-O bond (Scheme 2). The combined information
suggested that the compound might represent 1-cyano
methano phosphocholine. The structure assignment is
also consistent with the results obtained from similar
ESI-MS/MS analysis of the corresponding ions after H-
D exchange. The results indicate the presence of one
exchangeable hydrogen and an analogous loss of DCN
is also observed (data not shown).
generating system. At the end of the incubation period
the reaction products were analyzed after derivatization
with PFB hydroxylamine utilizing GC–MS. Fig. 7 shows
two peaks generated from the syn- and the anti-isomers
Fig. 7. MPO-derived RCS targets S1P: S1P (100 ␮g) was incubated
with MPO-derived RCS generating reagents, which includes MPO
(1U), H2O2 (1 mM) and NaCl (100 mM) in 20 mM phosphate buffer
(pH 4) for 15 min. At the end of the incubation period the reaction prod-
ucts were derivatized by PFB hydroxylamine and analyzed by GC–MS
as detailed under Section 2. Figure shows the total ion chromatogram
generated by the derivatized reaction products of S1P incubated with
MPO-derived RCS (trace a), in the absence of H2O2 (trace b) or in the
absence of MPO (trace c). The anti- and syn-isomers of PFB oxime of
2-hexadecenal are labeled (trace a).
To confirm the presence of the cyano group in the
compound we also performed an acid methanolysis
reaction using methanolic HCl on the crude reac-
tion product extracts followed by structural analysis

V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
79
Fig. 8. Identification of 1-cyano methano phosphocholine. SPC (100 ␮g) was incubated with MPO-derived RCS generating reagents, which includes
MPO (1U), H2O2 (1 mM) and NaCl (100 mM) in 20 mM phosphate buffer at pH 4. The products were extracted and analyzed by ESI-MS/MS; (A)
Product-ion spectra of m/z 223 [M + H]+; (B) Product-ion spectra of m/z 245 [M + Na]+; (C) Product-ion spectra of m/z 164 generated by skimmer
CID. The extracts were also derivatized by acid methanolysis and analyzed by ESI-MS/MS. (D) Shows the product-ion spectra for methyl ester of
1-cyano methano phosphocholine at m/z 270 [M]+ species.
with ESI-MS/MS. Indeed, a molecular species and its
product-ion spectra that are consistent with the structure
of a methyl ester of 1-cyano methano phosphocholine
were observed (Fig. 8D, Scheme 3). Having identified
the fragment ions, we utilized HPLC-MS/MS selected
reaction monitoring technique (Fig. 9) to confirm that
1-cyano methano phosphocholine is generated with a
corresponding reduction in SPC when MPO-derived
RCS reacts with SPC. In contrast, 1-cyano methano
phosphocholine is not formed in the absence of either
MPO or H2O2.
(Winterbourn et al., 1992). Since the sphingosine base
in SPC is also unsaturated we addressed the possibility
of monochlorinated and chlorohydrin product formation
from MPO-derived RCS attack on SPC. SPC was incu-
bated with MPO-derived RCS generating reagents as
describedearlierandthereactionproductswereanalyzed
with ESI-MS/MS. Fig. 10 shows the product-ion spectra
of the monochlorinated SPC (m/z = 499) (Fig. 10A) and
SPC chlorohydrin (m/z = 517) (Fig. 10B). The product-
ion spectra of the [M + H]+ at m/z 499 shows the base
peak at m/z 184 along with ions at m/z 440 and 316
which arise from neutral losses of 59 and 183, respec-
tively. Additionally, an ion at m/z 463, arising from the
neutral loss of 36 (loss of HCl) was also observed. The
Previously hypochlorous acid has been shown
to attack –C C– bonds in phosphatidylcholine to
form monochlorinated and chlorohydrin products

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V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
Scheme 2. Fragmentation scheme for 1-cyano methano phosphocholine.
spectrum suggested that the compound is a monochlo-
rinated SPC. The product-ion spectra of the [M + H]+
ion at m/z 517 contains ions at m/z 499 (517—H2O) and
463 (499—H35Cl) consistent with the suggested struc-
ture as a chlorohydrin. It should be mentioned however,
we did not confirm the positions assigned for chlorine
and hydroxyl group in these structures. Moreover, the
possibility that this monochlorinated SPC is a chlo-
ramine cannot be excluded. Although, on incubating
sphinganylphosphorylcholine, which has a free amino
Scheme 3. Fragmentation scheme for methyl ester of 1-cyano methano phosphocholine.

V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
81
group similar to SPC but does not possess the –C C–
in the sphingosine base, with MPO-derived RCS gen-
erating system does not yield the corresponding ion at
m/z = 501 (data not shown).
Since in vitro experiments revealed the production
of 2-hexadecenal from SPC and S1P, further stud-
ies were performed to determine the susceptibility of
HDL-associated lysosphingolipids to RCS attack. In
these studies, we used GC–MS selected ion monitor-
ing for the quantification of PFB-oxime derivatives of
2-hexadecenal with synthetic 2-[d5]-hexadecenal as an
internal standard. Base peaks observed at m/z 413 and
418 in the NICI spectra for the PFB oxime derivative
of 2-hexadecenal and 2-[d5]-hexadecenal, respectively
were used for quantification of 2-hexadecenal. A lin-
ear relationship is observed between the ratio of the
integrated peak area of m/z 413 and m/z 418 using
SIM GC–MS (data not shown). We have previously
used a similar technique for quantification of 2-chloro-
hexadecanal in biological tissue (Thukkani et al., 2002).
The generation of 2-hexadecenal was then quanti-
fied after HDL was treated with MPO and H2O2 in
phosphate-buffered saline at pH 4.0, using SIM GC–MS
for m/z 413 (from PFB oxime of 2-hexadecenal) and
m/z 418 ions (from PFB oxime of 2-[d5]-hexadecenal).
As shown in Fig. 11, 2-hexadecenal was increased
when HDL was treated with complete reaction mix-
ture capable of generating RCS. In contrast a similar
increase in 2-hexadecenal was not detected when MPO
or H2O2 is not present in the reaction mixture. Taken
together with studies using SPC, this leads us to con-
clude that MPO-derived RCS can attack HDL-associated
lysosphingolipids.
Fig. 9. LC/ESI-MS/MS of 1-cyano methano phosphocholine and SPC.
SPC (100 ␮g) was incubated with or without MPO/RCS generat-
ing reagents, which includes MPO (2U), H2O2 (1 mM) and NaCl
(100 mM) in 20 mM phosphate buffer at pH 4 as described in Sec-
tion 2. The reaction products were subjected to HPLC/ESI-MS/MS
analysis as described in Section 2 with SRM of 223 → 136.9 (A) for
1-cyano methanophosphocholine and the 465 → 184.1 (B) for SPC as
described in Section 2. The chromatograms shown are representative
of three independent experiments.
Fig. 10. Identification of monochlorinated SPC and SPC chlorohy-
drin. SPC (100 ␮g) was incubated with MPO-derived RCS generating
reagents, which includes MPO (1U), H2O2 (1 mM) and NaCl
(100 mM) in 20 mM phosphate buffer at pH 4. The products were
extracted and analyzed by ESI-MS/MS in positive ion mode; (A)
Depicts the fragmentation spectrum of monochlorinated SPC (m/z 499
[M + H]+); (B) Depicts the fragmentation spectrum of SPC chloro-
hydrin (m/z 517 [M + H]+). The insets show putative fragmentation
schemes for respective compounds.
Fig. 11. HDL (200 ␮g) protein was incubated in the presence or
absence MPO/RCS generating reagents including MPO (2U), H2O2
(1 mM), NaCl(100 mM)in20 mMphosphatebufferatpH4for25 mins
at 37 ◦C. For each condition, 2-hexadecenal was quantified follow-
ing derivatization with pentafluorobenzyl hydroxylamine and analysis
by NICI GC–MS using 2-[d5]-hexadecenal as internal standard. Each
value represents the mean S.E.M of three independent experiments.

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V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
4. Discussion
HDL-associated lysosphingolipids have been shown
to partly account for the atheroprotective effects of HDL
(Kimura et al., 2001; Nofer et al., 2003; Nofer et al.,
2001; Nofer et al., 2004). Lysosphingolipids also dis-
play other atheroprotective actions such as inhibition of
thrombin generation (Deguchi et al., 2004) and inhi-
bition of platelet activation (Altmann et al., 2003). It
is already known that MPO-derived RCS can attack
HDL-apolipoprotein A-I in the arterial wall and affect
cholesterol transport (Bergt et al., 2004; Zheng et al.,
2004). Herein, we show that MPO-derived RCS can
attackHDL-associatedlysosphingolipids. HDLhasbeen
reported to contain 1.2 and 4.7 ␮g/mg protein of SPC and
lysosulfatide, respectively (Nofer et al., 2001). It is likely
that these are the primary HDL sphingolipids attacked by
RCS. It is also possible that HDL S1P and sphingosine
are attacked by RCS as well. Taken together these data
suggest that during vascular wall injury, MPO-derived
RCS can attack HDL-associated lysosphingolipids and
alter the atheroprotective potential of HDL.
MPO-derived RCS are generated as a part of host
defense mechanism (Albrich et al., 1981), however they
are also known to attack host tissue (Weiss, 1989)
and may be involved in pathogenesis of atherosclerosis
(Podrez et al., 2000). In this study we describe a pos-
sible mechanism through which MPO-derived RCS can
react with HDL associated lysosphingolipids to gener-
ate 2-hexadecenal, an ␣,␤-unsaturated aldehyde. First,
we identified the products that can be generated from
MPO-derived RCS attack on SPC as 2-hexadecenal
and 1-cyano methano phosphocholine. Structural con-
firmation for 2-hexadecenal was obtained by NMR
and GC–MS studies, while 1-cyano methano phospho-
choline was confirmed with ESI-MS/MS studies of the
parent compound, after H-D exchange and after acid
methanolysis derivatization. We propose the formation
of 2-hexadecenal and 1-cyano methano phosphocholine
from MPO-derived RCS attack of SPC as shown in
Scheme 4. The initial attack of hypochlorous acid would
form a chloramine leading toformation of2-hexadecenal
as a stable intermediate with loss of HCl. Secondary
attack by hypochlorous acid then leads to formation
of 1-cyano methano phosphocholine through an imine
intermediate.
Lysosphingolipids may be attacked by MPO-derived
RCS in vivo and MPO-derived RCS oxidation might
provide an alternative pathway of lysosphingolipid
degradation under these conditions. It is likely that 2-
hexadecenal is metabolized by cellular enzymes such as
2-alkenal reductase and aldehyde dehydrogenase (van
Scheme 4. Reaction mechanism.

V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
83
Veldhoven and Mannaerts, 1993). It is also possible
that, 2-hexadecenal, being an ␣,␤-unsaturated aldehyde
forms Schiff base adducts and Michael adducts with
primary amino and thiol groups similar to 4-hydroxy-
nonenal and might affect protein function (Crabb et al.,
2002; Okada et al., 1999).
Recently, it has been shown that S1P/S1P lyase prod-
ucts have similar proliferative actions as S1P (Kariya et
al., 2005). Moreover, a role for a ceramide metabolite in
cell toxicity has also been suggested (Tserng and Griffin,
2004). Thus, it is likely that 2-hexadecenal and 1-cyano
methano phosphocholine possess biological activity and
it would be of interest to learn these in an effort to under-
stand the pathogenesis of atherosclerosis.
Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction
and purification. Can. J. Biochem. Physiol. 37, 911–917.
Carr, A.C., van den Berg, J.J., Winterbourn, C.C., 1998. Differential
reactivities of hypochlorous and hypobromous acids with puri-
fied Escherichia coli phospholipid: formation of haloamines and
halohydrins. Biochim. Biophys. Acta 1392, 254–264.
Cech, P., Papathanassiou, A., Boreux, G., Roth, P., Miescher, P.A.,
1979. Hereditary myeloperoxidase deficiency. Blood 53, 403–411.
Crabb, J.W., O’Neil, J., Miyagi, M., West, K., Hoff, H.F., 2002.
Hydroxynonenal inactivates cathepsin B by forming Michael
adducts with active site residues. Protein Sci. 11, 831–840.
Deguchi, H., Yegneswaran, S., Griffin, J.H., 2004. Sphingolipids as
bioactive regulators of thrombin generation. J. Biol. Chem. 279,
12036–12042.
Edwards, S.W., Nurcombe, H.L., Hart, C.A., 1987. Oxidative inac-
tivation of myeloperoxidase released from human neutrophils.
Biochem. J. 245, 925–928.
Hampton, M.B., Kettle, A.J., Winterbourn, C.C., 1998. Inside the
neutrophil phagosome: oxidants, myeloperoxidase, and bacterial
killing. Blood 92, 3007–3017.
Acknowledgements
Harrison, J.E., Schultz, J., 1976. Studies on the chlorinating activity of
myeloperoxidase. J. Biol. Chem. 251, 1371–1374.
This research was supported by NIH grants HL 74214
(DAF) RR00954 (Washington University Mass Spec-
trometry Resource) and RR19232 (DAF) as well as
Grant-in-Aid 0650044Z (DAF) from the American Heart
Association.
Hazen, S.L., Hsu, F.F., Duffin, K., Heinecke, J.W., 1996. Molecular
chlorine generated by the myeloperoxidase-hydrogen peroxide-
chloride system of phagocytes converts low density lipoprotein
cholesterol into a family of chlorinated sterols. J. Biol. Chem. 271,
23080–23088.
Hollenberg, P.F., Rand-Meir, T., Hager, L.P., 1974. The reaction of
chlorite with horseradish peroxidase and chloroperoxidase. Enzy-
matic chlorination and spectral intermediates. J. Biol. Chem. 249,
5816–5825.
Hsu, F.F., Hazen, S.L., Giblin, D., Turk, J., Heinecke, J.W., Gross, M.L.,
1999. Mass spectrometric analysis of pentafluorobenzyl oxime
derivative of reactive biological aldehydes. Int. J. Mass Spectr.
187, 795–812.
Jiang, Q., Griffin, D.A., Barofsky, D.F., Hurst, J.K., 1997. Intraphago-
somal chlorination dynamics and yields determined using unique
fluorescent bacterial mimics. Chem. Res. Toxicol. 10, 1080–1089.
Kariya, Y., Kihara, A., Ikeda, M., Kikuchi, F., Nakamura, S.,
Hashimoto, S., Choi, C.H., Lee, Y.M., Igarashi, Y., 2005. Prod-
ucts by the sphingosine kinase/sphingosine 1-phosphate (S1P)
lyase pathway but not S1P stimulate mitogenesis. Genes Cells 10,
605–615.
Kimura, T., Sato, K., Kuwabara, A., Tomura, H., Ishiwara, M.,
Kobayashi, I., Ui, M., Okajima, F., 2001. Sphingosine 1-phosphate
may be a major component of plasma lipoproteins responsible
for the cytoprotective actions in human umbilical vein endothelial
cells. J. Biol. Chem. 276, 31780–31785.
Kitahara, M., Eyre, H.J., Simonian, Y., Atkin, C.L., Hasstedt, S.J.,
1981. Hereditary myeloperoxidase deficiency. Blood 57, 888–893.
Lampert, M.B., Weiss, S.J., 1983. The chlorinating potential of the
human monocyte. Blood 62, 645–651.
Lehrer, R.I., Cline, M.J., 1969. Leukocyte myeloperoxidase deficiency
and disseminated candidiasis: the role of myeloperoxidase in resis-
tance to Candida infection. J. Clin. Invest. 48, 1478–1488.
Mancuso, A.J., Huang, S.-L., Swern, D., 1978. Oxidation of long-chain
and related alcohols to carbonyls by dimethyl sulfoxide “activated”
by oxalyl chloride. J. Org. Chem. 43, 2480–2482.
References
Albert, C.J., Crowley, J.R., Hsu, F.F., Thukkani, A.K., Ford, D.A.,
2001. Reactive chlorinating species produced by myeloperoxidase
target the vinyl ether bond of plasmalogens: identification of 2-
chlorohexadecanal. J. Biol. Chem. 276, 23733–23741.
Albrich, J.M., McCarthy, C.A., Hurst, J.K., 1981. Biological reactivity
of hypochlorous acid: implications for microbicidal mechanisms
of leukocyte myeloperoxidase. Proc. Natl. Acad. Sci. USA 78,
210–214.
Alewijnse, A.E., Peters, S.L., Michel, M.C., 2004. Cardiovascu-
lar effects of sphingosine-1-phosphate and other sphingomyelin
metabolites. Br. J. Pharmacol. 143, 666–684.
Altmann, C., Meyer Zu Heringdorf, D., Boyukbas, D., Haude, M.,
Jakobs, K.H., Michel, M.C., 2003. Sphingosylphosphorylcholine,
a naturally occurring lipid mediator, inhibits human platelet func-
tion. Br. J. Pharmacol. 138, 435–444.
Asselbergs, F.W., Reynolds, W.F., Cohen-Tervaert, J.W., Jessurun,
G.A., Tio, R.A., 2004. Myeloperoxidase polymorphism related to
cardiovascular events in coronary artery disease. Am. J. Med. 116,
429–430.
Ayanoglu, E., Wegmann, A., Pilet, O., Dean Marbury, G., Ronald
Hass, J., Djerassi, C., 1984. Mass spectrometry of phospholipids.
Some applications of desorption chemical ionization and fast atom
bombardment. J. Am. Chem. Soc. 106, 5246–5251.
Baumann, W.J., Schmid, H.H., Mangold, H.K., 1969. Oxidative cleav-
age of lipids with sodium metaperiodate in pyridine. J. Lipid Res.
10, 132–133.
Bergt, C., Pennathur, S., Fu, X., Byun, J., O’Brien, K., McDonald,
T.O., Singh, P., Anantharamaiah, G.M., Chait, A., Brunzell, J.,
Geary, R.L., Oram, J.F., Heinecke, J.W., 2004. The myeloperoxi-
dase product hypochlorous acid oxidizes HDL in the human artery
wall and impairs ABCA1-dependent cholesterol transport. Proc.
Natl. Acad. Sci. USA 101, 13032–13037.
Marsche, G., Heller, R., Fauler, G., Kovacevic, A., Nuszkowski, A.,
Graier, W., Sattler, W., Malle, E., 2004. 2-Chlorohexadecanal
derived from hypochlorite-modified high-density lipoprotein-
associated plasmalogen is a natural inhibitor of endothelial

84
V.V. Brahmbhatt et al. / Chemistry and Physics of Lipids 145 (2007) 72–84
nitric oxide biosynthesis. Arterioscler. Thromb. Vasc. Biol. 24,
2302–2306.
McMillen, T.S., Heinecke, J.W., LeBoeuf, R.C., 2005. Expression of
human myeloperoxidase by macrophages promotes atherosclerosis
in mice. Circulation 111, 2798–2804.
Nikpoor, B., Turecki, G., Fournier, C., Theroux, P., Rouleau, G.A.,
2001. A functional myeloperoxidase polymorphic variant is associ-
ated with coronary artery disease in French-Canadians. Am. Heart
J. 142, 336–339.
Nofer, J.R., Levkau, B., Wolinska, I., Junker, R., Fobker, M.,
von Eckardstein, A., Seedorf, U., Assmann, G., 2001. Suppres-
sion of endothelial cell apoptosis by high density lipoproteins
(HDL) and HDL-associated lysosphingolipids. J. Biol. Chem. 276,
34480–34485.
ing neutrophil activation target tissue plasmalogens: production
of the chemoattractant, 2-chlorohexadecanal. J. Biol. Chem. 277,
3842–3849.
Thukkani, A.K., McHowat, J., Hsu, F.F., Brennan, M.L., Hazen, S.L.,
Ford, D.A., 2003. Identification ofalpha-chlorofatty aldehydes and
unsaturated lysophosphatidylcholine molecular species in human
atherosclerotic lesions. Circulation 108, 3128–3133.
Tohru Hino, K.N., Mikio Taniguchi, Masako, Nakagawa, 1986. A
new route to racemic erythro-sphingosine and ceramides. The 1,2-
versus 1,4-addition reaction of hexadec-2-enal with 2-nitroethanol.
J. Chem. Soc., Perkin Trans. 1, 1687–1690.
Tserng, K.Y., Griffin, R.L., 2004. Ceramide metabolite, not intact
ceramide molecule, may be responsible for cellular toxicity.
Biochem. J. 380, 715–722.
Nofer, J.R., Geigenmuller, S., Gopfert, C., Assmann, G., Buddecke, E.,
Schmidt, A., 2003. High density lipoprotein-associated lysosphin-
golipids reduce E-selectin expression in human endothelial cells.
Biochem. Biophys. Res. Commun. 310, 98–103.
van Veldhoven, P.P., Mannaerts, G.P., 1993. Sphingosine-phosphate
lyase. Adv. Lipid Res. 26, 69–98.
Weil, I., Morris, J., 1949. Kinetic studies on the chloramines. I. The
rates of formation of monochloramine, N-chlormethylamine and
N-chlordimethylamine. J. Am. Chem. Soc. 71, 1664–1671.
Weiss, S.J., 1989. Tissue destruction by neutrophils. N. Engl. J. Med.
320, 365–376.
Weiss, S.J., Lampert, M.B., Test, S.T., 1983. Long-lived oxidants
generated by human neutrophils: characterization and bioactivity.
Science 222, 625–628.
Winterbourn, C.C., van den Berg, J.J., Roitman, E., Kuypers, F.A.,
1992. Chlorohydrin formation from unsaturated fatty acids reacted
with hypochlorous acid. Arch. Biochem. Biophys. 296, 547–555.
Zgliczynski, J.M., Selvaraj, R.J., Paul, B.B., Stelmaszynska, T.,
Poskitt, P.K., Sbarra, A.J., 1977. Chlorination by the myelo-
peroxidase-H2O2–Cl-antimicrobial system at acid and neutral pH.
Proc. Soc. Exp. Biol. Med. 154, 418–422.
Nofer, J.R., van der Giet, M., Tolle, M., Wolinska, I., von Wnuck Lip-
inski, K., Baba, H.A., Tietge, U.J., Godecke, A., Ishii, I., Kleuser,
B., Schafers, M., Fobker, M., Zidek, W., Assmann, G., Chun, J.,
Levkau, B., 2004. HDL induces NO-dependent vasorelaxation via
the lysophospholipid receptor S1P3. J. Clin. Invest. 113, 569–581.
Okada, K., Wangpoengtrakul, C., Osawa, T., Toyokuni, S., Tanaka,
K., Uchida, K., 1999. 4-Hydroxy-2-nonenal-mediated impairment
of intracellular proteolysis during oxidative stress. Identifica-
tion of proteasomes as target molecules. J. Biol. Chem. 274,
23787–23793.
Podrez, E.A., Abu-Soud, H.M., Hazen, S.L., 2000. Myeloperoxidase-
generated oxidants and atherosclerosis. Free Radic. Biol. Med. 28,
1717–1725.
Sullards, M.C., Merrill Jr., A.H., 2001. Analysis of sphingosine 1-
phosphate, ceramides, and other bioactive sphingolipids by high-
performance liquid chromatography-tandem mass spectrometry.
Sci. STKE 2001, PL1.
Thukkani, A.K., Hsu, F.F., Crowley, J.R., Wysolmerski, R.B., Albert,
C.J., Ford, D.A., 2002. Reactive chlorinating species produced dur-
Zheng, L., Nukuna, B., Brennan, M.L., Sun, M., Goormastic, M., Set-
tle, M., Schmitt, D., Fu, X., Thomson, L., Fox, P.L., Ischiropoulos,
H., Smith, J.D., Kinter, M., Hazen, S.L., 2004. Apolipoprotein A-I
is a selective target for myeloperoxidase-catalyzed oxidation and
functional impairment in subjects with cardiovascular disease. J.
Clin. Invest. 114, 529–541.