0022-3565/01/2992-611–619$3.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics
JPET 299:611–619, 2001
Vol. 299, No. 2
4140/938684
Printed in U.S.A.
Vicinal Nitrohydroxyeicosatrienoic Acids: Vasodilator Lipids
Formed by Reaction of Nitrogen Dioxide with Arachidonic Acid
MICHAEL BALAZY, TAKAFUMI IESAKI, JAMES L. PARK, HOULI JIANG, PAWEL M. KAMINSKI, and MICHAEL S. WOLIN
Departments of Pharmacology and Physiology, New York Medical College, Valhalla, New York
Received May 15, 2001; accepted July 20, 2001
This paper is available online at http://jpet.aspetjournals.org
NO formed in vascular tissue can be oxidized by several
pathways, which lead to formation of reactive products, particularly in situations when other free radicals are also generated, as is often the case in many pathophysiological conditions of the cardiovascular system (Ignarro, 2000; Wolin,
2000). Many of the biologically important pathways of NO
oxidation lead to formation of significant amounts of the
reactive free radical NO2. These pathways include the direct
oxidation of NO by O2, various pathways of peroxynitrite
decomposition, and the reaction of peroxidases with the NO
decomposition product nitrite (Byun et al., 1999; Radi et al.,
2001). It has been known for some time that NO2 induces
lipid peroxidation via reactions with components of biological
membrane lipids (Pryor and Lightsey, 1981). Much of the
work in this area originated from the hypothesis that because
This study was supported by National Institutes of Health Grants R01
GM62453, S10 RR12993 (to M.B.); HL31069, HL43023, and HL66331 (to
M.S.W.); and a grant from the American Heart Association New York State
Affiliate (9850104 to M.B.). This work was presented at the 72nd Scientific
Sessions of the American Heart Association in Atlanta GA [(1999) Circulation
100:I814].
spectrometry to be eight isomers having molecular weight of
367 and a fragmentation pattern indicative of arachidonic acid
derivatives containing nitro and hydroxy groups and consistent
with the structures of vicinal nitrohydroxyeicosatrienoic acids.
These lipids spontaneously released NO (183 ⫾ 12 nmol NO/15
min/␮mol) as detected by head space/chemiluminescence
analysis. Mild alkaline hydrolysis of total lipids extracted from
bovine cardiac muscle followed by isotopic dilution gas chromatography/mass spectrometry analysis detected basal levels
of nitrohydroxyeicosatrienoic acids (6.8 ⫾ 2.6 ng/g tissue; n ⫽
4). Thus, the oxidation product of NO, NO2, reacts with arachidonic acid to generate biologically active vicinal nitrohydroxyeicosatrienoic acids, which may be important endogenous mediators of vascular relaxation and sGC activation.
NO2 is a major urban air pollutant, lipid peroxidation within
the lung could be a contributing factor to lung injury and the
development of diseases such as cancer (Ichinose and Sagai,
1992). Previous work has identified conjugated dienes, thiobarbituric acid reactive species, peroxides, and other products after exposure of animals to air containing ppm levels of
NO2 (Ichinose and Sagai, 1982; Sevanian et al., 1982) as well
as cells (Jiang et al., 1999), physiological fluids (Halliwell et
al., 1992), and isolated lipids to NO2 gas (Pryor and Lightsey,
1981; Lai and Finlayson-Pitts, 1991; Gallon and Pryor, 1993).
Biological membranes are likely to be a particular target for
NO2 because phospholipids play an important role in the
oxidative chemistry of NO. Both NO and NO2 are highly
lipophylic and NO oxidation is accelerated more than 8-fold
when phospholipids are added to aqueous NO solution (Liu et
al., 1998). Additionally, about 90% of NO oxidation by O2
takes place within the hydrophobic phospholipid bilayer (Liu
et al., 1998). Thus, biomembrane phospholipids are likely to
react with NO2 not only because they concentrate NO and
accelerate its oxidation but also because the hydrophobic
ABBREVIATIONS: NO, nitric oxide; NO2, nitrogen dioxide; PG, prostaglandin; ODQ, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one; NO2AA,
nitroeicosatetraenoic acid; HPLC, high-performance liquid chromatography; GC/MS, gas chromatography/mass spectrometry; ESI, electrospray
ionization; LC/MSn, liquid chromatography/tandem mass spectrometry; PFB, pentafluorobenzyl; TMS, trimethylsilyl; BCA, bovine coronary artery;
sGC, soluble guanylate cyclase; NO2AAOH, vicinal nitrohydroxyeicosatrienoic acids (mixture of isomers); EET, epoxyeicosatrienoic acids;
diHETrE, vicinal dihydroxyeicosatrienoic acids.
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ABSTRACT
Nitric oxide (NO)-derived species could potentially react with
arachidonic acid to generate novel vasoactive metabolites. We
studied the reaction of arachidonic acid with nitrogen dioxide
(NO2), a free radical that originates from NO oxidation. The
reaction mixture contained lipid products that relaxed endothelium-removed bovine coronary arteries. Relaxation to the lipid
mixture was inhibited ⬃20% by indomethacin and ⬃70% by a
soluble guanylate cyclase (sGC) inhibitor (ODQ). Thus, novel
lipid products, which activate sGC presumably through a
mechanism involving NO, appeared to have contributed to the
observed vasorelaxation. Lipids that eluted at 9 to 12 min
during high-performance liquid chromatography fractionation
accounted for about one-half of the vasodilator activity in the
reaction mixture, which was inhibited by ODQ. Lipid products in
fractions 9 to 12 were identified by electrospray tandem mass
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Experimental Procedures
Materials. Nitrogen dioxide (purity ⬎97%) was from Matheson
Tri-Gas, Inc. (Parsippany, NJ). Arachidonic acid, phenylephrine hydrochloride, indomethacin, and EDTA were from Sigma Chemical
(St. Louis, MO). U46619, 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1one (ODQ), and arachidonic acid-d8 (purity ⱖ98 atom % D) were from
Cayman Chemicals (Ann Arbor, MI). [1-14C]Arachidonic acid (specific activity, 50 mCi/mmol) was from PerkinElmer Life Science Products (Boston, MA). All solvents were of highest chromatographic
grade. Other chemicals were of analyzed reagent grade and were
obtained J. T. Baker (Phillipsburg, NJ).
Reaction of Arachidonic Acid with NO2. NO2 was prepared
freshly before reaction with lipids as described (Jiang et al., 1999).
Briefly, helium was used as a carrier gas to evaporate liquid NO2
from a capped test tube into a solution of arachidonic acid by using
Teflon tubing. NO2 was bubbled at a rate of about 0.1 ml/min
through a solution of arachidonic acid in hexane (0.33 mM) for 3 to 5
min. The specific activity of arachidonic acid used for this reaction
was 0.03 mCi/mmol. Aliquots of the reaction mixture were taken at
1-min time intervals and analyzed by HPLC to establish the progress
of the reaction. The total reaction mixture was finally washed several times with water to remove NO2. The final extract was concentrated under nitrogen and dissolved in ethanol for bioassay experiments. The concentration of the lipid products was calculated from
the amount of the radioactive 14C tracer remaining in crude reaction
mixture. The concentration of the lipid products applied to tissues
was calculated from measurements of radioactivity by scintillation
counting based on specific activity of the original arachidonic acid
used for preparation of lipid products (0.03 mCi/mmol). The amount
of bioactive lipids in the crude mixture was calculated from the
radioactivity of material in fractions 9 to 18 after HPLC fractionation. This procedure was also used for preparation of deuteriumlabeled lipids from arachidonic acid-d8 (specific activity 0.3 mCi/
mmol) as internal standards for isotopic dilution GC/MS analysis. In
some experiments, the total reaction mixture was cooled on ice and
an aliquot was directly analyzed by electrospray tandem mass spectrometry. Arachidonic acid (100 ␮g; 30.4 ␮Ci/mmol) was also suspended in 500 ␮l of aqueous sodium nitrite solution (0.1 mM), which
was titrated with HCl solution (0.1 mM) until pH ⬇ 3. The reaction
continued for additional 10 min and was terminated by extraction of
lipids with ethyl acetate followed by HPLC chromatography and
GC/MS analysis.
HPLC Analysis. HPLC analyses were performed on an HP1100
system (Hewlett Packard, Palo Alto, CA) by using a C18 column
(250 ⫻ 4.6 mm; Beckman Coulter, Inc., Fullerton, CA) and a UV
diode array detector. Samples were analyzed with a gradient of
acetonitrile in water (62.5% increased to 100% in 50 min). UV absorbance of the effluent at 205, 234, and 320 nm was monitored
during the HPLC run. Radiolabeled products originating from
[1-14C]arachidonic acid were also detected by an on-line radioactivity
monitor (Packard Instrument Co., Meriden, CT) and an Ecolite(⫹)
scintillation cocktail (ICN Biomedicals, Cleveland, OH). A Gilson FC
203B fraction collector was used to collect fractions at 1-min intervals. The solvent was evaporated under vacuum and the residue was
dissolved in ethanol. Aliquots (100 ␮l) were taken for scintillation
counting to establish the concentration of radiolabeled lipids in each
fraction. Based on radioactivity measurements and specific activity
of arachidonic acid, the final concentration of radiolabeled lipids in
each fraction was adjusted to 10⫺2 M with ethanol. For bioassay
experiments, aliquots (10 ␮l) of the ethanolic solution of fractions 1
to 30 were added to tissue bath containing rat aorta in 10 ml of
Krebs’ buffer.
Mass Spectrometry. Electrospray ionization (ESI) tandem mass
spectrometry (LC/MSn; n ⫽ 1–3) was performed using a Brucker
Daltonics (Billerica, MA) Esquire ion trap mass spectrometer. The
capillary exit potential was ⫺45 V and the skimmer 1 potential was
⫺30 V for negative ion polarity ESI LC/MSn scans (scan range was
m/z 50 –700). The nebulizer pressure was 20 psi and the nitrogen
flow was adjusted to 2 l/min for flow sample injection and 5 l/min for
HPLC sample introduction. The temperature of dry gas was 300°C.
The ion accumulation time was 50 to 100 ms in the ESI LC/MS2
experiments. Helium was used as collision gas and its pressure was
adjusted to 6 ⫻ 10⫺6 mbar above base pressure. For flow injection
sample introduction, lipids were dissolved in 100 ␮l of acetonitrile/
water (50:50, v/v) and injected via a syringe pump (Cole-Parmer
Instrument, Chicago, IL) at 1.5 ␮l/min to the ESI source. For HPLC
sample introduction, the samples were injected on a reverse phase
HPLC column (250 ⫻ 1 mm, 5 ␮m; Phenomenex, Torrance, CA) and
eluted with a gradient of water in acetonitrile (62.5% increased to
100% in 50 min) at a flow of 100 ␮l/min. The GC/MS analysis was
performed on a Hewlett Packard 5989A mass spectrometer as described previously (Balazy, 1991). Briefly, lipids were converted into
pentafluorobenzyl (PFB) trimethylsilyl (TMS) derivatives and analyzed on a capillary fused silica gas chromatographic column (DB-1,
10 m, 0.25 mm i.d., 0.25-␮m film thickness; J & W Scientific, Folsom,
CA). The mass spectrometer operated in the chemical ionization
mode with negative ion detection by using methane as a moderating
gas (2.8 torr) (Balazy, 1991). Relative retention time (C value) of
derivatized lipids on analysis by GC/MS was established from a plot
of retention time of a series of saturated fatty acids (PFB esters)
versus their carbon chain length (18 –24 carbons). The regression
analysis produced a formula for a correlation line (r2 ⫽ 0.999) that
allowed converting retention times of analyzed compounds into their
C values.
Measurement of Changes in Force in Bovine Coronary Arteries (BCAs). Bovine left circumflex coronary arterial rings were
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milieu may slow down NO2 hydrolysis. Several studies have
also identified processes other than peroxidation that appear
to involve NO2-mediated lipid nitration (Lai and FinlaysonPitts, 1991; Gallon and Pryor, 1993; Gallon and Pryor, 1994).
NO2 generates a mixture of products from linoleic acid, which
includes allylic nitrite and nitro linoleates (Gallon and Pryor,
1993; O’Donnell et al., 1999). The work of Lai and FinlaysonPitts (1991) has described nitrated phospholipids originating
from treatment of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine by NO2 and N2O5. These studies indicate that NO2
may not only abstract the allylic hydrogen but also may bind
to the fatty acid double bond.
Several studies have suggested that vasoactive peroxidation products of arachidonic acid such as isoprostaglandins
are formed by oxidative stress in vascular tissue by a mechanism involving hydroxyl radical (Morrow and Roberts, 1997;
Reilly et al., 1998). It has been thus proposed that these lipid
products function as signaling molecules of oxidative stress
largely because the major isoprostaglandin molecule (8-isoPGF2␣) is a potent vasoconstrictor (Lahaie et al., 1998). We
hypothesized that novel signaling lipid mediators could also
be produced by the reaction of arachidonic acid with NO2,
which appears to be favorable kinetically (Prütz et al., 1985),
but structural characterization of such lipid products and
their potential involvement in vascular signaling processes
remain to be investigated. Our previous work has identified
cis-trans isomerization of arachidonic acid as a major process
initiated by NO2 that leads to formation of trans-arachidonic
acids (Jiang et al., 1999; Boulos et al., 2000). In this study we
observed that the reaction of arachidonic acid with NO2
generates potent vasorelaxing lipid products. A major component of these lipids was characterized by the electrospray
tandem mass spectrometry to be a mixture of unique ␤-nitro
alcohols that release NO.
Nitrogen Dioxide and Arachidonic Acid
Results
Characterization of Vasorelaxation. The crude reaction mixture of lipid products obtained from the reaction of
arachidonic acids with NO2 in hexane was washed with wa-
ter to remove NO2. The extract was concentrated, dissolved
in ethanol, and aliquots were applied to BCA rings precontracted with iso-osmotic KCl (25 mM). The reaction mixture
contained potent vasorelaxing lipids, which relaxed BCA
smooth muscle in a dose-dependent manner (Figs. 1 and 2).
The vasorelaxing effect (55 ⫾ 4% relaxation of BCA; n ⫽ 19)
of the lipid mixture (10⫺6 M) was sensitive to indomethacin
(42 ⫾ 3% relaxation of BCA; n ⫽ 12), presumably due to the
metabolism of arachidonic acid present in the total mixture
to prostaglandins (PGI2, PGE2) by cyclooxygenase in BCA
(Fig. 2). Based on the data in Fig. 1 showing the absence of a
relaxation to arachidonic acid in the presence of indomethacin, the unreacted arachidonic acid present in NO2-generated
reaction mixture does not contribute to the indomethacinindependent relaxation caused by this mixture. The vasodilation was inhibited by 10 ␮M ODQ (19 ⫾ 3% relaxation of
BCA; n ⫽ 17), and treatment of BCA rings with ODQ plus
indomethacin produced the greatest inhibitory effect (13 ⫾
3% relaxation of BCA; n ⫽ 12) (Fig. 2). Thus, lipid products
that activated sGC presumably through a mechanism involving NO appeared to mediate the observed vasorelaxation
(Figs. 1 and 2). To identify the vasorelaxant lipids, the crude
reaction mixture was fractionated by HPLC on a reverse
phase chromatographic column (Fig. 3), and the activity of
each fraction was tested in phenylephrine precontracted rat
aortic rings preincubated with indomethacin (Fig. 3). The
vasorelaxant lipids appeared in fractions 9 to 18 (Fig. 3),
which contained 10% of total product radioactivity. Further
experiments with BCA rings confirmed that purified fractions 9 to 12 contained potent vasorelaxant lipids (Fig. 4).
The vasodilation (35 ⫾ 3% relaxation of BCA; n ⫽ 4) caused
by these lipids was inhibited by 10 ␮M ODQ (11 ⫾ 5%
relaxation of BCA; n ⫽ 4). Thus, lipids in fractions 9 to 12
appeared to be major nitrated lipids of the arachidonic acid/
NO2 product mixture. Because the vasorelaxant response
was sensitive to ODQ, which suggested that NO might be
involved in the relaxing response, we further studied the
Fig. 1. Relaxation of BCA rings by nitrated lipids generated from the
reaction of arachidonic acid (AA) with NO2. The effect of AA on ring
tension is compared with the effect of lipid mixture generated from AA
treated by NO2 in hexane and washed with water. Rings were treated
with indomethacin (10 ␮M) and precontracted with iso-osmotic KCl solution (25 mM). The concentrations (log M) in the bottom panel represent
the amount of the AA and lipid products, as measured by scintillation
counting of the 14C-labeled tracer.
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prepared from bovine hearts and the measurements of changes in
isometric force were done essentially as described previously (Mohazzab et al., 1996). Briefly, the rings (without endothelium) were
incubated in thermostated (37°C) glass tissue baths (10 ml; Metro
Scientific, Farmingdale, NY) for 2 h at an optimal passive tension of
5 g in Krebs’ bicarbonate buffer, pH 7.4, containing 118 mM NaCl,
1.5 mM CaCl2, 25 mM NaHCO3, 1.1 mM MgSO4, 1.2 mM KH2PO4,
and 5.6 mM glucose. The solution was bubbled with 5% CO2 in air
throughout the experiment. After a 2-h equilibration period and a
brief depolarization with KCl (123 mM), the BCA rings were reequilibrated for 30 min before treatment with test compounds. The vessels
were precontracted either with iso-osmotic KCl (25 mM) or a thromboxane mimetic, U46619 (0.1 ␮M), and after the stable tone was
achieved, aliquots of the total lipid mixture from the arachidonic
acid/NO2 reaction or fractionated lipids purified by HPLC were applied to the rings. Iso-osmotic solutions were generated by replacing
25 mM NaCl with 25 mM KCl. The lipids were dissolved in ethanol
(10 ␮l, final concentration 0.1%) and injected with a Hamilton syringe. The relaxation response was studied in the absence or presence of a specific inhibitor of soluble guanylate cyclase (sGC), ODQ
(10 ␮M), and/or cyclooxygenase inhibitor indomethacin (10 ␮M)
(Iesaki et al., 1999). Control injections of ethanol (10 ␮l) did not cause
a vascular response. Relaxation was measured as the percentage of
change of a steady level contraction after injection of test lipids or
control compounds.
Analysis of Tissue Lipids. Bovine cardiac papillary muscle was
isolated from slaughterhouse-derived hearts used for coronary artery
studies and cut into thin slices (⬃0.6 g). The freshly prepared cardiac
muscle slices were incubated in Krebs’ bicarbonate buffer gassed
with an air ⫺5% CO2 mixture for 30 min before freezing and extraction. Tissue was homogenized in chloroform/methanol (1:2) by using
a tissue homogenizer and total lipids were extracted as described
previously (Bednar et al., 2000). The lipid extract was hydrolyzed
with 0.1 N KOH, and the hydrolyzate was mixed with 100 ng of
octadeuterium-labeled nitrohydroxyeicosatrienoic acid (NO2AA) as
internal standard. Samples were purified by HPLC and fractions 9 to
12 were collected, derivatized, and analyzed by GC/MS. The amount
of nitrohydroxyeicosatrienoic acids was determined from a standard
curve by using ions at m/z 438 and 446 corresponding to the endogenous molecule and internal standard, respectively. Compounds
were detected in fresh tissue, which was not exposed to any treatments.
Measurement of Changes in Force in Rat Aorta. Male Wistar
rats were anesthetized (50 mg/kg pentobarbital i.p.), and descending
thoracic aorta was excised and cut into rings (⬃4 mm in length).
Endothelium was mechanically removed by gentle rubbing. The rat
aortic rings were incubated in a manner identical to BCA but a
passive tension was 3 g and the Krebs’ bicarbonate buffer contained
0.03 mM EDTA. The vessels were precontracted with phenylephrine
(0.1 ␮M) and then aliquots of HPLC fractions were applied to test
their potential vasorelaxant effect in the presence of indomethacin.
Chemiluminescence Assay of NO. NO generation was measured as described previously (Balazy et al., 1998). Briefly, lipids
were dissolved in Krebs’ buffer, briefly sonicated, incubated, and an
aliquot of the accumulated headspace gas was injected into a chemiluminescence NO analyzer (Sievers Instruments, Boulder, CO).
Statistical Analysis. Results are expressed as mean ⫾ S.E.M.,
with n equal to the number of animals used. Comparisons between
groups were made by analysis of variance and Student’s t test with
a Bonferroni correction for multiple comparisons. A value of p ⬍ 0.05
was used to determine statistical significance.
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Balazy et al.
Fig. 4. Bioassay of products from fractions 9 to 12 on BCA (precontracted
with 0.1 ␮M U46619) in the presence and absence of 10 ␮M ODQ (n ⫽ 4).
Fig. 5. Detection of NO from products in fractions 9 to 12 by headspace/
chemiluminescence assay in the presence and absence of ⬃300 mg of
BCA.
Fig. 3. Identification of vasoactive lipids by HPLC, mass spectrometry,
and bioassay. Top, chromatogram was generated by the arachidonic
acid/NO2 reaction products having UV absorbance at 205 nm. The horizontal bars indicate fractions that contain lipids characterized by mass
spectrometry: iso-PG, isoprostaglandins; HETE, hydroxyeicosatetraenoic
acids; NOxAA, nitrated arachidonic acids. Lipids were analyzed on a
reverse phase C18 column (250 ⫻ 4.6 mm) and separated with a gradient
of acetonitrile in water (62.5–100% in 50 min). Bottom, relaxation of lipid
products in fractions 1 to 30 was detected by rat aorta precontracted with
0.1 ␮M phenylephrine and containing indomethacin (10 ␮M). The vertical
bars show the magnitude of the vasorelaxant response produced by
radiolabeled lipid products in individual fractions.
release of NO from lipid products in fractions 9 to 12 by a
chemiluminescence detector. The lipids in fractions 9 to 12
released detectable amounts of NO after incubation in Krebs’
buffer (Fig. 5). The maximal production of NO from lipids in
fractions 9 to 12 suspended in Krebs’ buffer was 183 ⫾ 12
nmol NO/15 min/␮mol of lipid products (n ⫽ 3). Addition of
vascular tissue to these lipids resulted in the production of
150 ⫾ 18 nmol NO/15 min/␮mol (n ⫽ 3) as detected by
headspace/chemiluminescence analysis. After 24 h at room
temperature the samples without and with tissue produced
35 ⫾ 2 and 18 ⫾ 8 nmol NO/15 min/␮mol (n ⫽ 3), respectively
(Fig. 5).
Structural Characterization of Lipid Products. Figure 3 provides a summary of structural identification of lipid
products by mass spectrometry, which involved GC/MS and
ESI LC/MSn techniques. The major product of the reaction
eluted after the peak of arachidonic acid and was identified
as a mixture of its trans isomers as described previously
(Jiang et al., 1999). The focus of this study was on lipids in
fractions 9 to 12 (Figs. 6 and 7) that induced vasorelaxation
via a mechanism involving activation of sGC. These lipid
products were further purified by HPLC fractionation prior
to mass spectrometric analysis and bioassay.
Characterization of Vicinal Nitrohydroxyeicosatrienoic Acids (NO2AAOH). Lipid products collected in HPLC
fractions eluting at 9, 10, 11, and 12 min (Fig. 3) were
initially analyzed by GC/MS as PFB and TMS derivatives
(Fig. 6). The lipids in these four fractions produced several
peaks on analysis by capillary gas chromatographic column
that had a relative retention time (C value) of 22.8 to 24.6
and displayed similar mass spectra that showed a molecular
anion at m/z 438 (M-PFB, relative abundance 50 –100%). The
fragment ions were at m/z 348 (M-PFB-TMSOH, 28 –100%),
m/z 391 (M-PFB-HNO2, 4 –5%), m/z 319 (M-PFB-TMS-NO2
2–3%), m/z 303 (M-PFB-TMSO-NO2, 2–3%), and m/z 301 (m/z
348-HNO2, 1%) (Fig. 6). These spectra suggested that fractions 9 to 12 contained a mixture of new lipid isomers having
one nitro group and one hydroxy group bound to the arachidonic acid carbon chain. Total ion chromatograms obtained
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Fig. 2. Effect of inhibitors on BCA relaxation by the total mixture of
arachidonic acid/NO2 products. Indo, indomethacin (10 ␮M); AA, arachidonic acid (n ⫽ 12–19). BCA rings were precontracted with KCl (25 mM).
Nitrogen Dioxide and Arachidonic Acid
615
from GC/MS analysis revealed that no other isoeicosanoids
were present in fractions 9 to 11 (Fig. 6). Fraction 12 contained a small amount of a component showing a mass spectrum with a molecular anion at m/z 348 and likely to have the
structure of an NO2AA. This component had a relative retention time (C value) of 22.8 and was well separated from an
NO2AAOH isomer in that fraction (C value, 23.5). Comparison of the ion current intensities revealed that the ion at m/z
348 from NO2AA constituted about 10% of the ion current
generated from NO2AAOH (m/z 438) in fraction 12 and ⬃2%
in combined fractions 9 to 12. The NO2AA in fraction 12 was
not further characterized. Structural analysis was also performed with aliquots of unmodified lipids from individual
fractions 9 to 12. The lipids were slowly injected in acetonitrile/water (50:50, v/v) to the ESI source of the mass spectrometer operating in the negative ionization mode. The
mass spectra revealed abundant carboxylic molecular anions
at m/z 366. Collisional activation of these anions produced
mass spectra that revealed more structural details (Fig. 7).
The common features of these mass spectra were the fragment ions at m/z 348 (loss of H2O) and m/z 301 (loss of H2O
and HNO2). These spectra confirmed that fractions 9 to 12
contained isomers of NO2AAOH. Additional prominent and
characteristic ions resulted from the cleavage of NO2-CC-OH carbon bonds, suggesting that these lipids were ␤-nitro
alcohols. The characteristic fragmentation allowed identifying all eight possible isomers of vicinal NO2AAOH (Fig. 7).
The fragmentation appeared to be driven by proton transfer
Fig. 7. A, electrospray tandem mass spectrum and structures of products
in fraction 9. B, electrospray tandem mass spectrum and structures of
products in fraction 10. C, electrospray tandem mass spectrum and
structures of products in fractions 11 and 12.
from the hydroxyl to the nitro group and was followed by the
cleavage of the carbon-carbon bond, resulting in two types of
carboxylic anion fragments (Fig. 7; Scheme 1). Type a ions
were likely to have the structure of a nitronic acid (having a
protonated nitro group as in the aci form; Smith and March,
2001), whereas ions type b that of an aldehyde (Scheme 1).
These two ions identified a pair of isomers (presumably racemic) having nitro and hydroxy substituted carbons at each
double bond. Thus, ion at m/z 266 originated from 14-nitro15-hydroxyeicosatrienoic acid, whereas ion at m/z 235 from
fragmentation of 14-hydroxy-15-nitroeicosatrienoic acid (Fig.
7A). Because it was not possible to separate these isomers by
chromatography, composite mass spectra were obtained for
mixtures of two or more isomers that appeared in fractions 9
to 12 (Fig. 7). Collisional activation of the ion at m/z 366 from
lipids in fraction 10 generated a mass spectrum (Fig. 7B)
showing ions at m/z 226 and 195 that originated from the
cleavage of the C-11,C-12 bond from a mixture of isomers,
11-nitro-12-hydroxyeicosatrienoic acid and 11-hydroxy-12nitroeicosatrienoic acid. Finally, the mass spectrum of products in fractions 11 and 12 contained fragment ions at m/z
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Fig. 6. Determination of purity of lipid products in fractions 9 to 12 by
GC/MS. The chromatograms (left) show total ion chromatograms of lipids
in fractions indicated and derivatized as PFB, TMS. The mass spectra
(right) are average spectra acquired between 4.7 and 5 min.
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Balazy et al.
group (R1R2CAN-OH). Additionally, these fractions also contained hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acid (EET) products. Because of the complexity of this
NOxAA lipid mixture and difficulty to obtain nitrated lipids
in a purified form for biological experiments, these fractions
were not further characterized.
Efforts were also directed to elucidate the mechanism of
formation of NO2AAOH. The reaction products were rapidly
cooled on ice and analyzed by mass spectrometry without
extraction from water. The analysis revealed a spectrum that
is consistent with the structure of one isomer of nitro nitrite
derivative of arachidonic acid (Fig. 9).
Discussion
186 and 155 from the cleavage of the C-8,C-9 bond that were
consistent with a mixture of 8-nitro-9-hydroxyeicosatrienoic
acid and 8-hydroxy-9-nitroeicosatrienoic acid (Fig. 7C). Relatively weak fragment ions at m/z 146 and 115 in the same
spectrum also suggested the occurrence of the nitrohydroxy
products generated at C-5,C-6 bond: 5-nitro-6-hydroxyeicosatrienoic acid and 5-hydroxy-6-nitroeicosatrienoic acid. Further studies were directed to analyze fragment ions by a
higher level of mass spectrometry (LC/MS3) and confirmed
the proposed fragmentation (Scheme 1). For example, decomposition of carboxylate anion at m/z 266 (Scheme 1; Fig. 7A)
generated a mass spectrum having fragment ions at m/z 219
(100%, loss of HNO2) and m/z 175 (20%, loss of HNO2 ⫹ CO2),
which confirmed its structure with nitronic acid function at
C-15 (Scheme 1). Decomposition of carboxylate anion at m/z
195 (Fig. 7B) produced a mass spectrum with fragment ions
at m/z 177 (100%, loss of H2O) and m/z 167 (26%, loss of CO)
that was consistent with a C-11 aldehyde. NO2AAOH compounds were also obtained by treatment of arachidonic acid
with acidified sodium nitrite solutions (data not shown).
We developed an isotopic dilution GC/MS assay for quantitative analysis of NO2AAOH in biological material. Cardiac
muscle total lipids were extracted and saponified by mild
alkaline hydrolysis. Selected monitoring of ions at m/z 438
and 446 was used to detect endogenous NO2AAOH and the
octadeuterium-labeled internal standard, respectively. The
analysis revealed that cardiac muscle contained 6.8 ⫾ 2.6 ng
of NO2AAOH/g of tissue (n ⫽ 4) (Fig. 8).
The vasoactive lipid products in fractions 13 to 18 contained a complex mixture of lipid products (Fig. 3) that was
partially characterized by mass spectrometry. The nitrated
lipids (NOxAA; Fig. 3) that were likely to cause vasorelaxation via a mechanism sensitive to ODQ inhibition appeared
to be diene NO2 adducts and likely to be isomers of mono
NO2AA. Another group of lipids appeared to contain oxime
Fig. 8. Detection of NO2AAOH in cardiac muscle phospholipids by GC/
MS. Ion chromatograms corresponded to endogenous NO2AAOH (m/z
438) and internal standard (NO2AAOH-d8, m/z 446). The analysis revealed 6.8 ⫾ 2.6 ng NO2AAOH/g cardiac tissue (n ⫽ 4). Injection of the
NO2AAOH-d8 in the absence of cardiac muscle phospholipid extract did
not produce detectable signal at m/z 438 (data not shown).
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Scheme 1.
Experiments reported in the current study have detected
the presence of vasodilator metabolites originating from the
exposure of arachidonic acid to NO2. Because the majority of
the vasodilator activity in the crude mixture was inhibited by
the ODQ probe, it appears that this mixture contains nitrovasodilator lipids, which function through stimulating sGC.
Purification and characterization of the vasoactive lipids resulted in the identification of novel NO2AAOH metabolites as
the source of about one-half of the vasodilator activity that
was detected. The purified NO2AAOH metabolites spontaneously released NO and showed potent vasodilator activity
that was inhibited by ODQ. In addition, these metabolites
were detected in bovine cardiac muscle under basal conditions.
Characterization of the crude mixture reaction products of
NO2 with arachidonic acid, which caused vascular relaxation
through mechanisms independent of prostaglandins, detected vasoactive lipids in HPLC fractions 9 to 18, with the
NO2AAOH metabolites appearing in fractions 9 to 12. The
Nitrogen Dioxide and Arachidonic Acid
Fig. 9. Identification of a nitro nitrite derivative of arachidonic acid by
electrospray tandem mass spectrometry.
NO2AAOH during purification and storage. Although we attempted to minimize this effect by cold storage of samples
and by performing bioassay experiments shortly after purification, some loss of activity may have occurred. The reaction of NO2 with arachidonic acid also generated a mixture of
isoeicosanoids (iso-PGs, HETEs, and EETs) that have been
known as products of oxygen free radical-mediated peroxidation of arachidonic acid. Thus, the interaction of NO2 with
biological membrane lipids is an alternative pathway for
isoeicosanoid formation. Although several members of isoeicosanoid family have been known to cause vasorelaxation,
mechanisms other than activation of sGC have been suggested to explain the vasorelaxing effect. Because we are not
aware of previous reports showing vasorelaxation via sGC
activation by lipids modified by NO2, the NO2AAOH metabolites appear to be novel NO-derived vasoactive lipids.
The spontaneous release of NO from NO2AAOH could originate from intramolecular rearrangement. Previous studies
have established that ␤-nitro alcohols such as 2-nitroethanol
assume a favored guche conformation (Scheme 2) in the gas
phase and probably in aqueous solution (Marstokk and Møllendal, 1996). Additionally, the gauche conformation of ␤-nitro alcohols shows a hydrogen bond between the hydroxyl
hydrogen and one of the oxygens of the nitro group (Marstokk
and Møllendal, 1996). Analysis of molecular models suggests
that the gauche conformation with a hydrogen bond should
be also favorable for NO2AAOH (␤-nitro alcohols of arachidonic acid) (Scheme 2). Such bond would facilitate formation
of prominent fragment ions that appear in the LC/MS2 mass
spectra of NO2AAOH. We have also obtained evidence that
2-nitroethanol and other vicinal alkyl nitro alcohols induce
ODQ-sensitive vasorelaxation (M. Balazy, P. M. Kaminski,
and M. S. Wolin, unpublished observations), and thus have
similar properties as NO2AAOH. However, the mechanism
by which this new group of nitrovasodilators releases NO is
not known. One potential mechanism may involve the spontaneous rearrangement of a ␤-nitro alcohol to nitrous acid
and an epoxide (Scheme 2). Nitrous acid exists in equilibrium
with its anhydride, N2O3 (Williams, 1983), which spontaneously dissociates to NO and NO2 in aqueous solutions at
physiological pH (Jones, 1973). In the presence of vascular
tissue, N2O3 is also known to react with thiols to form Snitrosothiols (Williams, 1996), which release NO. Our previous studies have also established that nitration of thiols by
NO2 is an important vasodilatory mechanism that involves
generation of NO from S-nitroglutathione (Davidson et al.,
1996, 1997; Balazy et al., 1998). The detection of spontaneous
release of NO from NO2AAOH and a relaxation response
inhibited by ODQ are consistent with a role for NO-mediated
stimulation of cGMP production in the vasorelaxation caused
by these active lipid metabolites.
Prütz et al. (1985) have observed nitroarachidonyl radicals
by the electron paramagnetic resonance spectroscopy after
treatment of arachidonic acid with NO2. Thus, the initial
NO2 adduct to arachidonate double bonds is likely to have a
structure of a ␤-nitroarachidonyl radical (Scheme 3). It also
appears that the rates favor formation of nitroarachidonyl
radicals over the reaction of NO2 with water (Prütz et al.,
1985). The rearrangement of such radical followed by elimination of NO2 is known to produce trans bonds (Lai and
Finlayson-Pitts, 1991; Jiang et al., 1999). Both O2 and a
second NO2 may also attach to NO2AA radical and both
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
GC/MS study did not detect other isoeicosanoids in fractions
9 to 11, and only a small amount of NO2AA was present in
fraction 12. Although it is possible that this small amount of
NO2AA could have contributed to the vasorelaxing activity of
fractions 9 to 12, it seems unlikely that it would have produced a significant effect. NO2AA but not NO2AAOH or other
nitrated products were also found in fraction 13. However,
this fraction produced only a minor vasorelaxing effect (Fig.
3). Decomposition of the carboxylate molecular anions produced LC/MS2 mass spectra that revealed eight isomers of
NO2AAOH. Because two stereogenic centers are present in
each isomer this free radical reaction is expected to generate
a racemic mixture of NO2AAOH stereoisomers. Structural
identification was facilitated by the appearance of characteristic fragment ions that resulted from a strong influence of
the nitro group on fragmentation. One limitation of our study
is that we were unable to resolve chromatographically all of
the bioactive lipids, and we relied on mass spectrometric
analysis of the mixture of isomers that appeared in fractions
9 to 12. A group of lipids that might be expected to generate
similar ions as NO2AAOH is vicinal dihydroxyeicosatrienoic
acids (diHETrE), which originate from the hydrolysis of
EETs. EETs are products of cytochrome P450 epoxygenase,
which have also been detected among peroxidation products
of arachidonic acid (Nakamura et al., 1997). Wheelan et al.
(1996) have reported the LC/MS2 mass spectra of diHETrE
isomers, some of which showed ions similar to aldehyde ions
type b (Scheme 1); however, whereas NO2AAOH revealed
strong b ions, the diHETrE fragmented mostly by other
mechanisms, which resulted in weak b-like ions. Thus,
NO2AAOH metabolites appear to be the vasoactive lipids
detected in HPLC fractions 9 to 12, and the vasoactive substances in fractions 13 to 18 remain to be identified.
NO2AAOH isomers in HPLC fractions 9 to 12 appear to be
important vasodilator components present in the crude mixture, because in the presence of indomethacin the activity of
purified isomers is similar to the crude mixture (e.g., 35 ⫾ 5
versus 42 ⫾ 3% relaxation at 1 ␮M; Figs. 2 and 4). However,
it should be noted that the crude mixture also contained
other substances in fractions (13–18), which appear to be
nitroeicosanoids, and these lipids potentially have a vasodilator activity, which could be similar to NO2AAOH. However,
the complexity of the lipid mixture in fractions 13 to 18 would
require additional studies to isolate the vasoactive components in a purified form. It is also possible that some loss of
activity was due to spontaneous slow release of NO from
617
618
Balazy et al.
Scheme 3.
processes may lead to NO2AAOH (Scheme 3). The second
NO2 may attach to nitro alkyl radicals via bonding through
oxygen or nitrogen to generate either a nitro nitrite or dinitro
intermediate (Lai and Finlayson-Pitts, 1991). Detection of
vicinal nitro nitrite derivatives of arachidonic acid among the
reaction products suggests that they could be involved in
formation of NO2AAOH. At low levels of NO2 that may be
produced in biological systems an alternative mechanism
might produce NO2AAOH. O2 might add to NO2AA radical to
form a nitro peroxide molecule, which would be enzymatically reduced to NO2AAOH. We also observed formation of
NO2AAOH from arachidonic acid treated with acidified aqueous nitrite solution, which is known to generate NO2 (Jones,
1973). Recent reports have described formation of nitrated
lipids (O’Donnell et al., 1999), including vicinal nitrohydroxy
analogs (Napolitano et al., 2000), from linoleic acid esters by
treatment with acidified nitrite solution. Thus, NO2AAOH
could be important biologically active metabolites derived
from the reaction of arachidonic acid with NO2, and environments that influence the conformation of this fatty acid could
enhance the formation of specific isomers.
The present study identified NO2AAOH as NO-releasing
vasodilator metabolites of arachidonic acid resulting from its
reaction with NO2 (Scheme 4). Because the majority of arachidonic acid is found in an esterified form within cellular
membrane phospholipids, NO2AAOHs are likely to be formed
as esters of glycerophospholipids after exposure of biological
membranes to NO2. The storage of NO2AAOHs in phospholipids and their release by phospholipases could be a mechanism that regulates NO generation and other signaling
actions of these biologically active lipids. The observed occurrence of NO2AAOH in cardiac phospholipids suggests that a
NO-mediated free radical mechanism is likely to be involved
in generation of these compounds within cardiac phospholipids under more physiological conditions where NO2 is produced. This could occur in regions of tissues exposed to elevated levels of NO through its oxidation in membrane
environments to NO2, or as a result of NO2 formation from
peroxynitrite (in the absence or presence of CO2), or the
Scheme 4.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 15, 2017
Scheme 2.
oxidation of nitrite by peroxidase enzyme reactions under
conditions where reactive oxygen species are generated
(Scheme 4). The latter two mechanisms could be of particular
importance in inflammatory responses. Because the inhalation of NO2 has previously been reported to suppress pulmonary host resistance, to induce inflammation (Chauhan et al.,
1998) and cardiac arrhythmia (Peters et al., 2000) among
numerous other effects (Ehrlich, 1966), NO2 may have actions that modulate multiple biological signaling systems.
Although many of these effects have been attributed to lipid
peroxidation processes (Pryor and Lightsey, 1981; Sevanian
et al., 1982), our findings also suggest that nitration of lipids
by NO2 and formation of NO2AAOH could be an additional
mechanism by which NO2 modifies membrane lipids. Similar
mechanisms may form NO2AAOH in the lung after inhalation of air containing increased levels of NO2 (polluted urban
air, cigarette smoke). The acidic environment of the stomach
combined with a diet containing nitrite (frequently used as a
food preservative) and arachidonic acid (Taber et al., 1998)
may also stimulate formation of NO2AAOH. Thus, the oxidation product of NO, NO2, reacts with arachidonic acid to
generate biologically active NO2AAOH metabolites, which
may be important mediators of vascular relaxation, sGC
activation, and other pathophysiological processes.
Nitrogen Dioxide and Arachidonic Acid
Acknowledgments
We thank Dr. Haisong Tan for analysis of samples by mass spectrometry.
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