Nitrogen Dioxide Induces
cis-trans-Isomerization of Arachidonic Acid
within Cellular Phospholipids
DETECTION OF TRANS-ARACHIDONIC ACIDS IN
VIVO*
Houli
Jiang
,
Nathan
Kruger
,
Debarshi Rana
Lahiri
,
Dairong
Wang
,
Jean-Michel
Vatèle§, and
Michael
Balazy
¶
From the
Department of Pharmacology, New York Medical
College, Valhalla, New York 10595 and the § Laboratoire
de Chimie Organique 1, Université Claude Bernard,
69622 Villeurbanne Cédex, France
 |
ABSTRACT |
Oxygen free radicals oxidize arachidonic acid to
a complex mixture of metabolites termed isoeicosanoids that share
structural similarity to enzymatically derived eicosanoids. However,
little is known about oxidations of arachidonic acid mediated by
reactive radical nitrogen oxides. We have studied the reaction of
arachidonic acid with NO2, a free radical generated
by nitric oxide and nitrite oxidations. A major group of products
appeared to be a mixture of arachidonic acid isomers having one
trans-bond and three cis-double bonds. We have
termed these new products trans-arachidonic acids. These
isomers were chromatographically distinct from arachidonic acid and
produced mass spectra that were nearly identical with mass spectra of
arachidonic acid. The lack of ultraviolet absorbance above 205 nm and
the similarity of mass spectra of dimethyloxazoline derivatives
suggested that the trans-bond was not conjugated with any
of the cis-bonds, and the C=C bonds were located at carbons 5, 8, 11, and 14. Further identification was based on comparison of
chromatographic properties with synthetic standards and revealed that
NO2 generated 14-trans-eicosatetraenoic acid
and a mixture containing 11-trans-, 8-trans-,
and 5-trans-eicosatetraenoic acids. Exposure of human
platelets to submicromolar levels of NO2 resulted in a
dose-dependent formation of
14-trans-eicosatetraenoic acid and other isomers within
platelet glycerophospholipids. Using a sensitive isotopic dilution
assay we detected trans-arachidonic acids in human plasma
(50.3 ± 10 ng/ml) and urine (122 ± 50 pg/ml). We proposed a
mechanism of arachidonic acid isomerization that involves a reversible
attachment of NO2 to a double bond with formation of a
nitroarachidonyl radical. Thus, free radical processes mediated by
NO2 lead to generation of trans-arachidonic
acid isomers, including biologically active
14-trans-eicosatetraenoic acid, within membrane
phospholipids from which they can be released and excreted into urine.
 |
INTRODUCTION |
Arachidonic acid is one of the most abundant polyunsaturated fatty
acids found in the cellular membrane phospholipid bilayer. A
characteristic structural feature of this fatty acid is a 20-carbon chain containing four cis-double bonds that form a molecule
of 5Z,8Z,11Z,14Z-eicosatetraenoic
acid. These double bonds are homoconjugated resulting in three
bis-allylic methylene groups. Abstraction of a single
hydrogen from one of these methylene groups via a homolytic cleavage of
a C-H bond is a fundamental process of arachidonic acid metabolism by
enzymatic as well as nonenzymatic reactions. Enzymatic processes
lead to a family of biologically active lipids such as prostaglandins
and leukotrienes, known collectively as the eicosanoids (1). Syndromes
of oxidative stress elevate levels of free radicals that can directly
target arachidonic acid bound to phospholipids. This generates a
complex mixture of oxidized products, known as isoeicosanoids, that can
be cleaved off by phospholipases, circulated, and excreted in urine.
Isoprostaglandins (2, 3) and isoleukotrienes (4) are structurally
similar to the native eicosanoids, and some of them display potent
biological activity. Hydroxyl radical is a potent activator of
polyunsaturated fatty acid peroxidation due to its high intrinsic
oxidation potential. The oxidations of fatty acids are somehow limited
by its short reactive half-life (~10
9 s) and occur at
the diffusion controlled rates within a close distance to the site of
OH radical formation.
Relatively less is known about transformations of polyunsaturated fatty
acids induced by free radical nitrogen oxides. Nitric oxide reacts very
slowly with olefins but quite fast with lipid peroxy and alkoxy
radicals, which leads to unstable nitro and oxonitro derivatives of
linoleic and linolenic acids, and by this mechanism nitric oxide is
thought to terminate progression of lipid peroxidation (5).
NO2 is a toxic free radical found in biological systems as
a product of spontaneous oxidation of NO and enzymatic oxidations of
nitrite (6). NO2 is also an air pollutant and has been
implicated to cause pulmonary edema and fibrosis, bronchitis, asthma,
and possibly cancer (7). NO2 is a potent oxidant that
causes lipid peroxidation (8, 9); however, the reaction of
NO2 with arachidonic acid has not been characterized.
Oxidation of nitric oxide to NO2 is significantly
accelerated within the hydrophobic phase of cellular phospholipid
bilayer (10). This intramembrane reaction is facilitated by the much
higher solubility of nitric oxide in hydrophobic layer of phospholipids
than in the aqueous phase. Thus, it is possible that a significant
amount of NO2 may be formed under aerobic conditions within
the cellular phospholipid bilayer. These observations raise the
possibility of novel nonenzymatic, free radical pathways involved in
arachidonate transformations by NO2. In this study, we
established the chemical structures of major products formed from this
reaction, and we found that the predominant process mediated by
NO2 leads to a new group of lipids, which we have termed
trans-arachidonic acids.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Arachidonic acid was from Sigma.
[1-14C]Arachidonic acid (specific activity, 57 Ci/mmol)
was from NEN Life Science Products. [5,6,8,9,11,12,14,15-d8]Arachidonic acid (isotopic
purity, >98%) was from Biomol (Plymouth Meeting, MI). Nitrogen
dioxide was from Matheson (E. Rutherford, NJ). All solvents were of
highest chromatographic grade.
Reaction of NO2 with Arachidonic Acid--
In
a typical experiment, arachidonic acid (100 µg) was dissolved in 1 ml
of hexane, and sodium arachidonate (100 µg) was dissolved in 1 ml of
phosphate buffer (0.5 mM, pH 7.4).
d8-Archidonic acid (10 µg) was mixed with
[1-14C]arachidonic acid (10,000 cpm) and was used to
prepare the internal standards for quantitative analyses. Nitrogen
dioxide was prepared shortly before reaction with arachidonic acid as
described (11). Briefly, about 1 ml of liquid was collected from the
original NO2 tank. NO2 gas was delivered into
arachidonate solutions either via bubbling using helium as a carrier
gas (~0.1 ml/min) or was sampled using a 50-µl gas-tight syringe.
The final concentrations of NO2 were 43-430
µM. The reaction was carried for additional 3-5
min, and the lipids were isolated by extraction with organic solvents.
The extracts were dissolved in small volume of methanol and analyzed by
HPLC.1 In some experiments
the lipid extracts were treated with sodium borohydride to reduce hydroperoxides.
Reaction of PTSA with Arachidonic Acid--
PTSA was prepared
from its sodium salt (Aldrich) as described (12). Briefly, the PTSA
sodium salt (5 g) was dissolved in water (5 ml) and acidified with 6 N H2SO4. The precipitated sulfinic acid was filtered, washed with ice-cold water and hexane, and dried. An
equimolar amount of PTSA was added to arachidonic acid in dry
tetrahydrofuran, and the reaction was carried out at 100 °C for 40 min. The solvent was evaporated under nitrogen, the residue was mixed
with water, and lipids were extracted with ethyl acetate. The extract
was dried and analyzed by HPLC.
HPLC Analyses--
HPLC analyses were performed on a HP1050
system (Hewlett-Packard) using C18 column (250 × 4.6 mm, Beckman
Instruments). Samples were eluted with a gradient of acetonitrile in
water (62.5% increased to 100% in 60 min), and the effluent was
analyzed by an on-line UV diode array detector. Fractions were
collected by a Gilson FC 203B fraction collector. In the experiments
where [1-14C]arachidonic acid was used as a substrate for
NO2, the effluent was also analyzed by the on-line
radioactivity monitor to detect radiolabeled products.
Preparation of Derivatives--
Pentafluorobenzyl (PFB)
and methyl esters were prepared as described (13). Dimethyloxazoline
(DMOX) derivatives were prepared as described (14) by treatment of
fatty acids with 50 µl of 2-amino-2-methylpropanol (Aldrich) in a
microvial at 150 °C for 1 h. After cooling, samples were dried
under a stream of nitrogen, mixed with water, extracted with ethyl
acetate, and finally purified by HPLC. The DMOX derivatives of compound
I and arachidonic acid eluted at 28 and 23.5 min, respectively.
N,O-Bis(trimethylsilyl)trifluoroacetamide was
used to convert hydroxyl groups into trimethylsilyl (TMS) derivatives.
Samples were finally dissolved in n-decane, and 1-µl aliquots were analyzed by GC/MS. The samples were hydrogenated by
bubbling hydrogen gas through a solution of esterified samples of
compounds I and II in hexane containing catalytic amounts of rhodium
adsorbed on alumina (~1 mg) as described (15).
Mass Spectrometry--
Electrospray tandem mass
spectrometric analyzes were performed on an Esquire ion trap instrument
(Brucker Daltonics, Billerica, MA). Samples (1 µg/ml) were injected
using a syringe pump into a mass spectrometer operating in the negative
ion polarity with a capillary exit voltage of
65 V, a skimmer voltage
of
26 V, a nebulizer pressure of 16.2 psi, and a dry gas temperature
of 352 °C.
GC/MS analyses were performed on an HP 5890A instrument
(Hewlett-Packard). Samples were analyzed using a DB-1 fused silica gas
chromatographic column (10 m, 0.25-mm internal diameter, 0.25-µm film
thickness, J and W Scientific, Folsom, CA) and a temperature program of
150 °C (held for 1 min after injection) to 250 °C at a rate of
8 °C/min. The temperature of the injector, transfer line and ion
source was 250 °C. Relative retention times (C values) were
established from a plot of retention time of a series of saturated
fatty acids (PFB or methyl ester derivatives) versus their
carbon chain length (18-24 carbons). The regression analysis produced
a formula for a correlation line (r2 = 0.999)
that allowed conversion of retention times of analyzed compounds
into their C values.
Preparation of trans-Arachidonic Acids--
Epoxyeicosatrienoic
acids (EET) were prepared from arachidonic acid and
m-chloroperoxybenzoic acid as described (16). 14,15-EET and,
separately, a mixture of 5,6-, 8,9-, and 11,12-EET (200-400 µg) were
dissolved in dry tetrahydrofuran (100 µl) and mixed with a solution
of triphenylphosphine in terahydrofuran (final concentration 0.1 mM) (17). The reaction was carried out in a glass tube, under nitrogen, in a block heated to 100 °C for 40 min. The products were extracted with ethyl acetate and purified by HPLC.
5Z,8Z,11Z,14E-eicosatetraenoic acid (14E-AA) was synthesized as described (18). Briefly,
this synthesis involved a Wittig reaction between
(Z)-7-(t-butyldiphenylsilyloxy)hept-3-enal and
the ylide of
(3Z,6E)-dodeca-3,6-dienyl-triphenylphosphonium bromide. The C19 tetraenoic ether was isolated and transformed in three
steps to a methyl ester of 14E-AA in 81% overall yield (18). The 14E-AA methyl ester was hydrolyzed with lithium
hydroxide (0.1 N) in tetrahydrofuran/water (10:1). The
stereoisomeric purity of 14E-AA (PFB derivative) was >99%
as established by GC/MS.
Determination of trans-Arachidonic Acids in Human
Platelets--
A concentrate of fresh human platelets was obtained
from Hudson Valley Blood Bank (Elmsford, NY), and platelet suspensions in phosphate buffer were prepared as described (13). Platelets were
exposed to NO2 as described (11). Briefly, stirred platelet suspensions in 1 ml (6.8×109 cells/ml) of phosphate buffer
(0.5 mM, pH 7.4) were mixed with NO2 solution
in helium (1-20 µl; final concentration, 0.08-0.7 µM)
delivered with a gas-tight syringe, and the cells were stirred for an
additional 3-5 min at room temperature. Total platelet lipids were
extracted with chloroform/methanol using a Bligh and Dyer protocol
without acidification. The lipid extracts were dried under nitrogen and
hydrolyzed in 1 N NaOH at 60 °C for 2 h. Fatty acids were then extracted with ethyl acetate. Prior to hydrolysis, 5 ng
of d8-trans-arachidonic acids was added as
internal standard. Lipids were purified by HPLC, and the fractions
containing trans-arachidonic acids were collected and dried.
The residue was derivatized with PFB bromide and analyzed by GC/MS.
Ions at m/z 303 and 311, corresponding to
endogenous trans-arachidonic acids and deuterium-labeled
internal standard, were monitored. The amount of the
trans-arachidonic acids formed in human platelets following
exposure to NO2 was calculated from a standard curve.
Determination of trans-Arachidonic Acids in Human Plasma
and Urine--
Plasma samples (250 µl) were prepared from blood of
four healthy donors who have not taken any medication and were
supplemented with 1 ng of deuterium-labeled
trans-arachidonic acids. The samples were mixed with
methanol (1.25 ml) and centrifuged. The methanolic solution was
evaporated to near dryness, dissolved in 1 ml of water, and extracted
with ethyl acetate. The lipid extracts were purified by HPLC and
analyzed by GC/MS as described above. Urine samples from three healthy
donors (10 ml) were supplemented with 5 ng of deuterium-labeled
trans-arachidonic acids and equilibrated at room temperature
for 20 min. Lipids were extracted using ToxElute 3210 columns (Varian)
and methylene chloride. The extracts were purified by HPLC and analyzed
by GC/MS as described above.
 |
RESULTS |
Arachidonic acid reacted readily with NO2 in a
dose-dependent manner generating two major compounds (I and
II) and a complex mixture of less abundant products (Fig.
1). Compounds I and II eluted after the
peak of arachidonic acid, suggesting that they were relatively less
polar than arachidonic acid. Purified material in peaks I and II did
not show UV light absorbance above 205 nm. Table
I shows that compounds I and II accounted
for 57.3% of total products when the reaction was carried out in
hexane and for 18.3% when the reaction was carried out in phosphate
buffer. The combined yield of other metabolites was 11-35% of the
total NO2-derived arachidonate products. Similar
chromatograms were obtained when NO2 was either injected or
bubbled through a solution of arachidonic acid. Removal of oxygen from
arachidonic acid solutions prior to addition of NO2
increased the relative intensity of peaks I and II by about 15%.
Addition of catalytic amounts of copper chloride had no effect on the
formation of compounds I and II. A similar profile of metabolites was
obtained from the treatment of arachidonyl phosphatidylcholine with
NO2 in phosphate buffer followed by a mild alkaline
hydrolysis (not shown).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 1.
Representative chromatogram showing detection
of products from the NO2/arachidonic acid reaction by
HPLC. In this experiment, 100 µg of arachidonic acid was
dissolved in hexane and bubbled with NO2 (470 µM) in helium for 3 min. Lipids were analyzed on a C18
column (250 × 4.6 mm) and separated with a gradient of
acetonitrile in water (62.5-100% in 50 min). The inset
shows a tandem electrospray mass spectrometry of product I following
collision-induced decomposition of the molecular anion at
m/z 303. Material in peaks labeled AA,
I, and II produced similar spectrum and was
identified as arachidonic acid and a mixture of
trans-arachidonic acids, respectively.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Relative abundance of products generated from the reaction of nitrogen
dioxide with arachidonic acid
Shown are the percentages of total peak area of products absorbing at
205 nm from chromatograms obtained by HPLC analyses as shown in Fig. 1
(n = 2-5). Compounds I and II were identified as a
mixture of arachidonic acid isomers having one or more
trans-double bonds, whereas oxidized products contained
prostaglandin F, hydroxyeicosatetraenoic acids, epoxyeicosatrienoic
acids, nitrohydroxyeicosatrienoic acids, and nitroeicosatetraenoic
acids.
|
|
Electrospray mass spectrometry of compounds I and II produced strong
anions at m/z 303. Collisional activation of ion
m/z 303 revealed major fragment ions at
m/z 285 (loss of H2O), 259 (loss of
CO2), and 205 (loss of C7H14) (Fig.
1). GC/MS analyses of compounds I and II (PFB derivatives) produced
prominent ions at m/z 303 and revealed a
characteristic pattern of peaks having retention time 0.1-0.45 min
longer than the PFB ester of arachidonic acid (Fig.
2). This retention time difference
corresponded to an increase of C value by 0.3-0.5 relative to
arachidonic acid (21.3). The mass spectrometric data suggested that
compounds I and II contained several isomers having the molecular mass
of 304 units and were likely to be isomers of arachidonic acid having altered double bond location and/or configuration. Catalytic reduction of I and II with hydrogen gas and rhodium revealed a single
chromatographic peak showing a mass spectrum with an ion at
m/z 311. Thus, reduction of double bonds in I and
II produced a compound indistinguishable from saturated arachidonic
acid, eicosanoic acid.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 2.
Comparison of chromatograms obtained by GC/MS
analyses of PFB esters of AA and products I and II via a selected ion
monitoring of anion m/z
303.
|
|
The location of C=C bonds in compound I was established by GC/MS
analysis of DMOX derivatives. These derivatives have been useful in
establishing the double bond position in fatty acids (14), including
arachidonic acid (19). The DMOX derivative of compound I eluted at the
relative retention time (C value) of 21.66, e.g. 0.33 units
more than the DMOX derivative of arachidonic acid. The spectra
contained ions characteristic for DMOX derivatives at
m/z 113 (base peak) and 126, and the molecular
ion appeared at m/z 357. The location of the C=C
bonds was established by analysis of mass differences in a series of
characteristic ions. Sequential cleavage of each of the carbon-carbon
bonds directed by the ionized dimethyloxazoline moiety led to a series
of ions differing by 14 units (CH2). An advance by 26 units
(CH=CH) occurred when the carbon of a double bond was encountered. The
ions critical for establishing C=C bond location in compound I are
summarized in Table II. The mass spectra
revealed no essential differences between compound I and arachidonic
acid, suggesting that the double bonds in compound I were located at
the same carbons as in arachidonic acid, e.g. at carbons 5, 8, 11, and 14. To confirm that compounds I and II contained
trans-double bonds, arachidonic acid was reacted with PTSA,
a compound known to induce a
cis-trans-isomerization of olefins and methyl
linoleate (20, 21). PTSA generated compounds that were detected after
the peak of arachidonic acid and appeared to have chromatographic (Fig.
3) and mass spectrometric (Fig. 4) properties very similar to those of
compounds I and II obtained from the NO2/arachidonic acid
reaction.
View this table:
[in this window]
[in a new window]
|
Table II
Comparison of mass spectra of arachidonic acid and compound I
Shown are the dimethyloxazoline derivatives. Intensity is relative to
ion m/z 113.
|
|

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 3.
Comparison of UV chromatograms (205 nm) from
HPLC analyses of trans-arachidonic acids obtained via
the reaction of arachidonic acid with NO2
(top) or PTSA (middle); the analysis
of synthetic standard 14E-AA is shown for comparison
(bottom).
|
|

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 4.
Identification of
trans-arachidonic acids by GC/MS. The
chromatograms were obtained via a selected monitoring of ion
m/z 303 of the following compounds (from
top to bottom): synthetic 14E-AA, a
reaction product from a mixture of epoxyeicosatrienoic acids with
triphenylphosphine containing a mixture of 11E-,
8E-, and 5E-AA, products obtained from the
reaction of AA and NO2, and products obtained from the
reaction of AA and PTSA.
|
|
Standard samples of trans-arachidonic acid isomers were
prepared form arachidonate epoxides (EET) by reaction with
triphenylphosphine in tetrahydrofuran. 14E-AA prepared from
14,15-EET as well as via full synthesis (18) coeluted with the product
of the NO2/arachidonic acid (Figs. 3 and 4). A mixture of
three arachidonate epoxides (11,12-, 8,9- and 5,6-EET) treated with
triphenylphosphine showed a product at 11.6 min, which had the same
retention time as a broadened peak from the NO2/archidonic
acid reaction (Fig. 4). Coelution experiments were performed by mixing
the synthetic 14E-AA with product I obtained either from the
reaction with NO2 or PTSA followed by GC/MS analyses. These
experiments resulted in selective increases of the intensity of the
peak eluting at 11.35 min. The symmetrical shape of this peak suggested
that the synthetic 14E-AA was not separable from the
11.35-min isomer of compound I (not shown). The C value differences and
the reduction to eicosanoic acid were consistent with trans
rather than branched isomers of arachidonic acid. Thus, by comparison
of chromatographic and mass spectroscopic properties, we identified a
major product of arachidonic acid/NO2 reaction as a
mixture of four mono trans-arachidonic acids (Scheme
I).
Mass spectra of material in fractions eluting at 8-10 min (Fig. 1)
revealed a compound having molecular mass of 367 units that is likely
to have nitro and hydroxyl groups attached to the arachidonyl chain.
Mass spectrum of a major component (PFB and TMS derivative) revealed
ions at m/z 438 (M-PFB, relative abundance 100%), 391 (M-PFB-HNO2, 2%), 348 (M-PFB-TMSOH, 38%), and
301 (m/z 348-HNO2, 1%). Electron
ionization mass spectrum of this derivative revealed characteristic
ions at m/z 619 (M+, 1.4%), 572 (M+-HNO2, 1%), 529 (M+-TMSOH,
10%), 232 (M+-NO2CHCH(OTMS)C5H11,
9%), 173 (TMSOCH(CH2)4CH3, 29%),
and 181 (C6F5CH2+,
100%) (not shown). This spectrum was consistent with the structure of
14-nitro-15-hydroxy-eicosatrienoic acid. Minor products of the
NO2/arachidonic acid reaction were identified as having
structures consistent with isomers of hydroxyeicosatetraenoic acids,
EET, prostaglandin F, and nitroeicosatetraenoic acids and were not analyzed further.
Development of a sensitive quantitative assay enabled us to
investigate the occurrence of trans-arachidonic acids in
cells exposed to NO2 and in vivo. Analyses of
human platelets exposed to NO2 (0.08-0.7 µM)
revealed that trans-arachidonic acids were formed within
platelets in a dose-dependent manner (Fig.
5). The arachidonic acid isomers from
platelets coeluted with deuterium-labeled and synthetic
trans-arachidonic acids standards. NO2 induced
formation of 14E-AA and, relatively, 2.5-fold more of other
mono trans-arachidonic acids, possibly a mixture of
11E-, 8E-, and 5E-AA. Basal levels of
trans-arachidonic acids in human platelets were 2.9-4.2
ng/106 cells. Hydrolysis of lipid extracts was essential to
detect trans-arachidonic acids, indicating that these
isomers were formed in esterified form, possibly from arachidonic acids
bound to platelet membrane phospholipids. Human plasma levels of
trans-arachidonic acids were 50.3 ± 10 ng/ml
(n = 4), whereas human urine levels were 122 ± 50 pg/ml (n = 3) (Fig.
6).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 5.
Identification of
trans-arachidonic acids in human platelets exposed to
NO2. The chromatogram shows detection of ions
m/z 303 and 311 corresponding to endogenous
trans-arachidonic acids and the internal standard,
d8-trans-arachidonic acids, respectively. In
this experiment 6 × 109 platelets were exposed to 0.7 µM of NO2, and the phospholipids were
extracted, mixed with 5 ng of
d8-trans-arachidonic acids, and hydrolyzed with
NaOH as described under "Experimental Procedures." The
chromatograms were normalized to the highest peak. The graph on the
right shows the dependence of trans-arachidonic
acids formation in platelets on the dose of NO2.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 6.
Selected ion chromatograms obtained from
GC/MS negative ion chemical ionization analysis of
trans-arachidonic acids in human plasma
(A) and urine (B) using
deuterium-labeled analogs as internal standard (ion
m/z 311). Compounds were
analyzed as pentafluorobenzyl esters on a 11-m GC column. Levels of the
endogenous trans-isomers ranged in plasma from 22.9 to 88.8 ng/ml and in urine from 40 to 203 pg/ml. Arachidonic acid was partially
removed during HPLC purifications.
|
|
 |
DISCUSSION |
Oxidations of arachidonic acid by reactive oxygen radicals
generate a complex family of oxidized lipids known as isoeicosanoids. Initially formed arachidonate hydroperoxy radicals or hydroperoxides have been detected as intermediate products in formation of
isoprostanoids and isoleukotrienes. We describe here a new unique
family of free radical-generated lipids derived from
NO2-mediated isomerization of the arachidonic acid double
bonds that does not appear to involve hydroperoxides. Formation of
trans-arachidonic acids within cellular membranes is a new
aspect of NO2 biochemistry and may have profound influence
on cellular membrane properties. Several isomers having distinct
chromatographic mobility were observed and appeared to have one
trans-bond and three cis-bonds. Thus, four such
isomers of arachidonic acid can potentially be generated (Scheme I).
Therefore, we have termed the arachidonic acid isomers having a single
trans-double bond (E configuration) as
trans-arachidonic acids. Analytical data provided evidence
that NO2 changed arachidonate double bond geometry without
rearrangement. Analyses by GC/MS detected one sharp peak that had the
retention time identical with the synthetic 14E-AA and
another, broadened peak that appeared to contain several components and
coeluted with a mixture of trans-arachidonic acids prepared
from epoxyeicosatrienoic acids and triphenylphosphine. NO2
also caused formation of smaller amounts of material (compound II) that
probably contained arachidonate isomers having more than one
trans-bond. Although the relative proportion of the
arachidonate trans-isomers remains to be established, the
14E-AA was separated from arachidonic acid and other
trans-isomers on a gas capillary column. This isomer
cochromatographed with two synthetic standards, and comparison of the
retention times allowed identification of 14E-AA as a
product from the reaction of NO2 with arachidonic acid. We
observed that the profile of trans-arachidonic acids produced by NO2 was nearly identical to that from the
reaction of arachidonic acid with PTSA, a reagent known to induce
cis-trans-isomerization of olefins that does not
rearrange double bonds (21).
Several mechanisms may be involved in the formation of a
trans-bond in arachidonic acid. The similarity with PTSA
product profile suggested that generation of the
trans-arachidonic acids by NO2 may occur via a
free radical mechanism. It is possible that NO2 initially
attaches to a double bond and forms a nitroarachidonyl radical. The
rearrangement of this radical followed by elimination of
NO2 is likely to form a trans-bond (Fig.
7, arrow a). One piece of
evidence supporting formation of the nitroarachidonyl radical comes
from detection of 14-nitro-15-hydroxyeicosatrienoic acid among the
products of the NO2/arachidonic acid reaction (Fig. 7).
This compound may originate from trapping of oxygen to the nitroarachidonyl radical or from attachment of the second molecule of
NO2. Hydrolysis of such a nitro nitrite intermediate would produce a nitro alcohol (Fig. 7, arrow b). This mechanism
appears to be more likely because we noticed that
nitrohydroxyarachidonic acids can be isolated without reduction of
samples by sodium borohydride. The NO2-mediated
isomerization of arachidonic acid appeared to be an efficient process
and exceeded formation of hydroperoxyeicosatetraenoic acids, which
accounted for only ~5% of total products. In aerobic solutions the
isomerization must compete with scavenging by oxygen. For the
trans-isomers to be formed at observed yields, the rate of
rotation of the nitroarachidonyl radical and elimination of NO2 would have to be greater than attachment of oxygen to
this radical. In addition, these reactions must be faster than
disproportionation of NO2. According to the work by
Prütz et al. (22), NO2 generates arachidonate radicals with a rate (~106
M
1 s
1) that is greater than the
disproportionation to nitrite and nitrate. Thus,
NO2-induced cis-trans-isomerization
of arachidonic acid is kinetically favorable in aerobic aqueous
solutions.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 7.
Proposed mechanism for a nitrogen
dioxide-mediated formation of 14-trans-arachidonic
acid and 14-nitro,15-hydroxyeicosatrienoic acid.
|
|
Studies describing the effects of NO2 on living systems
have focused almost exclusively on the toxicity of inhaled
NO2 (7). Many of these studies have established that the
toxic effects of NO2 could be correlated with increased
lipid peroxidation. Our findings suggest that formation of
trans-arachidonic acids within cellular phospholipids may
represent an additional aspect of NO2-induced toxicity.
Increased levels of trans-arachidonic acids could contribute
to changes of the membrane asymmetry and fluidity that have been noted
to occur following exposure to NO2 (23, 24). Fatty acids
with trans-bonds are known to have much different physical
properties, e.g. a higher melting point than analogous
cis-isomers. Our data suggest that isomerization of arachidonic acid is likely to occur in biological systems following exposure to NO2, which at low concentrations exists almost
exclusively as a monomer (22). In addition to changes of the membrane
biochemistry, the free trans-arachidonic acids are likely to
modulate the activity of cyclooxygenases and lipoxygenases. A recent
study has described inhibition of platelet aggregation by
14E-AA that coincides with inhibition of thromboxane
synthesis and generation of unique metabolites (25).
Although the origin of trans-arachidonic acids in
human urine remains to be determined, by analogy to the cyclooxygenase
derived prostaglandins as well as isoprostaglandins, these compounds
may derive at least in part from local formation in the kidney.
trans-Arachidonic acids have not been detected in human
plasma previously, and their rather high concentration warrants further
study. It has been known that trans-linoleic acids that are
found in processed foods, hydrogenated fats, and dairy products could
be desaturated and elongated to certain trans-arachidonic
acids in the rat liver (19). A diet enriched in fatty acids containing
trans-isomers has been suspected as a risk factor in
coronary artery disease and other disorders (26); however, the effects
of trans-fatty acids on health outcome are not fully
understood (27, 28). In particular, the possibility that
trans-arachidonic acids are formed within cells via a free
radical mechanism involving NO2 has not been explored.
Increased amounts of trans-isomers of arachidonic acid and
possibly of other polyunsaturated fatty acids may originate from
inhaled as well as endogenously formed NO2. Because
trans-fatty acids are not produced by the hydroxyl radical,
the detection and quantification of trans-arachidonic acids
in vivo may be used as a specific index to assess the degree
of cellular injury mediated by NO2. The present study
provides a basis for such an investigation. Studies into the mechanisms
of cellular activation and trans-arachidonic acids may
clarify their importance in vivo in syndromes such as inflammation, thrombosis, and ischemia-reperfusion injury in which damage to cellular membrane phospholipids coincides with oxidant stress.
 |
FOOTNOTES |
*
This work was supported by American Heart Association (New
York State Affiliate) Grant 9850104, National Institutes of Health Grant HL 34300, and National Institutes of Health Shared
Instrumentation Grant SO1 RR 12993 (to M. B.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed. E-mail:
michael_balazy{at}nymc.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
HPLC, high pressure
liquid chromatography;
PTSA, para-toluenesulfinic acid;
PFB, pentafluorobenzyl;
DMOX, 4,4-dimethyloxazoline;
TMS, trimethylsilyl;
GC/MS, gas chromatography/mass spectrometry;
EET, epoxyeicosatrienoic acid(s);
AA, arachidonic acid.
 |
REFERENCES |
-
Samuelsson, B.
(1991)
Z. Rheumatol.
50 (Suppl. 1),
3-6[Medline]
[Order article via Infotrieve]
-
Reilly, M. P.,
Lawson, J. A.,
and FitzGerald, G. A.
(1998)
J. Nutr.
128 (suppl.),
434-438[Abstract/Free Full Text]
-
Morrow, J. D.,
and Roberts, L. J.
(1997)
Prog. Lipid Res.
36,
1-21[CrossRef][Medline]
[Order article via Infotrieve]
-
Harrison, K. A.,
and Murphy, R. C.
(1995)
J. Biol. Chem.
270,
17273-17278[Abstract/Free Full Text]
-
Rubbo, H.,
Radi, R.,
Trujillo, M.,
Telleri, R.,
Kalyanaraman, B.,
Barnes, S.,
Kirk, M.,
and Freeman, B. A.
(1994)
J. Biol. Chem.
269,
26066-26075[Abstract/Free Full Text]
-
van der Vliet, A.,
Eiserich, J. P.,
Halliwell, B.,
and Cross, C. E.
(1997)
J. Biol. Chem.
272,
7617-7625[Abstract/Free Full Text]
-
Environmental Health Criteria 188.
(1997)
Nitrogen Oxides, World Health Organization, Geneva
-
Pryor, W. A.,
and Lightsey, J. W.
(1981)
Science
214,
435-437
-
Gallon, A. A.,
and Pryor, W. A.
(1994)
Lipids
29,
171-176[Medline]
[Order article via Infotrieve]
-
Liu, X.,
Miller, M. S.,
Joshi, M. S.,
Thomas, D. D.,
and Lancaster, J. R., Jr.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2175-2179[Abstract/Free Full Text]
-
Jiang, H.,
and Balazy, M.
(1998)
Nitric Oxide Biol. Chem.
2,
350-359[CrossRef][Medline]
[Order article via Infotrieve]
-
Kice, J. L.,
and Bowers, K. W.
(1962)
J. Am. Chem. Soc.
84,
605-610
-
Balazy, M.
(1991)
J. Biol. Chem.
266,
23561-23567[Abstract/Free Full Text]
-
Zhang, J. Y., Yu, Q. T.,
Liu, B. N.,
and Huang, Z. H.
(1988)
Biol. Environ. Mass Spectrom.
15,
33-44
-
Balazy, M.,
and Murphy, R. C.
(1986)
Anal. Chem.
58,
1098-1101[Medline]
[Order article via Infotrieve]
-
Balazy, M.,
and Nies, A. S.
(1989)
Biomed. Environ. Mass Spectrom.
18,
328-336[Medline]
[Order article via Infotrieve]
-
Wittig, G.,
and Haag, W.
(1955)
Chem. Ber.
88,
1654-1661
-
Berdeaux, O.,
Vatèle, J.-M.,
Eynard, T.,
Nour, M.,
Poullain, D.,
Noël, J.-P.,
and Sébédio, J.-L.
(1995)
Chem. Phys. Lipids
78,
71-80[CrossRef]
-
Ratnayake, W. M.,
Chen, Z. Y.,
Pelletier, G.,
and Weber, D.
(1994)
Lipids
29,
707-714[Medline]
[Order article via Infotrieve]
-
Gibson, T. W.,
and Strasburger, P.
(1976)
J. Org. Chem.
41,
791-793
-
Snyder, J. M.,
and Scholfield, C. R.
(1982)
J. Am. Oil Chem. Soc.
50,
469-470
-
Prütz, W. A.,
Mönig, H.,
Butler, J.,
and Land, E. J.
(1985)
Arch. Biochem. Biophys.
243,
125-134[Medline]
[Order article via Infotrieve]
-
Li, Y. D.,
Patel, J. M.,
and Block, E. R.
(1994)
Toxicol. Appl. Pharmacol.
129,
114-120[CrossRef][Medline]
[Order article via Infotrieve]
-
Patel, J. M.,
and Block, E. R.
(1986)
Am. Rev. Respir. Dis.
134,
1196-1202[Medline]
[Order article via Infotrieve]
-
Berdeaux, O.,
Chardigny, J. M.,
Sébédio, J.-L.,
Mairot, T.,
Poullain, D.,
Vatèle, J.-M.,
and Noel, J. P.
(1996)
J. Lipid Res.
37,
2244-2250[Abstract]
-
Feldman, E. B.,
Kris-Etherton, P. M.,
Kritchevsky, D.,
and Lichtenstein, A. H.
(1996)
Am. J. Clin. Nutr.
63,
663-670[Abstract]
-
Kritchevsky, D.
(1997)
Prostaglandins Leukotreines Essent. Fatty Acids
57,
399-402[Medline]
[Order article via Infotrieve]
-
Nelson, G. J.
(1998)
Nutr. Rev.
56,
250-252[Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.