Electrophilic fatty acid nitroalkenes are systemically transported and distributed upon esterification to complex lipids

Article abstract of DOI:10.1194/jlr.M088815

Electrophilic nitro-fatty acids [NO₂-FAs (fatty acid nitroalkenes)] showed beneficial signaling actions in preclinical studies and safety in phase 1 clinical trials. A detailed description of the pharmacokinetics (PK) of NO₂-FAs is complicated by the capability of electrophilic fatty acids to alkylate thiols reversibly and become esterified in various complex lipids, and the instability of the nitroalkene moiety during enzymatic and base hydrolysis. Herein, we report the mechanism and kinetics of absorption, metabolism, and distribution of the endogenously detectable and prototypical NO₂-FA, 10-nitro-oleic acid (10-NO₂-OA), in dogs after oral administration. Supported by HPLC-high-resolution-MS/MS analysis of synthetic and plasma-derived 10-NO₂-OA-containing triacylglycerides (TAGs), we show that a key mechanism of NO₂-FA distribution is an initial esterification into complex lipids. Quantitative analysis of plasma free and esterified lipid fractions confirmed time-dependent preferential incorporation of 10-NO₂-OA into TAGs when compared with its principal metabolite, 10-nitro-stearic acid. Finally, new isomers of 10-NO₂-OA were identified in vivo, and their electrophilic reactivity and metabolism characterized. Overall, we reveal that NO₂-FAs display unique PK, with the principal mechanism of tissue distribution involving complex lipid esterification, which serves to shield the electrophilic character of this mediator from plasma and hepatic inactivation and thus permits efficient distribution to target organs.—Fazzari, M., D. A. Vitturi, S. R. Woodcock, S. R. Salvatore, B. A. Freeman, and F. J. Schopfer. Electrophilic fatty acid nitroalkenes are systemically transported and distributed upon esterification to complex lipids.

Full text of DOI:10.1194/jlr.M088815

Electrophilic fatty acid nitroalkenes are systemically transported and distributed upon esterification to
complex lipids
Marco Fazzari1,2,†, Dario A. Vitturi2, Steven R. Woodcock2, Sonia R. Salvatore2, Bruce A. Freeman2,
Francisco J. Schopfer2
2
1Fondazione Ri.MED, Via Bandiera 11, 90133 Palermo, Italy; Department of Pharmacology and
Chemical Biology, University of Pittsburgh, 200 Lothrop Street, Pittsburgh, 15261, PA, U.S.A.
†
To whom correspondence should be addressed: Dr. Marco Fazzari, phone: (412) 648-9671, fax:
(412) 648-2229; email: maf167@pitt.edu, Department of Pharmacology and Chemical Biology,
University of Pittsburgh, 200 Lothrop Street, Pittsburgh, 15261, PA, U.S.A.
Supplementary key words: ● pharmacokinetics ● triglyceride ● nitro-fatty acids ● nitroalkene ●
mass spectrometry ● nitro-oleic acid
1

ABSTRACT
Electrophilic nitro-fatty acids (NO2-FA) showed beneficial signaling actions in preclinical studies,
and safety in Phase 1 clinical trials. Detailed understanding of the pharmacokinetics (PK) of NO2-FA
is complicated by the capability of electrophilic fatty acids to alkylate thiols reversibly, become
esterified in various complex lipids, and the instability of the nitroalkene moiety during enzymatic and
base hydrolysis. Herein, we report the mechanism and kinetics of absorption, metabolism, and
distribution of the endogenously-detectable and prototypical NO2-FA 10-nitro-oleic acid (10-NO2-
OA) in dogs after oral administration. Supported by liquid chromatography-high resolution-tandem
mass spectrometry (HPLC-HR-MS/MS) analysis of synthetic and plasma-derived 10-NO2-OA-
containing-triacylglycerides, we show that a key mechanism of NO2-FA distribution is an initial
esterification into complex lipids. Quantitative analysis of plasma free and esterified lipid fractions
confirmed time-dependent preferential incorporation of 10-NO2-OA into triglycerides when compared
to its principal metabolite 10-nitro-stearic acid (10-NO2-SA). Finally, new isomers of 10-NO2-OA
were identified in vivo, and their electrophilic reactivity and metabolism characterized. Overall, we
reveal that fatty acid nitroalkenes display unique pharmacokinetics, with the principal mechanism of
tissue distribution involving complex lipid esterification, which serves to shield the electrophilic
character of this mediator from plasma and hepatic inactivation and thus permits efficient distribution
to target organs.
2

INTRODUCTION
The knowledge of absorption, distribution, metabolism, and excretion (ADME) of new drug
candidates is important for safe and insightful development. The rate of drug development failures
attributable to ADME deficiencies exceeds 40% and, even after new drug approval, many drugs still
display ADME problems. Notably, ‘‘a chemical cannot be a drug, no matter how active nor how
specific its action, unless it is also taken appropriately into the body (absorption), distributed to the
right parts of the body, metabolized in a way that does not instantly remove its activity, and
eliminated in a suitable manner” (1). As a covalently-acting endogenous mediator and a class of new
drug candidates, electrophilic fatty acid nitroalkenes (NO2-FA) have displayed beneficial effects in
preclinical animal models of metabolic and inflammatory disease (2-7). A synthetic homolog of a
NO2-FA detected in plants and mammals (8, 9), (E)-10-nitro-octadec-9-enoic acid (10-nitro-oleic
acid, 10-NO2-OA) is now in Phase 2 clinical trials for treating focal segmental glomerulosclerosis and
will soon begin trials in pulmonary arterial hypertension and obese asthmatics.
NO2-FA are generated during inflammation and digestion by reactions between unsaturated fatty acids
−
and the nitric oxide (•NO) and nitrite (NO2 )-derived nitrating species nitrogen dioxide (•NO2) (10).
Endogenous formation of NO2-FA can be promoted by a) oxidative inflammatory reactions such as
inflammatory cell activation and myocardial ischemia/reperfusion (2, 5), and b) dietary
-
-
supplementation of conjugated linoleic acid and nitrite (NO2 ) or nitrate (NO3 ) (11). In the absence of
metabolic or inflammatory stimuli, plasma and urinary NO2-FA are normally present at ~1-3 and ~10
nM concentrations in humans, respectively (9, 11-13). Administration of NO2-FA has shown
pharmacologic benefits in animal models of atrial fibrosis (3), pulmonary hypertension (6),
inflammatory bowel disease (7), adriamycin-induced nephropathy (14) , cardiac ischemia/reperfusion
(2), cutaneous inflammation (4), and abdominal wall defect (15), among others. The signaling actions
of NO2-FA are attributed to the reversible Michael addition reaction between the electrophilic carbon
β of the vinyl nitro substituent and protein cysteine thiolates, thereby modulating adaptive gene
expression and enzymatic activities (16). For example, NO2-FA have been shown to activate Nrf2,
PPARγ, heat shock factor-1 dependent gene expression, inhibit NF-kB-dependent pro-inflammatory
3

gene expression and directly inhibit enzymes such as soluble epoxide hydrolase and xanthine
oxidoreductase (17-22).
Despite advances in understanding NO2-FA pharmacology and encouraging preclinical and clinical
responses, a complete pharmacokinetic profile is still missing, due to challenges associated with the
chemical reactivity and complex metabolic pathways of these mediators. For example, the
quantitative analysis and characterization of NO2-FA metabolism are complicated by reversible
reactions with low molecular weight and protein thiols (23, 24), esterification into complex lipids (25,
26), and inherent instability of the nitroalkene under conditions of enzymatic and base hydrolysis,
greatly limiting previous PK evaluations.
Recent studies have shed new light on the ADME of NO2-FA. Radiotracer analysis of orally
administered [14C] 10-NO2-OA in rats has shown a long-lasting accumulation of radioactivity in
adipose tissue (27, 28). In addition, NO2-FA-containing triacylglycerides (NO2-FA-TAG) have been
characterized in adipocytes and rat plasma after 10-NO2-OA administration (25). A better
understanding of NO2-FA distribution and metabolism in supplemented adipocytes and rat adipose
tissue was provided by the quantification of both the free acid and esterified 10-NO2-OA (28). These
studies confirmed that adipose tissue differentially stores electrophilic NO2-FA and their non-
electrophilic metabolites into complex lipids, a pool that is mobilized by lipase hydrolysis, to be
distributed to remote organs.
Dietary fatty acids are normally absorbed by intestinal enterocytes, esterified into TAG, packaged into
chylomicrons and secreted into the mesenteric lymph duct, to then reach the systemic circulation and
the peripheral tissues (29). The incorporation of lipophilic drugs into chylomicron TAG and their
lymphatic distribution protects from first-pass hepatic metabolism and can increase oral
bioavailability (30). In this regard, a clear understanding of the systemic distribution of NO2-FA is
lacking and relative plasma free versus esterified NO2-FA levels after oral supplementation has yet to
be defined.
4

Herein, we provide new insight into the PK of an exemplary lipid electrophile, 10-NO2-OA. We
reveal that the systemic distribution and targeted delivery of orally-administered NO2-FA occur as
complex lipid esterified species, following esterification and stabilization in triglycerides.
MATERIALS AND METHODS
Synthesis and spectrophotometric quantitation of (E)-10-nitro-octadec-9-enoic acid (10-NO2-OA),
(Z)-10-nitro-octadec-9-enoic acid ((Z)-10-NO2-OA) and the internal standard (E)-10-nitro[15N]-
octadec-9-enoic-15,15,16,16-[d4] acid ([15N]O2-[d4]OA) were performed as previously (27, 31, 32).
Synthesis of the labeled internal standard (E)-10-nitro[15N]-octadecanoic-15,15,16,16-[d4] acid
([15N]O2-[d4]SA) was performed by reducing [15N]O2-[d4]OA with sodium borohydride.
Recombinant human FLAG-tagged PtGR-1 was expressed in HEK-293T cells and purified as
described previously (33). All chemicals were purchased from Sigma (St. Louis, MO) unless
otherwise stated. Solvents used for extractions and mass spectrometric analyses were from Burdick
and Jackson (Muskegon, MI).
Animal study
Male Beagle dogs (7 months old, n=5 per group) were orally administered with 31.25 mg/kg 10-NO2-
OA (dissolved in sesame oil) twice a day, about 6 hr apart, or with vehicle (sesame oil) for 14 days.
Blood samples were collected at 0, 1, 4, 7, 10, 16, 24 hr at 1 and 14 days, centrifuged, and the
resultant plasma was stored at -80 ºC until used. Animal studies were performed in accordance with
the Guide for the Care and Use of Laboratory Animals published by the United States National
Institutes of Health (NIH Publication No.85-23, revised 1996).
5

Synthesis of 10-NO2-(t8,9)-18:1 standard
Synthesis of 10-nitro-octadec-trans-8-enoic acid (10-NO2-(t8,9)-18:1) was carried out charging a 25
mL round bottom flask with 9-acetoxy-10-nitrooctadecanoic ester (193 mg) (32), cesium carbonate
(73 mg, one-half equivalent) and benzene (10-15 ml). The solution was refluxed under nitrogen with
vigorous stirring under a Dean-Stark trap for azeotropic removal of water, 24 hr, at 80-90 ºC. After
completion, the solution was cooled to room temperature, partitioned with 5-10 ml 1 M hydrochloric
acid and 10 ml diethyl ether. After stirring the mixture was separated and the aqueous fraction
extracted three times with 10 ml aliquots of diethyl ether. The organic layers were combined and
washed once with water, once with saturated aqueous sodium chloride, and then dried over sodium
sulfate. The solution was filtered through a plug of silica gel/Celite and the solvent removed under
reduced pressure. The crude product was re-dissolved in 5 ml phosphate buffer/5ml methanol with
393 mg L-Cysteine (ten-fold excess) and stirred for 2 hr to remove nitroalkene-cysteine adducts, then
quenched with a few drops of acetic acid and extracted three times with ethyl acetate. The organic
layers were combined and washed twice with water, twice with saturated aqueous sodium chloride,
and dried over sodium sulfate. The final solution was evaporated under reduced pressure then
applied to a flash chromatography column (ethyl acetate-hexanes 0 to 5%) to yield 96 mg of 10-NO2-
1
(t8,9)-18:1 after de-esterification by standard techniques. Characteristic H NMR signals were
observed at δ 4.78 (q, J = 7.7 Hz), 5.54 (dd, J = 15.3, 9.0 Hz), and 5.77 (dt, J = 15.3, 6.7 Hz) ppm.
Synthesis of NO2-FA-containing TAG standards
Synthesis of NO2-FA-TAG was performed by esterification of the appropriate NO2-FA with 1,2-
dipalmitin. In a typical reaction, 66 mg of 10-NO2-OA (0.2 mmol) were charged to a vial along with
50 mg DCC (N,N’-dicyclohexylcarbodiimide, 0.24 mmol) and a catalytic amount (5 mg) of DMAP
(4-dimethylaminopyridine), then dissolved in CH2Cl2. The solution was stirred briefly before 114 mg
1,2-dipalmitin (0.2 mmol) was added and the solvent volume adjusted to 10 ml. The vial was sealed
under nitrogen and stirred overnight at room temperature. The final TAG was obtained by filtering
6

off the solids, removing the solvent by rotary evaporation and purifying the crude product via column
1
chromatography (82 mg, 48% yield). Products were analyzed by H-NMR and HPLC-HR-MS/MS
for structural confirmation as previously (25).
Plasma lipid processing
Analysis of non-esterified 10-NO2-OA and metabolites was performed by adding 250 µl acetonitrile
to 50 µl of plasma in the presence of 20 pmol [15N]O2-[d4]OA and [15N]O2-[d4]SA. Samples were
vortexed, centrifuged at 20,000 g for 10 min at 4 ºC, and the supernatant was analyzed. TAG were
extracted from 20 µl of plasma using the method of Bligh and Dyer method (34), dried under a stream
of nitrogen and reconstituted in 100 µl ethyl acetate.
β-mercaptoethanol reaction with 10-NO2-OA and metabolites
To confirm the electrophilic character of NO2-OA-derived metabolites, acetonitrile-precipitated
plasma samples (50 µl) were dried and reacted with β-mercaptoethanol (BME) by adding 100 µl
phosphate buffer 50 mM pH 7.4/ acetonitrile (9:1, v/v) in the presence of 500 mM BME followed by
90 min incubation at 37 ºC. Also, 10-NO2-OA, 10-NO2-(t8,9)-18:1, (Z)-10-NO2-OA and 10-NO2-SA
standards (20 pmol) were spiked with 10 pmol [15N]O2-[d4]OA and [15N]O2-[d4]SA mix of internal
standards and reaction with BME was performed as above but with an incubation time of 15 min.
Samples were analyzed by HPLC-ESI-MS/MS.
Acid hydrolysis of plasma
Plasma (20 µl) was spiked with 10 pmol [15N]O2-[d4]OA and [15N]O2-[d4]SA and acid hydrolysis was
performed with minor modifications as previously (28). Samples were incubated with 1 ml
acetonitrile/HCl (9:1, v/v) at room temperature to obtain the free acid levels of NO2-FA (before
hydrolysis condition) and 90 ºC for 1 hr for the total levels of NO2-FA (after hydrolysis condition).
7

After incubation, 1 ml 0.5 M phosphate buffer was added and extracted with 2 ml hexane. Then,
samples were vortexed, centrifuged at 1000 g for 5 min at 4 ºC, and the hexane phase recovered and
dried under a stream of nitrogen. Finally, NO2-FA were reconstituted in methanol for HPLC-MS/MS
analysis. The esterified level of NO2-FA were obtained by subtracting the free acid levels
(acetonitrile extracts, before hydrolysis) to the total levels (after hydrolysis condition).
Stability and recovery of synthetic 10-NO2-OA, 10-NO2-SA, 10-NO2-(t8,9)-18:1, (Z)-10-NO2-OA,
10-NO2-OA-containing TAG (10-NO2-OA-TAG), and 10-NO2-SA-containing TAG (10-NO2-SA-
TAG) standards (30 pmol) were assessed in presence of 0.5 mg triolein under the same acidic
hydrolysis conditions as above, with the only difference that the internal standard mixture was added
before the extraction with hexane. Relative levels before and after hydrolysis conditions were
reported as area ratio with the internal standards [15N]O2-[d4]OA and [15N]O2-[d4]SA, respectively
(Supplementary Fig. 3A-E). The extent of hydrolysis of the NO2-FA-containing-TAG standards (10-
NO2-OA-TAG and 10-NO2-SA-TAG) was further confirmed by TLC and iodine staining.
Prostaglandin reductase-1 activity analysis
The concentrations of (E)-10-NO2-OA and (Z)-10-NO2-OA were determined by UV-Vis spectrometry
by using the same extinction coefficient of 7.5 mM-1cm-1 at λ=257 nm in methanol. The
concentration of 10-NO2-(t8,9)-18:1 was adjusted by LC-MS/MS analysis using (E)-10-NO2-OA and
(Z)-10-NO2-OA standards as reference values. NADPH concentration in phosphate buffer was
calculated using an extinction coefficient (ε340) of 6.22 mM-1cm-1. Then, (E)-10-NO2-OA, (Z)-10-
NO2-OA and 10-NO2-(t8,9)-18:1 (500 nM) were incubated at room temperature with 30 ng PtGR-1
with 250 µM NADPH in 10 mM sodium phosphate buffer pH 7.0 supplemented with 100 µM DTPA
and 100 nM internal standard [15N]O2-[d4]SA. Aliquots were collected at 0, 1, 5, 15, 30 and 60 min,
and reactions were stopped by 10-fold dilution in methanol, and 10-NO2-SA formation and 10-NO2-
OA isomer consumption were quantified by HPLC-ESI-MS/MS.
8

Distribution of 10-NO2-OA among plasma lipid classes
Lipids were extracted from 100 µl of plasma using the Bligh and Dyer method chloroform phase dried
under a stream of nitrogen and dissolved in 0.5 ml hexane/methyl tert-butyl ether/acetic acid (HBA,
100:3:0.3 v/v/v). Lipid classes were chromatographically separated using solid phase extraction
Strata NH2 columns (100 mg/1 ml), preconditioned with 2 ml acetone/water (7:1, v/v) and
equilibrated with 2 ml hexane. The lipid extracts solubilized in HBA were loaded on the columns and
cholesterol ester (CE), TAG, diglyceride+monoglyceride (MAG+DAG), free fatty acid (FFA), and
phospholipid (PL) fractions were sequentially eluted with 1 ml of hexane, hexane/chloroform/ethyl
acetate (100:5:5, v/v/v), chloroform/2-propanol (2:1, v/v), diethylether/ 2% acetic acid, and methanol
respectively. For analysis, the TAG and MAG+DAG fractions were pooled together since some NO2-
FA-TAG eluted in the latter fraction. The lipid fractions were dried under a flow of N2, dissolved in
1.8 ml acetonitrile spiked with 40 pmol mix of labeled [15N]O2-[d4]OA and [15N]O2-[d4]SA, and
vortexed. Then, 0.9 ml were left at room temperature (free acid levels) and 0.9 ml were incubated at
90 ºC for 1 hr in presence of 100 µl HCl (total levels) and processed as above. Finally, 10-NO2-OA
and its main metabolites were quantified by HPLC-MS/MS.
HPLC-MS/MS analysis
Identification of NO2-FA and their BME adducts was performed by HPLC-ESI-MS/MS using an
analytical C18 Luna column (2 × 100 mm, 5 μm, Phenomenex) at 0.65 ml/min flow rate, with a
gradient solvent system consisting of water containing 0.1 % acetic acid (solvent A) and acetonitrile
containing 0.1 % acetic acid (solvent B). Samples were chromatographically resolved using the
following gradient program: 45-100 % solvent B (0-8 min); 100 % solvent B (8-10 min) followed by
2 min re-equilibration at initial conditions. Detection of 10-NO2-OA-glycerol was performed using a
C18 Luna column (2 × 20 mm, 5 μm, Phenomenex) at 0.7 ml/min flow rate with the same solvents
than above and the following gradient: 30-100 % solvent B (0-3 min); 100 % solvent B (3-4 min)
followed by 1 min re-equilibration at initial conditions.
9

To improve chromatographic resolution of 10-NO2-OA isomers, a Polaris amide-C18 column (2.1 x
150 mm, 3 µm, Agilent) was used with a 0.52 ml/min flow rate, the above gradient system and the
following gradient program: 65-84.2 % solvent B (0-9 min); 84.2 % solvent B (9-12 min) followed by
3 min re-equilibration at initial conditions. A QTRAP 6500+ triple quadrupole mass spectrometer
(Sciex, Framingham, MA) was used with the following parameters: declustering potential (DP) –50
V, entrance potential (EP) and collision cell exit potential (CXP) –5 V, and source temperature of 650
ºC. 10-NO2-OA and its isomers, 10-NO2-SA, [15N]O2-[d4]OA, and [15N]O2-[d4]SA were quantified in
negative ion mode (collision energy (CE) –42 eV) using the following MRM 326.2/46, 328.2/46,
331.2/47, 333.2/47 transitions. The analysis of BME adducts was performed in negative ion mode
with CE –15 eV, following the neutral loss of BME (MRM 404.2/326.2, and 409.2/331.2,
respectively). Quantitation of plasma NO2-FA was performed by stable isotopic dilution analysis
using 10-NO2-OA and 10-NO2-SA calibration curves in the presence of the [15N]O2-[d4]OA and
[15N]O2-[d4]SA internal standards, respectively. The analysis of 10-NO2-OA-glycerol was performed
in positive ion mode with CE 15, 25, 20, 25 eV, for the following MRM transitions 402.3/384.2,
402.3/328.2, 402.3/310.2, 402.3/292.2 respectively.
High-resolution mass spectrometry analysis of non-esterified 10-NO2-OA species was performed on a
Q-Exactive hybrid quadrupole-orbitrap mass spectrometer (Thermo Scientific) equipped with a
Vanquish (Thermo Scientific) UHPLC and a HESI II electrospray source and operated in negative ion
mode using the following parameters: auxiliary gas heater temperature 325 °C, capillary temperature
300 °C, sheath gas flow 45, auxiliary gas flow 15, sweep gas flow 2, spray voltage 4 kV, S-lens RF
level 60 (%). PRM (Parallel Reaction Monitoring) method following m/z 326.2337 was used for
identification and characterization.
Analysis of 10-NO2-OA-TAG was performed by HPLC-HR-MS/MS using a C18 Luna column (2 x
150 mm, 3 µm, Phenomenex) at a flow rate of 0.4 ml/min with a post-column infusion of 50 µl/min of
10 % ammonium acetate in acetonitrile (10 mM final). The gradient solvent system consisted of 10%
water in acetonitrile (solvent A) and ethyl acetate (solvent B). 10-NO2-OA-TAG were eluted using
the following gradient: 35-90 % solvent B (0-10 min); 90 % solvent B (10-13 min) to then reach the
10

initial conditions in 0.5 min and re-equilibrate for an additional 1.5 min. A Q-Exactive hybrid
quadrupole-Orbitrap mass spectrometer (Thermo Scientific) equipped with a HESI II electrospray
source was used to characterize 10-NO2-OA-TAG in positive mode. The following parameters were
used: auxiliary gas heater temperature 250 °C, capillary temperature 300 °C, sheath gas flow rate 20,
auxiliary gas flow rate 20, sweep gas flow rate 0, spray voltage 4 kV. Full mass scan analysis ranged
+
from 780 to 1000 m/z at 17500 resolution. Odd mass ions, corresponding to NH4 adducts of
molecules containing an additional odd number of nitrogen atoms, were selected and subjected to MS2
fragmentation (composition confirmed at the < 5 ppm level). A manufacturer’s recommended
calibration solution was used to calibrate the instrument.
Statistical analysis
Values are expressed as means ± standard deviation or error and two-way ANOVA plus Sidak’s
multiple comparison tests were used for statistical significance (P < 0.05).
RESULTS
Structural characterization of 10-NO2-OA-TAG
The TAG esterification of NO2-FA upon intestinal absorption could have a significant impact on NO2-
FA metabolism and distribution. To evaluate the formation of NO2-FA-containing TAG in vivo,
plasma extracts from dogs orally dosed with vehicle or 10-NO2-OA were analyzed by HPLC-HR-
MS/MS. The full MS chromatographic profiles (780-1000 m/z range) of samples from 10-NO2-OA
treated dogs presented a region with unique peaks between 6 and 7 min (Fig. 1A) that were absent in
non-treated animals (not shown), while common peaks corresponding to TAG were detected in the 7
min to 9 min region. Analytes showing RT 6-7 min were characterized as having odd m/z values,
contrasting the later eluting TAG that displayed even m/z values. As an example, MS analysis of peak
a (RT 6.5 min) showed the presence of two odd mass ions (m/z 919.77 and 945.78), which
corresponded to ammoniated nitro-fatty acid triglycerides [NO2-FA-TAG+NH4]+ with a total number
11

of carbons and double bonds of 52:3 and 54:4 respectively (Fig. 1B). In contrast, MS analysis of
peak-b, in the common region, at 7.4 min showed two even [TAG+NH4]+ mass ions m/z 872.70 and
898.78, consistent with 52:4 and 54:5 structures, respectively (Fig. 1B).
To better characterize NO2-FA-TAG present in plasma samples, a synthetic 16:0/10-NO2-OA/16:0-
TAG was synthesized and analyzed (Fig. 2A). It presented an RT of 6.9 min and an odd m/z value of
895.77. The MS2 analysis produced two even-mass fragments (m/z 622.5, containing 1 nitrogen
atom) and one odd-numbered fragment (m/z 551.5), which corresponded to the neutral losses of
palmitic acid and ammonia [M-16:0-NH3]+ and 10-NO2-OA and ammonia [M-10-NO2-OA-NH3]+,
respectively. Based on the information gathered with this standard, the odd species eluting in the 6-7
min region were further characterized. This analysis consisted of elemental composition verification
(at the 2 ppm level) and MS2 fragmentation (Table 1). As an example, the characterization of the odd
mass ion m/z 943.76 is shown (Fig. 2B) (25). This species eluted at RT 6 min and displayed an
elemental composition and isotopic distribution consistent with a NO2-FA-TAG. MS2 fragmentation
analysis showed the presence of three “diglyceride ions”: two even mass of m/z 646.50, which
corresponded to the neutral losses of linoleic acid and ammonia [M-18:2-NH3]+, and one with odd
mass of m/z 599.50, resulting from the neutral loss of 10-NO2-OA and ammonia [M-10-NO2-OA-
NH3]+. When this analytical approach was used on the remaining TAG species presenting a NO2
group, the majority of plasma NO2-FA-TAG were identified as 10-NO2-OA-TAG. The sn-1/sn-3 and
sn-2 fatty acid position on the glycerol backbone of this species was determined by comparing the ion
intensity of the MS2 fragments. This showed mono- and polyunsaturated fatty acids predominantly in
sn-1/sn-3 position and 10-NO2-OA in the sn-2 position (35-38). On the basis of the relative peak area,
18:1/10-NO2-OA/18:2-TAG was the most abundant species with an odd mass of m/z 945.77.
Assessment of the electrophilic character of 10-NO2-OA metabolites
The electrophilic nature of the vinyl nitro substituent of NO2-FA promotes Michael addition with
nucleophiles such as thiol-containing proteins and glutathione (GSH) (23). Plasma samples from 10-
NO2-OA treated dogs were analyzed before and after reaction with β-mercaptoethanol (BME) to
12

identify electrophilic versus non-electrophilic species (35). In this regard, the chromatogram for the
transition 326.2/46 before BME treatment (Fig. 3A left panel), showed a peak at 7.1 min, which co-
eluted with the synthetic 10-NO2-OA standard (Fig. 3B left panel), and a later peak at 7.3 min,
identified as (Z)-10-NO2-OA, its geometric isomer. As expected, the presence of deuterium in the
internal standard [15N]O2-[d4]OA (MRM 331.2/47) reduced binding to the stationary phase (36),
slightly decreasing its retention time (Fig. 3C left panel). After BME derivatization, the
corresponding BME-adducts for 10-NO2-OA and [15N]O2-[d4]OA (MRM 404.2/326.2 and
409.2/331.2) were detected at retention time (RT) 6 min, and complete loss of underivatized 10-NO2-
OA was observed (Fig. 3 B,C right panels). A peak for MRM transitions (326.2/46 and 331.7/47) was
obtained at the same retention time of the 10-NO2-OA-BME and [15N]O2-[d4]OA-BME adducts
respectively, corresponding to in-source β-elimination of BME from the adducts. When the
electrophilicity of plasma metabolites of 10-NO2-OA following 14 days of dosing was assessed, an
unreactive peak was detectable with a 326.2/46 MRM transition and a retention time (RT 7.1 min)
reflective of 10-NO2-OA (Fig. 3A right panel). Composition and fragmentation analysis by HR-
MS/MS confirmed the presence of a double bond, a nitro group on an 18-carbon fatty acyl chain
(Supplementary Fig. 1). This species did not display specific fragments corresponding to the presence
of a nitroalkene, thus a structure corresponding to a NO2-OA with a double bond migration to the 8-9
carbon was proposed.
Synthesis and characterization of 10-NO2-OA-derived isomers
To confirm the initial structural assignment of the newly detected species in plasma, a 10-NO2-(t8,9)-
18:1 standard was synthesized and characterized as described in Material and Methods. HPLC-
MS/MS analysis of (E)-10-NO2-OA, (Z)-10-NO2-OA, and 10-NO2-(t8,9)-18:1 standards (MRM
326.2/46), using a C18 reverse phase column, showed poor chromatographic resolution of the peaks
(Fig. 4 left panels) with the exception of (Z)-10-NO2-OA. Under these conditions, the (Z)-10-NO2-
OA standard showed an RT of 7.88 min, while the 10-NO2-(t8,9)-18:1 and (E)-10-NO2-OA standards
almost overlapped with RT of 7.71 min and 7.73 min, respectively (Fig. 4 A-D left panels). To
13

improve the chromatographic resolution of these isomers, a new method using a C18-amide reverse
phase column was developed that resolved all three isomers ((Z)-10-NO2-OA RT 6.25 min, 10-NO2-
(t8,9)-18:1 RT 5.75 min, and (E)-10-NO2-OA RT 5.96 min)) (Fig. 4 A-D right panels). The structural
assignment was further confirmed by comparing RT, HR-MS, MS2 fragmentation, and electrophilic
character between the standard and the plasma samples (Supplementary Fig. 1 and 2). The
electrophilic (Z)-10-NO2-OA and (E)-10-NO2-OA standards generated their corresponding BME
adducts, while 10-NO2-(t8,9)-18:1 was unreactive and no adduct formation was observed, paralleling
the observation made in plasma samples.
Enzymatic nitroalkene reduction
The appearance of 10-NO2-(t8,9)-18:1 and (Z)-10-NO2-OA isomers motivated their evaluation as
substrates of prostaglandin reductase-1 (PtGR-1) to form 10-NO2-SA, as this is the main metabolic
route for (E)-10-NO2-OA inactivation (33) (Fig. 5). (Z)-10-NO2-OA was a poor substrate and
underwent slow reduction to 10-NO2-SA in comparison with (E)-10-NO2-OA (Fig. 5A,B). As
predicted from the absence of a conjugated nitroalkene moiety, 10-NO2-(t8,9)-18:1 was not a
substrate for PtGR-1, yielded no 10-NO2-SA and was not consumed (Fig. 5A,B).
Stability of 10-NO2-OA isomers under acidic conditions
NO2-OA is unstable to hydrolysis conditions, given the reactivity of the nitroalkene group and the
possibility that changes in pH, organic solvent, temperature and reactions with nucleophiles may
induce (E)- to (Z)-nitroalkene isomerization (32, 37). Thus, isomerization of (E)-10-NO2-OA and (Z)-
10-NO2-OA was assessed under basal, acidic and acid plus temperature (hydrolysis) conditions. The
proportion of (Z)-10-NO2-OA in (E)-10-NO2-OA stock solution increased from (1.7% basal) to 4.7%
after addition of acidified acetonitrile and increased further to 8.3% at 90 ºC (Fig. 6A). This suggests
that the apparent 20% loss of (E)-10-NO2-OA after acidic hydrolysis could be in part attributable to
(E) to (Z) isomerization (Supplementary Fig. 3A). To further assess the stability of (Z)-10-NO2-OA
14

and confirm an acid-catalyzed re-equilibration between E and Z isomers, acidified (E)-10-NO2-OA
and (Z)-10-NO2-OA mixtures (100/0, 75/25, 50/50, 25/75, 0/100, v/v) were analyzed (Fig. 6B).
Regardless of the initial relative E and Z isomer composition, standards under acidic conditions
reached a new equilibrium characterized by ~10 % (Z)-10-NO2-OA (Fig. 6B,C).
The stability of 10-NO2-SA and 10-NO2-(t8,9)-18:1 was also assessed under the same conditions. 10-
NO2-SA was stable, while 10-NO2-(t8,9)-18:1 showed ~ 20% loss at 90 ºC to a product that was not
further characterized (Supplementary Fig. 3B,C).
Plasma metabolites after oral administration of 10-NO2-OA.
The PK of free plasma 10-NO2-OA and that esterified in complex lipids was evaluated after both 1
and 14 days of oral administration of 62.5 mg/kg/day of 10-NO2-OA (2 doses of 31.25 mg/kg given 6
hr apart daily). Notably, (E)-10-NO2-OA was preferentially esterified (>95%). (E)-10-NO2-OA
reached a maximum concentration (Cmax) of 20 ± 6.9 µM at 1 hr, followed by a 7.4-fold decrease at 4
hr (Fig. 7A), with levels doubling after the second daily administration to 5 ± 1.5 µM at 7 hr. In
contrast, un-esterified levels of 10-NO2-SA were significantly greater than 10-NO2-OA, both on day 1
and 14, while esterified levels were lower. After 14 days of treatment, un-esterified (E)-10-NO2-OA
levels were almost unchanged in comparison with day 1, while the esterified levels decreased ~ 4
fold, a behavior that was not evident for 10-NO2-SA (Fig. 7E).
Similar to the distribution of (E)-10-NO2-OA, (Z)-10-NO2-OA and 10-NO2-(t8,9)-18:1 were esterified
to a greater extent compared with the free acid and were ~10- and up to 100-times less abundant than
(E)-10-NO2-OA, respectively. The free acid 10-NO2-(t8,9)-18:1 displayed sustained levels at day 14
with little variation throughout the time course with an average concentration of ~ 0.06 µM.
In contrast to (E)-10-NO2-OA, its main metabolite 10-NO2-SA was predominantly non-esterified and
was the most abundant metabolite detected. Its levels accumulated in plasma from 1 to 16 hr with an
average of ~ 4 µM, then decreased to 0.9 ± 0.17 µM at 24 hr, displaying a similar profile after 14 days
(Fig. 7H). Triglyceride-esterified 10-NO2-SA levels showed a pharmacokinetic profile that was
15

similar to that of (E)-10-NO2-OA, with a 24 hr Cmax of 1.62 ± 0.7 µM, 10-fold lower than the Cmax of
esterified (E)-10-NO2-OA. Finally, esterified 10-NO2-SA concentrations slightly increased after 14
days (Cmax of 2.68 ± 0.3 µM).
Bio-distribution and metabolism of 10-NO2-OA in plasma lipids
The distribution of (E)-10-NO2-OA among different complex lipid classes was then determined. In
agreement with the observations of the PK study at 1 hr of day 1 and 14 (Fig. 7 A,E), (E)-10-NO2-OA
was preferentially detected in esterified forms, predominantly in TAG fraction with only minor
esterification in phospholipids (PL) (Fig. 8 A,C). Surprisingly, the total levels of (E)-10-NO2-OA
increased in the FFA fraction upon hydrolysis, indicating the contribution of an esterified species
present in this fraction. This species was identified as 10-NO2-OA-glycerol and was only detected in
FFA fractions by HPLC-MS/MS, using multiple MRM transitions (m/z 402.3/384.2, 402.3/328.2,
402.3/310.2, 402.3/292.2), transitions that correspond to the parent even mass ion [10-NO2-OA-
glycerol+H]+ and the corresponding even fragment ions [10-NO2-OA-glycerol+H-water]+, [10-NO2-
OA+H]+, [(10-NO2-OA)CO]+, [(10-NO2-OA)CO-water]+ respectively (Supplementary Fig. 4A,B).
Finally, the distribution of 10-NO2-SA, the main metabolite of (E)-10-NO2-OA, was greater in TAG
than CE and PL fractions, and comparable with the free nitroalkane levels in the FFA fraction (Fig. 8
B,C).
DISCUSSION
Fatty acid nitroalkenes will display a complex pharmacokinetic profile for multiple reasons. These
electrophilic species have a partial positive charge on the β carbon of the vinyl nitro substituent,
which supports reversible Michael addition reactions with nucleophilic thiols of GSH and cysteine-
containing proteins (16). By altering the structure and function of multiple thiol-containing
transcriptional regulatory proteins such as NF-kB p65 subunit, Keap1, stimulator of interferon genes
(STING) and PPAR-γ, NO2-FA modulate the expression of more than 300 genes crucial for
16

cytoprotective, metabolic and anti-inflammatory responses (38-40). In addition to the electrophilic
character of 10-NO2-OA, its PK is also influenced by a) esterification into phospholipids and TAG
(25, 41), b) inactivation upon prostaglandin reductase-1 reduction to the non-electrophilic nitroalkane
10-NO2-SA (33), c) β- and ω-oxidation reactions to yield shorter chain length dicarboxylate products
(27, 42), and d) isomerization to the corresponding geometric (Z)-isomer (43).
10-NO2-OA is undergoing Phase II clinical trials for the treatment of chronic pulmonary and renal
diseases, and the present study was designed to improve our understanding of the PK of this signaling
mediator and new drug candidate. We show: 1) TAG esterification as the primary mechanism of
systemic 10-NO2-OA distribution and 2) the detection and characterization of in vivo 10-NO2-OA
isomerization.
TAG esterification strongly influences the PK of 10-NO2-OA. For example, the whole body
distribution of radiolabeled [14C] 10-NO2-OA after oral administration in rats showed rapid
distribution to tissues that typically rely on fatty acid oxidation to supply energetic demand (28, 44).
Also, significant accumulation into adipose tissue was observed over two weeks, an observation
confirmed by lipidomic analysis (28). With improved analytical methods, we now show that in
orally-dosed dogs circulating plasma TAG contained high levels of 10-NO2-OA. This
compartmentalization can limit electrophilic reactions with nucleophiles and PtGR-1 and may ensure
better PK and safety profiles of lipid electrophiles, species that have typically been viewed as toxic.
The preferential distribution of plasma 10-NO2-OA in TAG (10-NO2-OA-TAG) (Fig. 1), as opposed
to free plasma (E)-10-NO2-OA levels (Fig. 7,8), suggest an incorporation into chylomicron TAG and
lymphatic delivery into the systemic circulation via the subclavian vein. In contrast, non-esterified
NO2-OA levels may be a consequence of absorption through the portal system and albumin dependent
transport. Importantly, absorption through the lymphatic system avoids first pass through the liver,
greatly reducing initial metabolism by phase I and II enzymes, thus enhancing oral bioavailability of
this reactive lipid electrophile (30). Also, after 14 days, a decrease of esterified (E)-10-NO2-OA
levels was observed when compared to day 1 (Fig. 7A,E), an effect that might be related to the
17

relatively high daily dose and have little or no effect at lower, more clinically-relevant doses. Further
studies are needed to assess if this is the case.
The preferential esterification of 10-NO2-OA in the sn2 position of TAG (Fig. 2 and Table 1) could
influence the species being delivered upon LPL hydrolysis in peripheral tissues. This enzyme
preferentially hydrolyzes TAG at sn-1 and sn-3 positions to generate sn-2 mono-glycerides (45)
(Supplementary Fig. 4). As a consequence, 10-NO2-OA-monoglycerides can be directly taken up by
tissues, could alter hepatic lipoprotein metabolism and phospholipid synthesis (46), and may induce
new signaling activities on the basis of its structural resemblance with the endocannabinoid 2-
arachidonylglycerol. Moreover, the detection of 10-NO2-OA-glycerol in plasma may support further
the role of LPL in tissue distribution.
The detection and characterization of geometrical and structural 10-NO2-OA isomers is now reported
in vivo. Previously, the profiling of nitro-oleic acid isomers had only been reported after biomimetic
nitrogen dioxide (•NO2) nitration of oleic acid, followed by pentafluorobenzyl
bromide/diisopropylethylamine (PFBBr/DIPEA) derivatization and GC/MS analysis (47). The lack of
reactivity of NO2-OA metabolites with BME now reveals the presence of 10-NO2-(t8,9)-18:1. This
isomer did not resolve by C18 reverse phase chromatography, thus a new rapid chromatographic
method with an amide-C18 column was developed to resolve and quantitate the formation of different
10-NO2-OA regioisomers (Fig. 4).
The mechanism involved in the in vivo isomerization of 10-NO2-OA into 10-NO2-(t8,9)-18:1 may be
a consequence of direct base-induced rearrangement, or related to the reversible formation of Michael
adducts (23, 48) (Scheme 1). These are addition products that preferentially regenerate the
nitroalkene group upon reversal. The first step in retro-Michael elimination is deprotonation of the α-
carbon, followed by the β-elimination of the thiolate group (Scheme 1 upper insert). However, base
catalyzed deprotonation of the significantly higher pKa γ-carbon followed by β-elimination would
result in the formation of 10-NO2-(t8,9)-18:1. This pathway may be more accessible in conditions
with conformational restrictions for elimination. Deprotonation at the γ-position could be spatially
favored in certain protein adducts, where close basic residues could participate in deprotonation and
18

simultaneous stabilization of the deprotonated γ-carbon by charge pairing, leading to the formation of
non-electrophilic 10-NO2-(t8,9)-18:1 upon elimination. In this mechanism, the irreversibility of this
isomerization reaction could result in 10-NO2-(t8,9)-18:1 accumulation, despite it being less
thermodynamically favored. A direct base-catalyzed isomerization, conversely, would be easily
reversible and thus tend towards an equilibrium mixture.
As for 10-NO2-(t8,9)-18:1, thiol-addition reactions could also be responsible for the isomerization to
(Z)-10-NO2-OA. While (E)-nitroalkenes are thermodynamically more stable than (Z)-isomers,
isomerization can be promoted with organic solvents, heat, photochemical exposure and catalytic
nucleophile concentrations to reach equilibrium (37, 43). Since (Z)-10-NO2-OA showed ~90%
isomerization to (E)-10-NO2-OA under acidic hydrolysis conditions (Fig. 6B), plasma free 10-NO2-
OA isomer levels were assessed after acetonitrile extraction to exclude any impact of low pH (Fig. 7).
Interestingly, oral preparations of (E)-10-NO2-OA contained ~1.7% of (Z)-10-NO2-OA (Fig. 6A), a
level that increased to ~10 % in plasma (Fig. 7A,C), indicating an in vivo geometrical isomerization of
the supplemented (E)-nitroalkene. Importantly, caution must be taken in the evaluation of esterified
(Z)-10-NO2-OA levels since enzymatic, basic and acidic hydrolysis methods all include at least one
destabilizing factor that would directly impact its levels.
The electrophilic vinyl nitro group of monounsaturated, bis-allylic, and conjugated nitroalkenes (NO2-
OA, NO2-LA, and NO2-CLA, respectively) is reduced by prostaglandin reductase-1 (PtGR-1) to a
non-electrophilic nitroalkane (33). A comparison of 10-NO2-OA isomers showed that they are
differentially metabolized by PtGR-1, with (E)-10-NO2-OA being a better substrate than (Z)-10-NO2-
OA and no catalytic effect towards 10-NO2-(t8,9)-18:1 (Fig. 5 A,B). The resistance of (Z)-10-NO2-
OA to PtGR-1 reduction could reveal new avenues for the development of novel geometrical
therapeutic signaling mediators having improved PK in vivo.
In conclusion, we established esterification of 10-NO2-OA in the sn-2 position of TAG as a
mechanism that could limit hepatic first-pass inactivation of electrophilic nitroalkenes and thus
promotes tissue distribution of an otherwise chemically reactive species. This will improve oral
bioavailability 10-NO2-OA and influence organ and cellular distribution. While these findings
19

originate from the administration of 10-NO2-OA to dogs and we have observed a similar metabolism
among different species and humans, the anatomic sites of incorporation of 10-NO2-OA into TAG in
humans remains to be investigated. This is a priority, as it will inform about the absorption and
biodistribution of synthetic 10-NO2-OA and both NO2-FA and other lipid electrophiles formed in the
gastric compartment during digestion. Finally, the detection, characterization, new analytical
methods, and profiling of 10-NO2-(t8,9)-18:1 and (Z)-10-NO2-OA in vivo reveals new directions for
better understanding fatty acid nitration during metabolism and inflammation and the development of
synthetic homologs lipid signaling mediators having improved PK characteristics.
Acknowledgments- This work was financially supported by Ri.MED Foundation (MF), NIH grants
R01-HL058115, R01-HL64937, P30-DK072506, P01-HL103455 (BAF), R01-GM125944 and R01-
DK112854 (FJS) and AHA 17GRN33660955 (FJS) and K01-HL133331 (DAV).
Declaration of interest
FJS, BAF, and DAV acknowledge financial interest in Complexa, Inc.
20

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40. Hansen, A. L., G. J. Buchan, M. Ruhl, K. Mukai, S. R. Salvatore, E. Ogawa, S. D. Andersen, M.
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23

Diaglyceride
product ion
[NO2-FA-TAG + NH4]+
Relative Retention
Intensity
%
Chemical
formula
Experimental Theoretical
peak
area
%
time
mass
mass
[M-R COOH-NH3]+
(min)
n
sn-2
sn-1/3
NO2-18:1a
C
57H107N2O8
+
+
947.7988
648.5184
603.5342
947.7988
650.5345
646.5032
603.5342
947.7988
650.5345
648.5184
601.5184
945.7818
648.5191
646.5033
601.5192
943.7683
646.5027
599.5022
921.7844
648.5185
622.5037
577.5188
919.7674
646.5027
622.5036
575.5034
947.8022
648.5198
603.5347
947.8022
650.5345
646.5041
603.5347
947.8022
650.5345
648.5198
601.519
21.4
18:1 18:1
+
100
75
NO2-18:1/18:1
18:1/18:1
C39H70NO6
+
C
39H71O4
NO2-18:1a
C
57H107N2O8
18:0 18:2
+
35
50
75
NO2-18:1/18:0
C39H72NO6
C39H68NO6
+
6.91
NO2-18:1/18:2
18:0/18:2
+
C
39H71O4
NO2-18:0a
C
57H107N2O8
+
18:1 18:2
+
35
100
20
NO2-18:0/18:1
C39H72NO6
C39H70NO6
+
NO2-18:0/18:1
18:1/18:2
+
C
39H69O4
+
945.7865
648.5198
646.5041
601.519
29.3
NO2-18:1 18:1 18:2
C
57H105N2O8
+
90
100
60
NO2-18:1/18:1
C39H70NO6
C39H68NO6
6.5
6.05
6.96
+
NO2-18:1/18:2
18:1/18:2
+
C
39H69O4
+
943.7709
646.5041
599.5034
921.7865
648.5198
622.5041
577.519
20.8
13.2
NO2-18:1 18:2 18:2
NO2-18:1 18:1 16:0
C
57H103N2O8
+
100
35
NO2-18:1/18:2
18:2/18:2
C39H68NO6
+
C
39H67O4
+
C
55H105N2O8
+
85
100
60
NO2-18:1/18:1
C39H70NO6
C37H68NO6
+
NO2-18:1/16:0
18:1/16:0
+
C
37H69O4
+
919.7709
646.5041
622.5041
575.5034
15.3
NO2-18:1 18:2 16:0
C
55H103N2O8
+
80
100
65
NO2-18:1/18:2
C39H68NO6
C37H68NO6
6.53
+
NO2-18:1/16:0
18:2/16:0
+
C
37H67O4
a
Order of notation does not connote regiochemistry
Table 1. Mass spectrometry characterization of the most abundant plasmatic NO2-FA-
containing TAG.
24

A
B
919.77
[NO2-FA-TAG+NH4]+
TAG
Peak-b
NO2-FA-TAG
Peak-a
(NO2-52:3)
945.78
[NO2-FA-TAG+NH4]+
898.78
[TAG+NH4]+
(54:5)
(NO2-54:4)
872.7
[TAG+NH4]+
(52:4)
Peak-a
Peak-b
5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
Time (min)
780
800
820
840
860
880
900
m/z
920
940
960
980 1000
Fig. 1. HPLC-high-resolution-massspectrometrycharacterizationof plasmatriglycerides.
(A) Chromatographic separation of NO2-FA-containing TAG and triglycerides. (B) MS
spectraof peak-aandpeak-b.
25

A
B
895.7708
943.7683
C53H103O8N2 = 895.7709
-0.0673 ppm
C57H103O8N2 = 943.7709
-2.7469 ppm
+
+
O
NO2
O
O
ONH4
NO2
O
ONH4
O
O
O
O
O
O
[M+NH4]+
[M+NH4]+
16:0/10-NO2-OA/16:0-TAG
18:2/10-NO2-OA/18:2-TAG
894 896 898 900
m/z
942 944 946 948
m/z
[M-16:0-NH3]+
622.5034
[M-18:2-NH3]+
646.5027
C37H68O6N = 622.5041
-1.1560 ppm
C39H68O6N = 646.5041
-2.1337 ppm
[M-10-NO2-OA-NH3]+
551.5024
[M-10-NO2-OA-NH3]+
599.5022
C35H67O4 = 511.5034
-1.7855 ppm
C39H67O4 = 599.5034
-2.0248 ppm
500 600 700
m/z
5.0 5.5 6.0 6.5 7.0
Time (min)
550 600 650 700
5
6
7
8
9
m/z
Time (min)
Fig. 2. Mass spectrometry characterization of
10-NO2-OA-containing
triglycerides. Chromatographic profile, isotopic distribution (insert), representative
chemical structure and fragmentation analysis of: (A) 10-NO2-OA-TAG standard,
(B)10-NO2-OA-TAGinplasma.
26

A
MRM 404.2/326.2
MRM 326.2/46
MRM 326.2/46
B
C
MRM 404.2/326.2
MRM 326.2/46
MRM 326.2/46
MRM 409.2/331.2
MRM 331.2/47
MRM 331.2/47
5.5 6.0 6.5 7.0 7.5 8.0 8.5
Time (min)
5.5 6.0 6.5 7.0 7.5 8.0 8.5
Time (min)
Fig. 3. Identification of non-electrophilic 10-NO2-OAisomers in vivo after oral
administration. MRM chromatograms before (left panels) and after BME
reaction (right panels) of: (A) plasma after 14 days dosing, (B) 10-NO2-OA
standard, (C)I.S.[15N]O2-[d4]OA.
27

A
*
*
MRM 326.2/46
MRM 326.2/46
B
C
MRM 326.2/46
D
MRM 331.2/47
7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3
Time (min)
5.5
5.7
5.9
6.1
6.3
6.5
Time (min)
Fig. 4. Chromatography separation of 10-NO2-OA isomeric
products. Chromatographic profile of (A*) (Z)-10-NO2-OA, (B) 10-
NO2-(t8,9)18:1, (C) (E)-10-NO2-OA, (D) I.S. [15N]O2-[d4]OA, using a
C18 reverse phase column (left panels) and C18-amide reverse phase
column(rightpanels).
28

A
100
80
60
40
20
0
(E)-10-NO2-OA
(Z)-10-NO2-OA
10-NO2-(t8,9)18:1
0
20
40
60
Time (min)
B
100
80
60
40
20
0
(E)-10-NO2-OA
(Z)-10-NO2-OA
10-NO2-(t8,9)18:1
0
20
40
60
Time (min)
Fig. 5. Reduction of 10-NO2-OA isomers by human recombinant PtGR-1. Time-
dependent enzymatic (A) reduction and (B) consumption of (E)-10-NO2-OA, (Z)-
10-NO2-OA, and 10-NO2-(t8,9)18:1. Traces are provided for illustration purposes
and were obtained by fitting the data to exponential association and decay
equations. Data shown are the mean (n=4)
representativeoftwo independentexperiments.
Standard deviation and are
29

A
C
(E)-10-NO2-OA
(Z)-10-NO2-OA
O
100
80
60
40
20
0
NO2
OH
(E)-10-Nitro-octadec-9-enoic acid
(E)-10-NO -OA
2
O
OH
NO2
Initial
Before
hydrolysis hydrolysis
(Acid)
(90oC+Acid)
After
(Z)-10-Nitro-octadec-9-enoic acid
(Z)-10-NO2-OA
B
100
80
60
40
20
0
(E)-10-NO2-OA
(Z)-10-NO2-OA
Standard mixture
composition
Before
hydrolysis
(Acid)
After
hydrolysis
(90oC+Acid)
Initial
Fig. 6. (A) Acid-catalyzed isomerization of (E)- to (Z)-10-NO2-OA. (B)
Relative composition of isomer mixtures under acidic hydrolysis conditions.
(C) (E)/(Z) 10-NO -OA isomer equilibrium in presence of hydrochloric acid.
2
Data shown are the mean (n=3)
experimentsforAandB, respectively.
SE of three and two independent
30