Synthesis and molecular targets of N-13-hydroxy-octadienoyl-ethanolamine, a novel endogenous bioactive 15-lipoxygenase-derived metabolite of N-linoleoyl-ethanolamine found in the skin and saliva

Article abstract of DOI:10.1016/j.bbalip.2021.158954

N-Arachidonoyl-ethanolamine (AEA) is an endocannabinoid (eCB) and endogenous lipid mimicking many of the effects of Δ⁹-tetrahydrocannabinol, notably on brain functions, appetite, pain and inflammation. The eCBs and eCB-like compounds contain fatty acids, the main classes being the monoacylglycerols and the N-acyl-ethanolamines (NAEs). Thus, each long chain fatty acid likely exists under the form of a monoacylglycerol and NAE, as it is the case for arachidonic acid (AA) and linoleic acid (LA). Following their biosynthesis, AA and AEA can be further metabolized into additional eicosanoids, notably by the 15-lipoxygenase pathway. Thus, we postulated that NAEs possessing a 1Z,4Z-pentadiene motif, near their omega end, would be transformed into their 15-lipoxygenase metabolites. As a proof of concept, we investigated N-linoleoyl-ethanolamine (LAE). We successfully synthesized LEA and LEA-d₄ as well as their 15-lipoxygenase-derived derivatives, namely 13-hydroxy-9Z,11E-octadecadienoyl-N-ethanolamine (13-HODE-EA) and 13-HODE-EA-d₄, using Novozyme 435 immobilized on acrylic resin and soybean lipoxygenase respectively. We also show that both human 15-lipoxygenase-1 and -2 can biosynthesize 13-HODE-EA. Co-incubation of LEA and LA with either human 15-lipoxygenase led to the biosynthesis of 13-HODE-EA and 13-HODE in a ratio equal to or greater than 3:1, indicating that LEA is preferred to LA by these enzymes. Finally, we show that 13-HODE-EA is found in human saliva and skin and is a weak although selective TRPV1 agonist. The full biological importance of 13-HODE-EA remains to be explored.

Full text of DOI:10.1016/j.bbalip.2021.158954

BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158954
Contents lists available at ScienceDirect
BBA - Molecular and Cell Biology of Lipids
journal homepage: www.elsevier.com/locate/bbalip
Synthesis and molecular targets of
N-13-hydroxy-octadienoyl-ethanolamine, a novel endogenous bioactive
15-lipoxygenase-derived metabolite of N-linoleoyl-ethanolamine found in
the skin and saliva
,
,
e
a,e
Francesco Tintoa e, Anne-Sophie Archambaulta , Elizabeth Dumais
,
´
Volatiana Rakotoariveloa e, Magdalena Kostrzewab, Cyril Martina e, Pier-Luc Plantec,
,
,
c
f
f
b
b
´
Yves Desjardins , Melissa Simard , Roxane Pouliot , Luciano De Petrocellis , Alessia Ligresti ,
Vincenzo Di Marzoa
e, Nicolas Flamanda
,
b,
c,
d,
,
e
,
*
a
´
´
´
´
´
´
´
Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de Quebec, Departement de medecine, Faculte de medecine, Universite Laval, Quebec City,
QC G1V 4G5, Canada
b Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche (CNR), Pozzuoli, Italy
c
´
´
´
Institut sur la Nutrition et les Aliments Fonctionnels, Centre NUTRISS, Ecole de nutrition, Faculte des sciences de l’agriculture et de l’alimentation, Universite Laval,
´
Quebec City, Canada
d
´
Joint International Unit between the CNR of Italy and Universite Laval on Chemical and Biomolecular Research on the Microbiome and its Impact on Metabolic Health
and Nutrition (UMI-MicroMeNu), Italy
e
´
´
Canada Excellence Research Chair on the Microbiome-Endocannabinoidome Axis in Metabolic Health (CERC-MEND), Universite Laval, Quebec City, Canada
f
´
´
´ `
´
´
´
´ ´ ´
Faculte de pharmacie de l’Universite Laval, Centre de Recherche en Organogenese Experimentale de l’Universite Laval/LOEX, Axe medecine regeneratrice, Centre de
´
´
´
recherche du CHU de Quebec-Universite Laval, Quebec City, Canada
A R T I C L E I N F O
A B S T R A C T
Keywords:
N-Arachidonoyl-ethanolamine (AEA) is an endocannabinoid (eCB) and endogenous lipid mimicking many of the
effects of Δ9-tetrahydrocannabinol, notably on brain functions, appetite, pain and inflammation. The eCBs and
eCB-like compounds contain fatty acids, the main classes being the monoacylglycerols and the N-acyl-ethanol-
amines (NAEs). Thus, each long chain fatty acid likely exists under the form of a monoacylglycerol and NAE, as it
is the case for arachidonic acid (AA) and linoleic acid (LA). Following their biosynthesis, AA and AEA can be
further metabolized into additional eicosanoids, notably by the 15-lipoxygenase pathway. Thus, we postulated
that NAEs possessing a 1Z,4Z-pentadiene motif, near their omega end, would be transformed into their 15-lip-
oxygenase metabolites. As a proof of concept, we investigated N-linoleoyl-ethanolamine (LAE). We success-
fully synthesized LEA and LEA-d4 as well as their 15-lipoxygenase-derived derivatives, namely 13-hydroxy-
9Z,11E-octadecadienoyl-N-ethanolamine (13-HODE-EA) and 13-HODE-EA-d4, using Novozyme 435 immobilized
on acrylic resin and soybean lipoxygenase respectively. We also show that both human 15-lipoxygenase-1 and -2
can biosynthesize 13-HODE-EA. Co-incubation of LEA and LA with either human 15-lipoxygenase led to the
biosynthesis of 13-HODE-EA and 13-HODE in a ratio equal to or greater than 3:1, indicating that LEA is preferred
to LA by these enzymes. Finally, we show that 13-HODE-EA is found in human saliva and skin and is a weak
although selective TRPV1 agonist. The full biological importance of 13-HODE-EA remains to be explored.
Endocannabinoid
15-Lipoxygenase
Inflammation
ALOX15
ALOX15B
Linoleic acid
Abbreviations: 13-HODE, 13-hydroxyoctadecadienoic acid; 13-HODE-EA, N-13-HODE-ethanolamine; AEA, N-arachidonoyl-ethanolamine; EA, ethanolamine; LA,
linoleic acid; LEA, N-linoleoyl-ethanolamine; NAE, N-acyl-ethanolamine; TRPV1, transient receptor potential cation channel subfamily V member 1.
´
* Corresponding author at: Centre de recherche de l’IUCPQ, 2725 chemin Ste-Foy, Office A3140, Quebec City, QC G1V 4G5, Canada.
E-mail address: Nicolas.Flamand@criucpq.ulaval.ca (N. Flamand).
https://doi.org/10.1016/j.bbalip.2021.158954
Received 31 December 2020; Received in revised form 2 April 2021; Accepted 22 April 2021
Available online 27 April 2021
1388-1981/© 2021 Elsevier B.V. All rights reserved.

F. Tinto et al.
BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158954
1. Introduction
LEA-d4, we essentially performed the same steps, but we replaced
ethanolamine with ethanolamine-d4. This led to the synthesis of LEA-d4
(2) in good yield (white solid, 85% yield).
The endocannabinoid N-arachidonoyl-ethanolamine (AEA) is an
endogenous lipid recognized to activate the cannabinoid receptors CB1
and CB2, as well as the transient receptor potential cation channel sub-
family V member 1 (TRPV1) receptor [1–4]. As such, AEA is involved in
numerous physiological responses, notably nociception, appetite and
inflammation [5–9]. In addition to AEA, N-acyl-ethanolamines (NAEs)
containing sixteen or plus carbons, together with their receptors
including other TRP channels and G-protein-coupled receptors as well as
peroxisome proliferator-activated receptors (PPARs), are part of the
endocannabinoidome [10].
2.1.2. Synthesis and purification of 13-HODE-EA and 13-HODE-EA-d4
The synthesis of 13-HODE-EA was previously achieved with the help
of Soybean lipoxygenase as previously documented [26]. We thus uti-
lized the same strategy with slight modifications. In brief, 13-HODE-EA
synthesis was done by incubating 0,01 mmol LEA (1) with 20 mg soy-
bean lipoxygenase in 100 ml of borate buffer (50 mM, pH 12) and stirred
for 15 min at room temperature. The hydroperoxide was then reduced
by the addition of 2 ml of a freshly prepared NaBH4 (1 M in EtOH) for 15
min at room temperature. The solution was next acidified by the drop-
Following the activation of its multiple cellular targets, NAEs can be
hydrolyzed to the corresponding fatty acids by specific hydrolases such
as the Fatty Acid Amide Hydrolases (FAAH)-1 and ꢀ 2 or the NAE acid
amidase [11–13]. AEA can also be metabolized into other bioactive
lipids, notably by eicosanoid biosynthetic enzymes. The most docu-
mented AEA metabolites arising from such metabolism are the
cyclooxygenase-2-derived prostamides, which elicit several functional
responses [14]. AEA can also be oxidized by CYP450 enzymes [15] and
via the 15-lipoxygenase (15-LO) pathway into N-15-hydroxy-eicosate-
traenoyl-ethanolamine (15-HETE-EA), notably by human neutrophils
and eosinophils [16–19] and into eoxamides by the Hodgkin lymphoma
cell line L1236 and platelets [20].
wise addition of glacial acetic acid (500 μl over 30 s) and gently stirred
until foaming stopped (~15 min). The lipids were next extracted with
300 ml of CHCl3, the organic phase was dried with 1 spoon of Na2SO4,
filtered, and concentrated in vacuo using a rotavap apparatus. The res-
idues were filtered over glass wool and then purified by RP-HPLC. In
brief, residues were diluted in 300
μ
l; MeOH, loaded onto an HPLC
column (Kinetex C18, 5
μ
m, 10 × 150 mm) at 3 ml/min and eluted using
a binary gradient involving solvent A (H2O + 0.1% AcOH) and solvent B
(MeCN +0.1% AcOH) as mobile phase. The gradient program was as
follows: 0–5 min: 45%–70% B; 5–20 min: 70% B; 20–21 min 70%–45%
B; 21–26 min: 45% B. The column was kept at 22 ◦C. UV detection was
set at 205 and 235 nm. 13-HODE-EA (3) and 13 HODE-EA–d4 (4) had a
retention time of 8.0 min while 13-HODE had a retention time of 11 min;
all the lipoxygenase metabolites had absorption at both UV wavelength
checked.
Interestingly, the 15-LO pathway can metabolize many long chain
fatty acids possessing a 1Z,4Z-pentadiene motif. As such, linoleic acid
(LA) is a recognized substrate of 15-LOs and is thus converted into 13-
hydroxyoctadecadienoic acid (13-HODE) [21,22]. Herein, we postu-
lated that the NAE containing LA, i.e. N-linoleoyl-ethanolamine (LEA),
would be metabolized by 15-LO enzymes into N-13-HODE-ethanol-
amine (13-HODE-EA). We thus report our strategy to synthesize LEA and
13-HODE-EA, as well as their deuterated derivatives. We also demon-
strate the ability of human recombinant 15-LO-1 and 15-LO-2 to syn-
thesize 13-HODE-EA, as well as the weak activity at TRPV1 and lack of
2.1.3. Characterization with 1D and 2D NMR experiments
All products and synthetic intermediate structures were assigned
using 1H, 13C, COSY, HSQC NMR experiments and the spectra were
recorded in 5 mm tubes on an Agilent 500 MHz DD2 system (Agilent
NMR System, Santa Clara, USA) with a OneNMR probe. 1H NMR and 13
C
activity at cannabinoid receptors and PPAR
α
and γ of this metabolite,
NMR chemical shifts are referenced to residual protons in deuterated
solvents. Multiplicities are reported as singlet (s), doublet (d), triplet (t),
quintet (q), multiplet (m), broad (br) or overlapping (ov). Chemical
shifts are reported in ppm. Coupling constants are reported in Hz. Pro-
tons were assigned using integrated 1H NMR and 2D 1H, 1H-gCOSY
spectra to detect correlation between neighboring protons. The quarte-
nary carbon was determined using the 1D 13C experiments. Protons were
then assigned to carbons using 2D 1H, 13C-HSQC experiments showing
single through-bond relationships and CH were discriminated from CH2
and CH3 with crosspeaks of different colors.
and its presence in selected tissue samples from humans.
2. Material and methods
2.1. Materials
LA was purchased from NU-CHEK PREP (Elysian, MN, USA). Etha-
nolammine-d4 was purchased from CDN Isotopes (Pointe-Claire, QC,
–
Canada). Soybean lipoxygenase Type I B, CDCl3, Novozym 435,
ethanolamine, Na2B4O7 and NaBH4 were purchased from Sigma Aldrich
and used without further purification. HPLC-grade acetonitrile and
glacial acetic acid were purchased from Fisher chemical. HPLC water
was purified with a Milli-Q system (Millipore). Human recombinant 15-
LOs were a kind gift of Prof. Ted Holman (USCS, Santa Cruz, USA), were
2.2. Characterization with HR-ESI-MS/MS spectrometry
High resolution mass spectrometry was performed on a Thermo
Orbitrap Fusion tribrid mass spectrometer (Thermo Scientific, Bremen,
Germany) using a Chemyx F100T2 syringe pump (Chemyx Inc) running
prepared and had comparable specific activity as in [23,24]. PPARα kits
were purchased from Eurofins DiscoverX (Fremont, California, USA) and
from Indigo Biosciences (State College, Pennsylvania, USA) while
PPARγ assay kits were from Eurofins DiscoverX (Fremont, California,
USA).
at 5 μl/min. The mass spectrometer was operated in positive or negative
ionization modes using an orbitrap detector with a resolution of 60,000.
For CID, the collisional energy was between 0 and 50 eV.
2.2.1. Enzymatic assays
2.1.1. Synthesis and purification of LEA and LEA-d4
Enzymatic assays were performed in a 100 μl volume. Human re-
For the synthesis of LEA, we followed a previously documented
strategy involving the enzyme Novozym 435 [25]. In brief, we mixed LA
and ethanolamine (0.20 mmol of each) in a glass vial containing 1 ml
hexane. The resulting mixture was placed in a thermoconstant girotory
shaker (New Brunswick Scientific, USA) and 50 mg of Novozym 435 was
then added. The mixture was shaken at 200 rpm overnight at 40 ◦C.
After cooling at room temperature, removal of the solvents was per-
formed under reduced pressure. Purification of LEA was achieved by
silica gel chromatography using CHCl3-MeOH (98:2, v/v) to give 54 mg
(0.17 mmol) of LEA (1) (white solid, 85% yield). For the synthesis of
combinant 15-LO-1 (1.4 μg) and human recombinant 15-LO-2 (5.6 μg)
were suspended in warm HEPES buffer (37 ◦C, 25 mM, pH = 7.4) con-
taining 0.01% Triton. Substrates (10 μM) were then added to the test
tubes for 5 min (15-LO-1) or 15 min (15-LO-2). 50 μl of a freshly pre-
pared 1 M NaBH4 solution was then added for 5 min at 37 ◦C. The re-
actions were next stopped by adding 0.5 ml of cold (20 ◦C) MeOH
containing 0.01% AcOH. Samples were then extracted using 1 ml of
CHCl3 containing 0.1 M AcOH, and vortexed for 1 min. The organic
phase (lower phase) was harvested, evaporated under a stream of ni-
trogen, and reconstituted with 25 μl of solvent A (H2O + 0.05% AcOH
2

F. Tinto et al.
BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158954
or positive ion mode using multiple reaction monitoring for the specific
mass transition of the metabolites, as shown in Table1. 15-LO metabo-
lites were quantified using calibration curves that were extracted as
described above.
Table 1
Mass transitions utilized for the quantification of the lipid mediators.
Compound
ISTD
Q1 → Q3
Ret. time
(min)
LLOQa
LEA
LEA-d4
324.30 → 62.05
328.00 → 66.10
322.00 → 62.20
10.771
10.743
7.147
25
–
2.3.1. Analysis of CB1 and CB2 binding
LEA-d4
–
13-HODE-EA
13-HODE-EA-
25
To establish whether our lipids bound to the CB1 and CB2 receptors,
competitive binding assays for the human CB1 and CB2 receptors were
performed as described before [28]. In brief, the membranes of HEK-293
cells transfected with either the human CB1 receptor (Bmax 5 pmol/mg
protein) or the human CB2 receptor (Bmax 10.4 pmol/mg protein) were
incubated with [3H]-CP 55,940 (0.4 nM for the CB1 receptor (kd of 0.18
nM) and 0.53 nM for the CB2 receptor (/kd of 0.1 nM)) as the high af-
finity ligand and displaced with increasing concentrations (0.1 nM – 10
d4
13-HODE-EA-
–
326.30 → 66.20
7.126
–
d4
LA
AA-d8
279.40 →
261.35
13.079
13.000
2500
AA-d8
–
311.20 →
267.25
–
13-HODE
13-HODE-d4
295.5 → 277.30
299.10 →
198.15
9.942
9.893
25
13-HODE-d4
–
–
μ
M) of the different lipids. IC50 values were determined for lipids
showing >50% displacement at 1 M. Nonspecific binding was defined
by 10 M of WIN 55,212-2 as the heterologous competitor (which has Ki
μ
a
LLOQ. Lowest limit of quantification in fmol.
μ
values of 9.2 nM for the CB1 and 2.1 nM for the CB2). Displacement
curves were obtained by incubating the lipids with [3H]-CP 55,940 for
90 min at 30 ◦C. Ki values were calculated by applying the Cheng-
Prusoff equation to the IC50 values (obtained using the GraphPad
Prism Software) for the displacement of the bound radioligand by
increasing concentrations of the lipids.
+0.01% NH4) and 25 μl of solvent B (95:5 MeCN:H2O + 0.05% AcOH
+0.01% NH4) and frozen (ꢀ 80 ◦C) until their analysis by LC-MS/MS.
2.3. Analysis of 13-HODE-EA by LC-MS/MS
The analysis of bioactive lipids in the saliva and skin of healthy do-
2.3.2. TRPV1 functionnal assay
´
´
nors was approved by the Comite d’ethique de la recherche de l’Institut
The activity of the lipids on TRPV1 was assessed using HEK-293 cells
stably over-expressing human recombinant TRPV1 as described previ-
´
´
universitaire de cardiologie et de pneumologie de Quebec – Universite
´
´
´
Laval and the Comite d’ethique de la recherche du CHU de Quebec-
ously [29]. In brief, HEK-293 cells in DMEM were loaded with 4 μM Fluo
´
Universite Laval.
4-AM (Molecular Probes, Invitrogen) in the dark for 60 min. Cells were
then washed twice in Tyrode’s buffer, re-suspended at 50,000 cells/ml
and transferred into the magnetically stirred quartz cuvette of the
spectrofluorometer (Perkin-Elmer, LS50B, Italy). Fluorescence was
monitored before and after the addition of the test compounds at exci-
tation and emission wavelengths of 488 and 516 nm, respectively, at
25 ◦C. Experiments were carried out by measuring cell fluorescence at
25 ◦C before and after the addition of the test compounds at various
concentrations. The efficacy of the agonists was determined by
Skin samples were obtained and processed exactly as described in
[27]. Saliva samples (400 μl) were thawed, mixed with 100 ul Tris buffer
(ph 7.4), acidified with 0.575% of AcOH and mixed with 500 μl meOH
containing 0.01 M AcOH. CHCl3 (1 ml) was then added to the samples
which were vortexed 1 min and centrifuged at 3000 ×g for 5 min. The
organic phase (lower phase) was harvested, dried under a stream of
nitrogen and reconstituted with 25
NH₄OH and 0.05% of AcOH) and 25
v/v containing 1 mM NH₄OH and 0.05% of AcOH). 40
μ
l of solvent A (H2O containing 1 mM
l of solvent B (MeCN/H2O, 95/5,
l of the samples
m,
μ
μ
comparing it to the analogous effect observed with 4 μM ionomycin. In
were injected onto an HPLC column (Kinetex C8, 150 × 2.1 mm, 2.6
μ
antagonism experiments, varying doses of PhAR was added 10 min prior
to 100 nM capsaicin. Data are expressed, for the agonists, as the con-
centration exerting a half-maximal effect (EC50), and, for the antago-
nists, as the concentration exerting a half-maximal inhibition (IC50),
both calculated using the Graphpad Prism Software.
Phenomenex) and eluted at a flow rate of 0.4 ml/min with a discon-
tinuous gradient from 35% to 75% of solvent B in 10 min, from 75% to
95% in 10 s and held to 95% for 5 min. The HPLC system was interfaced
with the electrospray source of a Shimadzu 8050 triple quadrupole mass
spectrometer and mass spectrometric analysis was done in the negative
Fig. 1. Strategy to synthesize LEA, LEA-d4 and the 15-LO metabolites 13-HODE-EA and 13-HODE-EA-d4. LA and ethanolamine are incubated with Novozym 435 to
yield LEA. LEA is then oxygenated by soybean lipoxygenase to yield 13-HODE-EA. 13-HODE-EA-d4 was obtained by incubating LA with ethanolamine-d4 instead of
ethanolamine.
3

F. Tinto et al.
BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158954
respectively incubating LEA and LEA-d4 with soybean lipoxygenase as
described in Methods. The incubation of LEA with soybean lipoxygenase
resulted in the production of compounds detectable at a UV wavelength
of 235 nm, according to the RP-HPLC analyses, suggesting the formation
of a conjugated diene. The yield of the reaction was ~45% for both 13-
HODE-EA and 13-HODE-EA-d4. 13-HODE-EA was easily separated by
RP-HPLC from LEA, LA and 13-HODE with retention times of ~8.0 min
for 13-HODE-EA and 13-HODE-EA-d4, ~11 min for 13-HODE, ~14.75
min for LEA and 20 min for LA (Fig. 2). The structure of the conjugated
diene of 13-HODE-EA and 13-HODE-EA-d4 was confirmed by NMR as
cis-trans based on the coupling constants between H-9 and H-12. Indeed,
the value of J9–10 was 11.0 Hz, indicating the presence of a cis config-
uration double bond, whereas the coupling constant of J11–12 was 15.2
Hz, characteristic of trans geometry (Table 2 for 13-HODE-EA and
Supplementary Figs. S5–S12 for the raw data). We next performed a
high-resolution mass scan of 13-HODE-EA. Positive electrospray ioni-
zation mass spectrometry (ESI+) of 13-HODE-EA yielded three main
cations. The protonated molecule with loss of water [M-H2O + H]+ was
found at m/z 322.2743. Additionally, two cations adducts, one with Na+
at m/z 362.2668 [M + Na]+ and the other one with K+ at m/z 378.2408
[M + K]+ were also identified (Fig. 3A). CID of 13-HODE-EA (m/z
322.2743, Fig. 3B) led to the dominant product ion of m/z 62.0600,
which is highly characteristic of NAEs [31–33] as it represents the
fragment of the protonated ethanolamine moiety ([HOCH2CH2NH3]+).
The other part of the molecule after the loss of the ethanolamine was
detected at m/z 261.2214, representing the ion m/z 279.2324 after loss
of water (Fig. 3B). Based on these data we quantitated 13-HODE-EA by
Fig. 2. RP-HPLC chromatogram of 13-HODE-EA and related derivatives. 13-
HODE-EA, 13-HODE, LEA and LA (~350 μg) were loaded onto the HPLC col-
umn and eluted as described in Material and Methods. The data represent the
combination of four different injections that are representative of at least three
independent injections.
3. Results
The first series of experiments consisted at developing a strategy
allowing the synthesis of 13-HODE-EA (3) and 13-HODE-EA-d4 (4). We
selected a strategy involving two enzymatic steps. First, we converted LA
into LEA (or LEA-d4) using Novozym 435, then we performed a lip-
oxygenation with soybean lipoxygenase as described in the Methods
(Fig. 1). We chose this strategy for two main reasons. First, Novozym
435 readily performs the amidation of fatty acids with an ethanolamine
moiety [25]. Second, soybean lipoxygenase can metabolize LA and LEA
into their 13-HODE derivatives [26]. The amidation of LA into LEA or
LEA-d4 was very efficient (~85% yield). 1H and 13C NMR (Supplemen-
tary Figs. 1–4 for the raw data) confirmed the molecular structure of
both compounds in accordance with the literature data for LEA [30].
We next synthesized 13-HODE-EA and 13-HODE-EA-d4 by
LC-MS/MS and by utilizing the MRM [M-H2O
+
H]+
→
[HOCH2CH2NH3]+ in the positive ion mode, in the remaining of the
paper.
We next assessed whether human 15-LO-1 and 15-LO-2 could bio-
synthesize 13-HODE-EA in response to LEA. In these experiments, the
LA-induced 13-HODE biosynthesis was used as positive control. Both 15-
LO-1 and 15-LO-2 biosynthesized 13-HODE following incubation with
10 μM LA (Fig. 4). The yield of the transformation of LA into 13-HODE
by either enzyme was between 25 and 30% (Fig. 4A,E). In this subset of
experiments, LEA and 13-HODE-EA were below detection limit. When
Table 2
1H NMR and 13C NMR data of 13-HODE-EA.
Carbon
No
13-HODE-EA
δ C (ppm)
δ H (ppm)
Features
J (Hz)
NA
1
175.25
36.55
25.51
28.86
28.69
30.32
29.04
27.48
132.77
128.62
125.69
135.93
72.86
37.35
29.26
31.78
22.62
14.07
42.45
63.7
NA
NA
m
m
m
m
m
m
m
m
t
2
2.21
1.64
1.31
1.31
1.31
1.38
2.17
5.43
5.98
6.49
5.67
4.16
1.53
1.31
1.31
1.31
0.89
3.43
3.72
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
1′
10.9
dd
dd
q
15.2, 11.1
15.2, 6.8
6.5
m
m
m
m
t
6.9
5
q
2′
t
4

F. Tinto et al.
BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158954
Fig. 3. Ionization and Collision-induced dissociation of 13-HODE-EA. A) Positive electrospray ionization mass spectrometry (ESI+) of 13-HODE-EA. Positive
electrospray ionization mass spectrometry (ESI+) of 13-HODE-EA yielded three main cations; [M-H2O + H]+ was found at m/z 322.2743, [M + Na]+ at m/z
362.2668 and [M + K]+ at m/z 378.2408. B) Collision-Induced Dissociation (CID) at Higher-energy collisional dissociation (HCD) of 20 eV of 13-HODE-EA. CID of
[M-H2O + H]+ at m/z 322.2743 led to the dominant product ion of m/z 62.0600, which is highly characteristic of NAEs. The second major peak was detected at m/z
261.2214 and representing the ion m/z 279.2324 after loss of water.
5

F. Tinto et al.
BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158954
Fig. 4. Formation of 13-HODE and 13-HODE-EA by human recombinant 15-lipoxygenases. Human recombinant 15-LO-1 and 15-LO-2 were incubated with 10
LA, LEA, or both (A–C, E–-G) or 1 M of both LA and LEA (D, H) for the indicated times. Samples then were processed and analyzed for 13-HODE and 13-HODE-EA
synthesis by LC-MS/MS as described in Material & methods. Results are the mean (±SEM) of three independent experiments.
μM
μ
Table 3
Table 5
Levels of 13-HODE-EA and related mediators in human samples.
Effect of 13-HODE-EA and related compounds on human recombinant TRPV1-
mediated intracellular calcium elevation.
Tissue
13-HODE-
EA
LEA
13-HODE
LA
Compound
Efficacy (% max
response)
Potency (EC50
in M)
Desensibilizationa (IC50
μ
in μM)
Saliva (n = 3)a
Epidermis (n =
3)b
0,22 ± 0.07
92.4 ± 7.1
5.2 ± 3.1
10,036 ±
2873
12.9 ± 6.1
573 ± 261
7496 ± 896
586.5 ± 144
AEA
66.6 ± 0.5
71.5 ± 0.3
<10
0.10 ± 0.01
0.14 ± 0.01
–
6.5 ± 0.1
0.17 ± 0.01
0.23 ± 0.02
>50
LEA
Dermis (n = 5)b
22.4 ± 4.93
1237 ± 826
3745 ±
155.7 ±
13-HODE
13-HODE-
EA
2473
65.3
58.8 ± 0.2
38.0 ± 4.3
a
b
Data are presented as pmol/ml saliva.
a
Data are presented as pmol/mg of tissue, except for LA (nmol/mg/tissue).
Desensibilization was assayed using 100 nM capsaicin.
13-HODE-EA/13-HODE ratio was 3.48 for 15-LO-1 (Fig. 4C) and 3.05
for 15-LO-2 (Fig. 4F).
Table 4
Affinity of 13-HODE-EA and related compounds for human recombinant CB1
and CB2 receptors.
We next screened several mouse tissues for their putative content in
13-HODE-EA. We could not detect this metabolite in the ileum, jejunum,
colon, gastrocnemius, brain or lung of mice, indicating that 13-HODE-
EA is not readily detectable in mice. However, we could find 13-
HODE-EA in human saliva samples, which were previously shown to
contain 15-LO metabolites [34,35] and NAEs [36]. Indeed, 13-HODE-EA
levels were in the mid nM range (Table 3), supporting the hypothesis
that this metabolite plays physiological roles. Furthermore, 13-HODE-
EA was also found in skin biopsies of healthy individuals, which are
also recognized to express numerous lipoxygenases that are very much
likely involved in the regulation of skin homeostasis [37].
Compound
CB1 receptor
CB2 receptor
IC50 Ki
Max tested (%
displacement)
IC50 Ki
Max tested (%
displacement)
(μ
M) M)
(
μ
(μ
M) M)
(μ
13-HODE
>10
>10
10
μ
M
>10
>10
10
(38.34 ± 5.4)
10
(47.29 ± 7.2)
μM
(7.56 ± 2.3)
13-HODE-
EA
>10
>10
10
μM
>10
>10
μM
(15.70 ± 5.3)
100 nM
AEAa
0.97
±
0.11
±
120
10
1 μM
(95.33 ± 4.9)
± 20
± 3
(87.40 ± 4.4)
0.14
3.20
±
0.01
0.35
±
LEA
a
10
μM
>10
>10
10
μM
We next sought to determine whether 13-HODE-EA could bind/
activate receptors linked to the endocannabinoidome [10], notably the
(103.00 ±
(46.87 ± 6.3)
1.7
0.2
3.0)
CB1, CB2, TRPV1, PPARα and PPARγ. While AEA bound specifically to
Values for AEA are in nM.
both cannabinoid receptors in membranes prepared from HEK 293 cells
overexpressing either CB1 or CB2 (Table 4), we did not detect any
incubated with 10
almost 100% of LEA into 13-HODE-EA, with 13-HODE-EA concentra-
tions approaching 10 M, and no biosynthesis of 13-HODE (Fig. 4B, F).
μM LEA, both 15-LO-1 and 15-LO-2 transformed
binding to these receptors for 13-HODE-EA up to 10 μM, despite the fact
that LEA did bind to the CB1 receptor (Ki of ~350 nM). In contrast, 13-
HODE-EA, but not 13-HODE, was able to induce TRPV1-mediated
elevation of intracellular calcium in HEK-293 cells overexpressing the
human orthologue of this receptor, although not to the same extent as
AEA or LEA (Table 5). Finally, 13-HODE-EA slightly, but not signifi-
μ
Interestingly, the combination of LA and LEA (1 or 10 μM each) did not
dramatically change the relative amounts of 13-HODE and 13-HODE-EA
being biosynthesized, with levels comparable to those observed when
LA and LEA were incubated individually (Fig. 4C,D,G,H). Moreover, the
cantly, activated PPAR-α in our luciferase assay, whereas GW590735
6

F. Tinto et al.
BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158954
Fig. 5. Impact of LEA and 13-HODE-EA on PPAR activity. Cells were incubated with 30
μ
M LEA, 13-HODE-EA or OEA overnight and PPAR activity was measured
using the PPAR assay kits according to the manufacturers’ instructions. GW 590735 (100 nM) and Rosiglitazone (1 μM) were utilized as positive controls for the A)
PPAR-α and B) PPAR-γ assays, respectively. Data represent the mean (±SEM) of three (PPAR-γ) or five (PPAR-α) experiments, each performed in triplicates.
and OEA, two documented PPAR-
α
agonists, were effective (Fig. 5A).
commercial scale synthesis of 13-HODE-EA and 13-HODE-EA-d4 will
likely necessitate other synthetic routes.
Unlike the PPAR-γ agonist rosiglitazone, 13-HODE-EA was also inef-
fective at activating this nuclear receptor (Fig. 5B).
Both human recombinant 15-LO-1 and 15-LO-2 were previously
documented as being able of lipoxygenating LA into 13-HpODE and/or
13-HODE [40,41]. We could confirm here this ability of the two enzymes
and additionally showed that they can use LEA as substrate to generate
13-H(p)ODE-EA (Fig. 4). In fact, the conversion of LEA was 3-fold that of
4. Discussion
The crystal structure elucidation of rabbit 15-LO established that AA
enters the catalytic pocket via its omega end [38,39]. Furthermore, 15-
LO enzymes are recognized to metabolize numerous polyunsaturated
fatty acid containing a 1Z,4Z-pentadiene motif aside from AA, notably
LA [22,40,41]. In addition to polyunsaturated fatty acids, 15-LO en-
zymes can metabolize other AA-containing lipids, notably the endo-
cannabinoid AEA [16,17,20]. This strongly suggested that other
polyunsaturated fatty acid-containing NAEs, notably LEA, might act as
substrates for 15-LO enzymes.
LA with 10 μM of either substrate. This suggests that LEA is a better 15-
LO substrate than LA. This assumption was further confirmed by co-
incubating 15-lipoxygenases simultaneously with LEA and LA, which
led to a 13-HODE-EA/13-HODE ratio of 3.48 for 15-LO-1 and 3.05 for
15-LO-2 (Fig. 4C,F), in agreement with the recent study from Ivanof and
colleagues in which they found that human 15-lipoxygenases better
oxidized AEA over AA [42]. Whether this substrate preference for LEA
over LA is also observed in 15-LO-expressing cells remains to be docu-
mented. The similar metabolism of LEA by 15-LO-1 and 15-LO-2 sug-
gests that in humans 13-HODE-EA might be found in several tissues,
notably in those in which 15-LO-1 and 15-LO-2 are strongly expressed,
provided that high endogenous levels of LEA are also present. In fact, we
could no detect 13-HODE-EA in the numerous mouse tissues we tested
(colon, ileum, brain, adipose tissue, gastrocnemius) despite a very high
sensitivity of the LC/MS-MS method (Table 1). However, we detected
13-HODE-EA in human saliva, a biological matrix previously shown to
contain several 15-LO metabolites and NAEs [34–36], as well as in the
skin, supporting the concept that 15-LO- and/or LEA-rich biological
matrices are susceptible to 15-LO-catalyzed metabolism and the related
biological effects of 13-HODE-EA, which need to be further explored.
Hamberg and Samuelsson established in 1967 that soybean LO
transformed LA and AA into 13(S)-H(p)ODE and 15(S)-H(p)ETE,
respectively [43]. Given that our synthetic route to obtain 13-HODE-EA
involved the same enzyme, and that both 15-LO-1 and -2 were capable of
biosynthesizing it, we believe that the reference compound we investi-
gated herein is 13(S)-HODE-EA. However, we could not confirm this as
we did not have pure, chemically synthetized isomers to validate this
using chiral chromatography.
The novel results presented in this study can be summarized as fol-
lows: 1) Novozym 435 readily allows the synthesis of LEA and LEA-d4;
2) soybean lipoxygenase allows the synthesis of 13-HODE-EA and 13-
HODE-EA-d4 from LEA and LEA-d4, respectively; 3) Human recombi-
nant 15-LO-1 and 15-LO-2 can generate 13-HODE-EA from LEA; 4) 13-
HODE-EA is found in the saliva and the skin of healthy volunteers, 5) 13-
HODE-EA does not bind to CB1 and CB2 receptors; 6) 13-HODE-EA can
activate the TRPV1 receptor, but not PPARα nor PPARγ.
Plastina et al. previously showed that LEA synthesis was successfully
achieved by performing an amidation using ethanolamine and LA [25].
Herein, we could reproduce this reaction with a comparable yield and
provide additional evidence that the amidation reaction is as efficient
when we replaced ethanolamine with ethanolamine-d4, which was key
for the synthesis of LEA-d4. This latter compound was used here for the
quantitative analysis of LEA by LC-MS/MS.
In addition, we provide compelling evidence that soybean lip-
oxygenase allows the synthesis of 13-HODE-EA and 13-HODE-EA-d4
from LEA and LEA-d4 respectively. This is in agreement with a previous
study from Van der Stelt et al. in which they found that this enzyme
converted N-linoleoylethanolamine (ODNHEtOH), N-linoleoylamine
(ODNH2), N-linoleoylmethylamine (ODNHMe) and N,N-linoleoyldime-
thylamine (ODN(Me)2 into 13-(S)-hydroperoxy-9Z,11E-octadeca-9,11-
dienoyl amides derivatives [26]. The yield of the reaction was moder-
ately good (~45%), under the experimental conditions described.
However, when we attempted to scale-up the reaction by increasing
starting material and adjusting volumes accordingly, the yield dramat-
ically diminished (data now shown). We cannot explain why we could
not increase the output of the reaction. However, this indicates that the
With regard to the possible biological activity of 13-HODE-EA, the
number of receptors being activated by endocannabinoids and related
lipids (the endocannabinoidome) has been steadily increasing over the
last decade [10]. For example, apart from being an endogenous agonist
of CB1 and CB2 receptors, AEA can activate TRPV1 channels, while OEA
activates PPAR
α as well as the orphan G-protein-coupled receptor
GPR119, which are also activated by LEA [3,10,44]. Of note, TRPV1 can
be also activated, although at higher concentrations than AEA, by 15(S)-
7

F. Tinto et al.
BBA - Molecular and Cell Biology of Lipids 1866 (2021) 158954
HETE-EA and, less potently by 9(S)-HODE, but not by racemic 13-HODE
[45]. Herein, we did not find any significant modulatory activity of LEA
or 13-HODE-EA on PPAR receptors. Furthermore, 13-HODE-EA did not
bind to the human CB1 (and CB2) receptor, in agreement with a previous
study from van der Stelt showing that it did not bind to the rat CB1 re-
ceptor [46]. However, we confirmed that LEA is a TRPV1 agonist, and
found that 13-HODE-EA, unlike 13-HODE, also activates this ligand-
activated channel, as efficaciously as, but less potently than, LEA.
Whether 13-HODE-EA participates to TRPV1-mediated responses in
vivo remains to be determined.
Acknowledgements
We thank Prof. Ted Holman for kindly providing human recombi-
nant 15-LO-1 and 15-LO-2.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbalip.2021.158954.
In conclusion, we showed that the chemical synthesis of LEA and 13-
HODE-EA can be achieved relatively easily although the large-scale
production of the latter compound could not be obtained with the
method used here. Furthermore, we showed that 13-HODE-EA is ob-
tained following the incubation with LEA of 15-LO enzymes, which
demonstrate a preference toward LEA vs. LA. Finally, the presence of 13-
HODE-EA in human saliva and skin samples as well as its interaction
with TRPV1, but not with cannabinoid or PPAR receptors, suggests that
13-HODE-EA, perhaps more selectively than other receptor-unselective
TRPV1-activating mediators of the endocannabinoidome [10], might be
involved in the several functions proposed for this channel in both pe-
ripheral and brain neurons and cells. It will be important to investigate
next if 15-LO-expressing cells, such as leukocytes and lung epithelial
cells, also produce 13-HODE-EA and if this mediator regulates some
physiological and pathological aspects of these cells.
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