Characterization and biological effects of di-hydroxylated compounds deriving from the lipoxygenation of ALA

Article abstract of DOI:10.1194/jlr.M035139

We have recently described a di-hydroxylated compound called protectin DX (PDX) which derives from docosahexaenoic acid (DHA) by double lipoxygenation. PDX exhibits anti-aggregatory and anti-inflammatory properties, that are also exhibited by similar molecules, called poxytrins, which possess the same E , Z , E conjugated triene geometry, and are synthesized from other polyunsaturated fatty acids with 22 or 20 carbons. Here we present new biological activities of di-hydroxylated metabolites deriving from α-linolenic acid (18:3n-3) treated by soybean 15-lipoxygenase (sLOX). We show that 18:3n-3 is converted by sLOX into mainly 13(S)-OH-18:3 after reduction of the hydroperoxide product. But surprisingly, and in contrast to DHA which is metabolized into only one di-hydroxylated compound, 18:3n-3 leads to four di-hydroxylated fatty acid isomers. We report here the complete characterization of these compounds using high field NMR and GC-MS techniques, and some of their biological activities. These compounds are: 9(R),16(S)-dihydroxy-10 E ,12 E ,14 E -octadecatrienoic acid, 9(S),16(S)-dihydroxy-10 E ,12 E ,14 E -octadecatrienoic acid, 9(S),16(S)-dihydroxy-10 E ,12 Z ,14 E -octadecatrienoic acid, and 9(R),16(S)-dihydroxy-10 E ,12 Z ,14 E -octadecatrienoic acid. They can also be synthesized by the human recombinant 15-lipoxygenase (type 2). Their inhibitory effect on blood platelet and anti-inflammatory properties were compared with those already reported for PDX. Copyright

Full text of DOI:10.1194/jlr.M035139

Characterization and biological effects of di-hydroxylated
compounds deriving from the lipoxygenation of ALA
Miao Liu,* Ping Chen,† Evelyne Véricel,* Moreno Lelli,§ Laetitia Béguin,§ Michel Lagarde,*
and Michel Guichardant1,*
Université de Lyon,* UMR 1060 Inserm (CarMeN), IMBL/INSA-Lyon, 69621 Villeurbanne, France; Tea
Science Department,† College of Agriculture and Biotechnology, Zhejiang University, 310029 Hangzhou,
China; and Centre de RMN à Très Hauts Champs,§ Université de Lyon (CNRS/ENS Lyon/UCB Lyon 1),
69100 Villeurbanne, France
Abstract We have recently described a di-hydroxylated com-
pound called protectin DX (PDX) which derives from doco-
sahexaenoic acid (DHA) by double lipoxygenation. PDX
exhibits anti-aggregatory and anti-inflammatory properties,
that are also exhibited by similar molecules, called poxytrins,
which possess the same E,Z,E conjugated triene geome-
try, and are synthesized from other polyunsaturated fatty
acids with 22 or 20 carbons. Here we present new biological
activities of di-hydroxylated metabolites deriving from
␣-linolenic acid (18:3n-3) treated by soybean 15-lipoxygenase
(sLOX). We show that 18:3n-3 is converted by sLOX into
mainly 13(S)-OH-18:3 after reduction of the hydroperox-
ide product. But surprisingly, and in contrast to DHA which
is metabolized into only one di-hydroxylated compound,
18:3n-3 leads to four di-hydroxylated fatty acid isomers.
We report here the complete characterization of these
compounds using high field NMR and GC-MS techniques,
and some of their biological activities. These compounds
are: 9(R),16(S)-dihydroxy-10E,12E,14E-octadecatrienoic
acid, 9(S),16(S)-dihydroxy-10E,12E,14E-octadecatrienoic
acid, 9(S),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic acid,
and 9(R),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic acid.
They can also be synthesized by the human recombinant
15-lipoxygenase (type 2). Their inhibitory effect on blood
platelet and anti-inflammatory properties were compared
with those already reported for PDX.—Liu, M., P. Chen, E.
Véricel, M. Lelli, L. Béguin, M. Lagarde, and M. Guichardant.
Characterization and biological effects of di-hydroxylated
compounds deriving from the lipoxygenation of ALA.
J. Lipid Res. 2013. 54: 2083–2094.
vivo many physiological functions. It is assumed that long-
chain n-3 PUFAs have beneficial effects on cardiovascular
and coronary artery diseases (1, 2), rheumatoid arthritis
(3–5), blood pressure control and heart hypertension (6,
7), and they might prevent multiple sclerosis and Alzheim-
er’s disease (8–11). More recently, new lipid mediators
deriving from eicosapentaenoic acid (EPA) and docosa-
hexaenoic acid (DHA) have been described. Among them,
resolvins are oxygenated molecules that derive from EPA
and DHA (12, 13) and are separated into two series: E-
series (from EPA) and D-series (from DHA) (14). In addi-
tion to the D-series resolvins, protectin D1 (PD1) is
produced from DHA via a mechanism involving an epox-
ide intermediate (15), as well as maresin which can be pro-
duced by macrophages (16). PD1 has been described as a
potent anti-inflammatory agent without an anti-aggrega-
tory effect (15, 17). In contrast, protectin DX (PDX), an
isomer of PD1, which derives from DHA via a double
lipoxygenation, reveals inhibitory effects on blood plate-
let aggregation. PDX inhibits platelet cyclooxygenase
(COX)-1 and antagonizes the thromboxane A2-induced
aggregation (18). We found that di-hydroxylated com-
pounds called poxytrins, synthesized from C20 and C22
PUFAs via the soybean 15-lipoxygenase (sLOX) and hav-
ing the same E,Z,E conjugated triene motif, including
PDX, are all anti-aggregatory agents (19). On the other
hand, very few data are available concerning the oxygen-
ated metabolism of ␣-linolenic acid (ALA) (18:3n-3). Inves-
tigations relating to ALA are particularly relevant because
Supplementary key words octadecatrienoic acid • 15-lipoxygenase •
platelet aggregation • anti-inflammatory activity • ␣-linolenic acid
Abbreviations: ALA, ␣-linolenic acid (9Z,12Z,15Z-octadecatrienic
acid); COX, cyclooxygenase; DHA, docosahexaenoic acid; EPA, eicosa-
pentaenoic acid; 9(S)-HOTE, 9(S)-hydroxy-10E,12Z,15Z-octadeca-
trienoic acid; 13(S)-HOTE, 13(S)-hydroxy-9Z,11E,15Z-octadecatrienoic
acid; 9(S)-HpOTE, 9(S)-hydroperoxy-octadeca-10E,12Z,15Z-trienoic
acid; 13(S)-HpOTE, 13(S)-hydroperoxy-9Z,11E,15Z-octadecatrienoic acid;
LOX, lipoxygenase; LTB4, leukotriene B4; PD1, protectin D1; PDX,
protectin DX; PGD2, prostaglandin D2; PGE2, prostaglandin E2;
RP-HPLC, reverse-phase high-performance liquid chromatography;
sLOX, soybean 15-lipoxygenase; 9,16-di-HOTE, 9,16-dihydroxyoctade-
catrienoic acid; UV, ultraviolet.
N-3 polyunsaturated fatty acids (PUFAs) have been well
studied during the last decades because they regulate in
This study was supported by Inserm and the French Ministry of Education and
Research. M.L. is a Ph.D. student granted from the China Scholarship
Council.
Manuscript received 18 December 2012 and in revised form 29 May 2013.
1 To whom correspondence should be addressed.
Published, JLR Papers in Press, June 5, 2013
DOI 10.1194/jlr.M035139
e-mail: michel.guichardant@insa-lyon.fr
Copyright © 2013 by the American Society for Biochemistry and Molecular Biology, Inc.
This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 54, 2013
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by using a linear solvent gradient: solvent A was a mixture of ac-
etonitrile/water acidified to pH 3 (10/90, v/v), and solvent B was
acetonitrile. The flow was set at 1 ml/min. Mono- and di-hydrox-
ylated fatty acids were detected with a diode array detector at 235
nm and 270 nm, respectively, and collected separately.
this fatty acid is taken in large amounts (1–2 g/day in
human adults) as a nutrient and hardly accumulates in
plasma, suggesting an active metabolism. Moreover, its
conversion into EPA and DHA is relatively limited. A con-
version of ALA into its long-chain product DHA was found
to be around 1% in men (20) and 2% in young women
(21). Formation of 9,16-dihydroxyoctadecatrienoic acid
(9,16-di-HOTE) isomers from ALA by sLOX-2 have already
been reported first in 1984 by Feiters et al. (22) without
establishing the double bond configuration or the stereo-
chemistry of the asymmetric carbons. Further studies pre-
sented by Sok and Kim (23, 24) reported four isomers, but
again their characterization was not fully established, espe-
cially that of the all trans isomers. In 1991 Grechkin et al.
(25) showed that potato tuber lipoxygenase (LOX) gener-
ated mainly 9(S)-hydroperoxy-octadeca-10E,12Z,15Z-trienoic
acid [9(S)-HpOTE] which is different from the sLOX
which produces mainly 13(S)-hydroperoxy-9Z,11E,15Z-
octadecatrienoic acid [13(S)-HpOTE]. Also, two com-
pounds after reduction by NaBH4 were characterized as
9,16-di-HOTE (10E,12Z,14E) except for the stereochemis-
try of the carbon 16 which remains undetermined and
trans isomers were not detected. Moreover, the biological
properties of the 9,16-di-HOTE isomers have not been in-
vestigated yet. Because ALA is the most abundant n-3 PUFA
in human diet, it is relevant to investigate more thoroughly
its oxygenated metabolism, especially by 15-LOX that is ex-
pressed in blood leukocytes and most endothelial cells, and
compare the biological activity of the metabolites to that of
PDX, the main 15-LOX product of DHA.
GC-MS analysis of mono- and di-hydroxylated fatty acids
Mono- and di-hydroxylated fatty acids isolated by RP-HPLC
were hydrogenated using PtO2 as a catalyst, and then derivatized
into methyl esters and TMS ethers. Samples were then analyzed
by GC-MS using the electron impact ionization (EI) mode in order
to localize the hydroxyl groups on the fatty chain (18).
NMR of 9,16-di-HOTEs
The NMR experiments were performed on a Bruker-Biospin
NMR spectrometer operating at 23.4 Tesla (1,000 MHz of Proton
Larmor frequency) and equipped with a Cryoprobe. All the spec-
tra were acquired in a CDCl3 solution, working at 298 K and ref-
erencing the chemical shifts to tetramethylsilane as an internal
standard. Standard sequences were used for 1D, DQF COSY
(26–28), and J-resolved (29–31) spectra. The 1H pulse lengths of
9.0 ␮s, 1 s, and 5 s were used as recycle delay for J-resolved and
COSY spectra. A weak selective decoupling of 270 Hz of power
during the acquisition time (900 ms) was used in 1D selective
homo-decoupling experiments to simplify some coupling pat-
terns. 2D DQF COSY was acquired with 8 scans and 2,048 × 1,024
real points (F2 × F1) with acquisition times of 114 ms and 57 ms
for the direct and indirect dimensions. J-resolved 2D was ac-
quired with 16 scans and 8,192 × 256 real points, acquiring 458 ms
in the direct dimension and 1,280 ms in the indirect J-coupling di-
mension. COSY spectra were processed with a 4,096 × 4,096 point
matrix using square cosine and square sine window function for
the direct and indirect dimensions. J-resolved 2D spectra were
processed with an 8,192 × 2,048 point matrix with square cosine
window function for both the dimensions.
Incubation of ALA, 9(S)-HOTE, and 9( )-HOTE with
human recombinant 15-LOX-2
MATERIALS AND METHODS
Materials
ALA, 9(S)-HOTE, and 9( )-HOTE were incubated separately
with human recombinant 15-LOX-2 in Tris-HCl buffer as previ-
ously described (32, 33). Resulting hydroperoxides were reduced
by NaBH4. Lipids were extracted after acidification to pH 3 with
acetic acid and di-hydroxylated metabolites were purified as de-
scribed above.
ALA, sLOX (E.C. 1.13.11.12, Type 1-B, 131,000 units/mg), plat-
inum oxide (PtO2), N,O-bis(trimethylsilyl)-trifluoroacetamide,
COX-1, and COX-2 (E.C. 1.14.99.1) were from Sigma-Aldrich.
Human recombinant 15-LOX-2 (0.23 units/mg), prostaglandin
D2 (PGD2)-d4, and prostaglandin E2 (PGE2)-d4 were from Cayman.
9(S)-hydroxy-10E,12Z,15Z-octadecatrienoic acid [9(S)-HOTE],
9(S)-HpOTE, and 13(S)-hydroxy-9Z,11E,15Z-octadecatrienoic
acid [13(S)-HOTE] were from Interchim. Organic solvents were
from Carlo-Erba. All chemicals used were reagent grade or the
highest quality available.
Preparation of cell suspensions
Platelet suspensions. Venous blood from healthy volunteers
who had not taken any medication for at least one week was col-
lected at the local blood bank (Etablissement Français du Sang).
Blood was drawn onto anticoagulant and centrifuged at 200 g for
15 min at 20°C to obtain the platelet-rich plasma upper phase,
and platelets were isolated as described previously (34). Briefly,
platelet-rich plasma was acidified to pH 6.4 with 0.15 M citric acid
and immediately centrifuged at 900 g for 10 min, and platelet
pellets were suspended into Tyrode-HEPES buffer (140 mM
NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 12 mM NaHCO3, 0.41 mM
NaH2PO4, 5 mM HEPES, and 5.5 mM glucose, pH 7.35).
Biosynthesis of 9,16-dihydroxy-ALA derivatives
ALA was incubated with sLOX (type 1-B) in sodium-borate
buffer. Synthesized hydroperoxides were reduced by sodium
borohydride (NaBH4) as previously described (18). After acidifi-
cation, mono- and di-hydroxylated fatty acids were extracted on a
C18 solid-phase cartridge as previously described (18). Commer-
cial 9(S)-HOTE, 9(S)-HpOTE, and the homemade racemic 9( )-
HOTE chemically produced by oxygen treatment of ALA and
isolated by reverse-phase high-performance liquid chromatogra-
phy (RP-HPLC) were further treated by sLOX in order to gener-
ate 9,16-di-HOTEs with defined stereochemistry.
Leukocyte suspensions. Leukocytes were isolated from blood
obtained from Etablissement Français du Sang, as previously de-
scribed (35). Dextran (2% final concentration) was added to the
lower phase obtained after the first blood centrifugation at 200 g.
Erythrocytes were then sedimented under gravity for 45 min at
15°C. The erythrocyte-depleted supernatant containing leukocytes
Purification of mono- and di-hydroxylated fatty acids
Mono- and di-hydroxylated fatty acids were analyzed by RP-
HPLC on a Waters XBridge C18 column (4.6 × 250 mm, 3.5 ␮m)
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Journal of Lipid Research Volume 54, 2013

was centrifuged at 400 g for 10 min. The resulting leukocyte-rich
pellets were resuspended into saline buffer, and layered over a
Ficoll-Paque Plus (density 1.077). After centrifugation, the frac-
tion above the erythrocyte layer was removed and centrifuged,
and resuspended in water for 20 s to lyse the contaminant eryth-
rocytes. Osmotic pressure was immediately restored with NaCl,
followed by centrifugation, which provided a white pellet consist-
ing of whole leukocytes that was resuspended into Tyrode-HEPES
buffer. The morphological examination and trypan blue exclu-
sion tests were performed to determine the cell count and purity,
and evaluate the viability of neutrophils.
to pH 3 with acetic acid. The oxygenated metabolites were then
extracted as previously described (18).
GC-MS measurement of purified COX-1 and COX-2
products
Commercial COX-1 (40 units) and COX-2 (40 units) were pre-
incubated for 15 min in a 0.1 M Tris-HCl buffer pH 8.1 with or
without 1 ␮M of different di-hydroxylated metabolites and incu-
bated for 10 min with 10 ␮M arachidonic acid used as a substrate.
Albumin (45 g/l) was added at the end of incubation to facilitate
the formation of PGD2 and PGE2 from PGH2 (37). After acidifica-
tion to pH 3 with acetic acid, PGE2 and PGD2 were extracted by
10 vol of diethyl ether stabilized by 10% ethanol. Deuterated
PGD2 and PGE2 used as internal standards were added for the
quantification. Prostaglandins were derivatized into methoxime,
pentafluorobenzyl esters, and TMS and further analyzed by neg-
ative-ion chemical ionization GC-MS. PGD2 and PGE2 were
measured using the SIM mode. Selected ions corresponding to
[M-181]Ϫ (loss of the pentafluorobenzyl group): m/z 524 for both
derivatized PGD2 and PGE2 and m/z 528 for their corresponding
deuterated internal standards (38, 39) were measured.
Measurement of platelet aggregation and incubation
with human leukocytes
Blood platelet aggregation was determined by the turbidimet-
ric method of Born (36) using a dual aggregometer (ChronoLog,
Havertown, PA). To measure changes in the light transmission
rate, 400 ␮l of platelet suspension was heated at 37°C for 1 min
with stirring, incubated in the presence or absence of di-hydrox-
ylated compounds (added in 1 ␮l of ethanol) for one more minute,
and then with collagen as a platelet aggregatory agent for 4 min
at 37°C. Concentrations of collagen were adjusted to obtain
nearly 75% aggregations for control platelets.
Statistical analysis
Isolated human leukocytes, suspended in a Tyrode-HEPES buffer
containing 2 mM Ca2+ were preincubated in the presence or ab-
sence of 1 ␮M of different 9,16-di-HOTE isomers for 3 min at 37°C,
and triggered with 1 ␮M ionophore A23187 plus 10 ␮M arachidonic
acid for 10 min. The reaction was stopped by acidifying the media
All results are expressed as means standard error of the mean
(SEM) or means standard deviation (SD). P < 0.05 was set as the
level of significance as determined by two-tailed Student’s t-tests.
One-way ANOVA analysis was used to assess differences between
groups.
Fig. 1. Typical RP-HPLC chromatogram of ALA metabolites. ALA was incubated with sLOX, and further treated by NaBH4 to reduce
hydroperoxides into hydroxylated derivatives. The lipid extract was then analyzed by RP-HPLC. Mono-hydroxylated fatty acids (A) and di-
hydroxylated fatty acids (B) were detected at ␭= 235 nm and 270 nm, respectively. On the right side of the chromatograms, the UV spectra
acquired on the top of chromatographic peaks are reported. mAU, milli absorbance units.
Di-hydroxylated metabolites from ALA
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Localization by GC-MS of hydroxyl groups on the
fatty chain
RESULTS
Characterization of metabolites obtained from ALA
incubated with sLOX
Mono- and di-hydroxylated compounds from ALA
mentioned above were hydrogenated and derivatized as
methyl esters and TMS ethers as described in Materials
and Methods and analyzed by GC-MS using the EI
mode.
The EI mass spectrum of the hydrogenated mono-
hydroxylated ALA derivative (Fig. 2A) shows character-
istic ions at: m/z 371 (M-15) loss of CH3, m/z 315 (M-71)
which corresponds to the loss of CH2-(CH2)3-CH3, and
m/z 173 [TMS-O-CH-(CH2)4-CH3]. Such a fragmenta-
tion pattern clearly indicates the presence of the hy-
droxyl group on carbon 13 of the fatty chain. So ALA
was mainly converted into 13(S)-HpOTE, further re-
duced by NaBH4 into hydroxylated product for analyti-
cal purposes.
All four hydrogenated di-hydroxylated compounds
from ALA exhibited the same mass spectra (Fig. 2B).
Their fragmentation pattern shows the following char-
acteristic ions: at m/z 459 (M-15, loss of CH3); at m/z 445
(M-29, loss of CH2-CH3); at m/z 317 [M-157, loss of
CH2-(CH2)6-COOCH3]; at m/z 259 [TMS-O-CH-(CH2)7-
COOCH3]; at m/z 131 (TMS-O-CH-CH2-CH3); and at
m/z 73 (TMS) which indicates an oxygenation at car-
bons 9 and 16.
From ALA incubated with sLOX as described in Materi-
als and Methods, hydroxylated derivatives analyzed by RP-
HPLC provided one main compound detected at 50 min
with a ␭max of 235 nm (Fig. 1A). Its retention time and its
ultraviolet (UV) spectrum were similar to that of commer-
cial 13(S)-HOTE (result not shown).
Regarding di-hydroxylated products (Fig. 1B), and in
contrast to DHA which is only converted into one major di-
hydroxylated metabolite called PDX, four di-hydroxylated
isomers were detected from ALA. They all exhibited charac-
teristic UV spectra with a maximum absorption (␭max) at
270 nm with two shoulder peaks at 260 nm and 280 nm,
indicating the presence of a conjugated triene. Moreover,
the UV spectra of the first two isomers eluted at 32.1 and
32.6 min are superimposable to that of commercial 12-epi-
all trans-leukotriene B4 (LTB4) with an E,E,E conjugated
triene geometry (result not shown), whereas the UV spectra
of the two other isomers eluted at 33.2 and 33.5 min are
superimposable to that of PDX, suggesting an E,Z,E geom-
etry of these conjugated trienes (Fig. 1B). The spectra of
these four isomers differ from those with a Z,E,E configura-
tion such as LTB4 and 12-epi-LTB4 (result not shown).
Fig. 2. Electron impact mass spectrum of the Me-TMS derivative of the hydrogenated HOTE (A) and
hydrogenated di-HOTE (B) synthesized from ALA by sLOX followed by reduction of hydroperoxides into
hydroxylated derivatives by NaBH4 treatment.
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Journal of Lipid Research Volume 54, 2013

Determination of carbon stereochemistry of
from those with ALA as a substrate (Fig. 4). As a matter of
fact, another major E,Z,E product (retention time 33.5 min,
see also Fig. 1B) is likely 9(R),16(S)-di-HOTE. In addition,
the same compound appeared when the racemic 9( )-HOTE
was incubated with sLOX (Fig. 3B, retention time 33.7 min).
However, we may assume that the two all trans diaste-
reoisomers synthesized from ALA, as this will be con-
firmed by NMR below (retention time 32.1 and 32.6 min)
(Fig. 1B), were likely to be all trans 9(R),16(S)-di-HOTE
and 9(S),16(S)-di-HOTE, respectively, according to the
separation of 8(R),15(S)- and 8(S),15(S)-all trans-diHETEs
reported in (41, 42). Such an elution order has also been
established for the separation of 5(S),12(R)- and 5(S),12(S)-
all trans LTB4 and 5(S),12(R)- and 5(S),12(S)-all trans
LTB5 diastereoisomers (43–45).
di-hydroxylated metabolites synthesized from ALA
Commercial 9(S)-HOTE and racemic 9( )-HOTE, pre-
pared as described in Materials and Methods, were incu-
bated separately with sLOX as described with ALA, and
further reduction by NaBH4. 9(S)-HOTE was metabolized
into a main compound eluted at 33.4 min (Fig. 3A). This
peak was attributed to 9(S),16(S)-di-HOTE because sLOX
produces almost exclusively the S enantiomer via a double
dioxygenation mechanism (40), although soybean LOX
generates almost exclusively 13(S)-HpOTE from ALA (40)
as it was already shown (Fig. 1A). On the other hand, the
9(R),16(S)-di-HOTE peak, eluting at 33.7 min, was also
formed (Fig. 3B) when racemic 9( )-HOTE was incubated
with sLOX, compared with 9(S)-HOTE. The two diastere-
oisomers 9(S),16(S)-di-HOTE and 9(R),16(S)-di-HOTE
eluted at 33.4 min and 33.7 min (Fig. 3B), respectively, ex-
hibited the same intensity in agreement with the equivalent
amounts of 9(S)- and 9(R)-HOTE present in the racemic
mixture. The latter chromatogram (Fig. 3B) is similar to that
presented in Fig. 1B, suggesting that the two peaks (retention
time 33.2 and 33.5 min, Fig. 1B) observed with ALA as a
substrate correspond to the two diastereoisomers 9(S),16(S)-
di-HOTE and 9(R),16(S)-di-HOTE, respectively.
NMR determination of the conjugated triene double
bound geometry
A NMR investigation was performed to determine the
E/Z configuration in the triene moiety. A change in the
1
E/Z configuration significantly modifies the H-1H J-cou-
plings among the protons bound to the sp2 carbons in C=C
double bonds, assuming values around ف17 Hz for E con-
figuration and around ف10 Hz for Z configuration (46).
Furthermore, molecules like 9(S),16(S)-dihydroxy-
10E,12Z,14E-octadecatrienoic acid (33.2 min, Fig. 1B) and
9(R),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic acid
This assignment was also confirmed by the fact that
chromatograms of di-hydroxylated isomers from 9(S)-
HpOTE and 9(S)-HOTE were almost identical, but different
Fig. 3. RP-HPLC profile of 9(S),16(S)-di-HOTE synthesized from 9(S)-HOTE by sLOX (A); RP-HPLC profile of 9(S),16(S)-di-HOTE and
9(R),16(S)-di-HOTE synthesized from 9( )-HOTE by sLOX (B). The intermediate hydroperoxide products were reduced by NaBH4 prior
to analysis. mAU, milli absorbance units.
Di-hydroxylated metabolites from ALA
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Fig. 4. RP-HPLC profile of 9,16-di-HOTEs synthe-
sized by sLOX from ALA (A); from 9(S)-HpOTE
(B); and from 9(S)-HOTE (C). The intermediate
hydroperoxide products were reduced by NaBH4
prior to analysis. mAU, milli absorbance units.
(33.5 min, Fig. 1B), which only differ for the inversion of
the stereochemical configuration at the chiral center of
position 9, should show very similar spectra with minor dif-
ferences in chemical shift and J-couplings, especially in
the triene region. On this basis, the inspection of the 1D
NMR spectra, and in particular of the triene resonance
region (4–7 ppm), allows us to rapidly distinguish among
the ensemble of molecules having the same triene config-
uration [e.g., the E,E,E compounds (32.1 min and 32.6
min, Fig. 1B) from the E,Z,E compounds (33.2 min and
33.5 min, Fig.1B)], as it can be seen in Figs. 5 and 6.The
resonance assignment has been determined by using DQF
COSY, while a large part of the J-couplings were measured
through the analysis of 2D J-resolved spectra (data not
shown). In such a way, it was possible to determine that the
1H-1H 3J-couplings between protons in positions 10-11
and 14-15 is around 15 Hz for all the examined cases, evi-
dencing that the protons are in trans position with re-
spect to the double bond. The determination of the 1H-1H
3J-coupling among protons 12 and 13 is complicated by
the quasi-equivalence of their chemical shifts (strong-cou-
pling condition) and by the presence of additional cou-
plings with the closer protons 11 and 14. To simplify the
problem, we observed the resonances of protons 12 and
13 in the 1D spectra of 9(R),16(S)-dihydroxy-10E,12Z,14E-
octadecatrienoic acid (33.5 min, Fig. 1B) upon selective
decoupling of the protons 11 and 14. This made a signifi-
cant simplification of the multiplet pattern making evident
a 3J12-13 coupling of ف10 Hz typical of Z configuration. The
same approach could not be done in the case of the E,E,E
molecules because of the strong overlap among the pro-
tons 11, 12, 13, and 14 that have a very similar chemical
shift. Nevertheless, because the determination of the con-
figuration in the E,Z,E molecules is demonstrated above,
we can safely assign by exclusion the configuration of the
E,E,E molecules on the basis of the marked differences
among the spectra of E,E,E and E,Z,E.
Metabolism of ALA by human recombinant 15-LOX-2
This experiment was done to check whether the human
recombinant 15-LOX type 2 is also able to synthesize such
metabolites. For this purpose ALA, 9(S)-HOTE, and 9( )-
HOTE were separately incubated with this enzyme. We
found that human recombinant 15-LOX-2 is able to con-
vert ALA into 13-HOTE as previously described with the
sLOX (result not shown). However, ALA was less markedly
metabolized into di-hydroxylated fatty acids, the main
ones being all trans 9,16-di-HOTE isomers. Interestingly
enough, the mono-hydroxylated derivatives 9(S)-HOTE
and 9(R)-HOTE were well converted into 9(S),16(S)-E,Z,E-
di-HOTE and 9(R),16(S)-E,Z,E-di-HOTE, respectively (re-
sult not shown) by the human recombinant LOX.
Inhibitory effect of 9,16-di-HOTEs on COX-1 and anti-
aggregatory effects
The inhibition of sheep COX-1 by different 9,16-di-
HOTE isomers was tested at 1 ␮M by measuring both PGD2
and PGE2 as described in Materials and Methods. Results
reported in Table 1 show that 1 ␮M of 9(S),16(S)-dihydroxy-
10E,12Z,14E-octadecatrienoic and 9(R),16(S)-dihydroxy-
10E,12Z,14E-octadecatrienoic acids decreased significantly
the synthesis of both PGD2 and PGE2 by around 28% and
38%, respectively. Such results are in agreement with pre-
vious data showing that poxytrins, characterized by a E,Z,E
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Journal of Lipid Research Volume 54, 2013

Fig. 5. 1D spectra and J-couplings for 9(S),16(S)-dihydroxy-10E,12E,14E-octadecatrienoic acid (A) and 9(R),16(S)-dihydroxy-10E,12E,
14E-octadecatrienoic acid (B). Resonance assignment and J-couplings were determined from the analysis of a DQF COSY, and 2D J-resolved
spectra (data not shown). Spectra were acquired with eight scans and 2 s of recycle delay, the acquisition time was 1.8 s. Spectra were pro-
cessed with exponential line broadening of 0.3 Hz as window function.
Di-hydroxylated metabolites from ALA
2089

Fig. 6. 1D spectra and J-couplings for 9(S),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic acid (A) and 9(R),16(S)-dihydroxy-10E,12Z,14E-
octadecatrienoic acid (B). Resonance assignments and J-couplings were determined from the analysis of a DQF COSY, and 2D J-resolved
spectra (data not shown). Spectra were acquired with eight scans and 2 s of recycle delay, the acquisition time was 1.8 s. Spectra were pro-
cessed with exponential line broadening of 0.3 Hz as window function.
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Journal of Lipid Research Volume 54, 2013

TABLE 1. Effect of 9,16-di-HOTEs on cyclooxygenase-1 and -2
Control
9(S),16(S)-di-HOTE E,Z,E
9(R),16(S)-di-HOTE E,Z,E
COX-1
(PGD2 + PGE2)
COX-2
(PGD2 + PGE2)
134.9 15.8
136.1 11.0
96.8 14.0b
124.5 8.8a
83.2 9.0b
107.4 7.2b
Purified COX-1 and COX-2 were incubated with or without 1 ␮M of different 9,16-di-HOTE isomers and with
10 ␮M arachidonic acid. PGD2 and PGE2 were measured by GC-MS. Results expressed in ng of PGD2 and PGE2
represent the mean SEM of four determinations. Only the inhibitory effects of E,Z,E isomers are shown.
aP < 0.05.
bP < 0.01 versus control.
triene geometry, are inhibitors of platelet COX-1 (19),
whereas the all trans isomers are inactive.
Effect of 9,16-di-HOTEs on the metabolism of arachidonic acid by
the human polymorphonuclear leukocyte 5-LOX. The inhibition
of the human 5-LOX pathway by 1 ␮M of 9,16-di-HOTE iso-
mers was assessed by measuring the following metabolites:
5(S)-HETE, LTB4 and its two all trans isomers. As reported in
Table 2, only the 9(R),16(S)-dihydroxy-10E,12Z,14E-octade-
catrienoic acid isomer significantly decreased the formation
of leukotrienes and 5(S)-HETE by around 10% and 20%, re-
spectively. All other isomers did not inhibit such a produc-
tion (results not shown). Such data suggest that the inhibition
concerns only 5-LOX but not leukotriene A4 hydroxylase.
Taken together, these data suggest that the 9(R),16(S)-dihy-
droxy-10E,12Z,14E-octadecatrienoic acid isomer may have a
potential anti-inflammatory effect.
Such results are in agreement with data relating to plate-
let aggregation induced by collagen. Platelet aggregation
was significantly decreased by 64% and 65% in presence
of 1 ␮M 9(S),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic
acid and 9(R),16(S)-dihydroxy-10E,12Z,14E-octadecatrie-
noic acid, respectively (Fig. 7). In addition, the aggrega-
tion started to be reversed from 2 min following the agonist
addition. As previously shown (19), all trans isomers had
no effect on platelet aggregation.
Anti-inflammatory effect of 9,16-di-HOTEs
After showing that 9,16-dihydroxy-10E,12Z,14E-octade-
catrienoic acid isomers possess anti-aggregatory properties
as those previously described for poxytrins (19), it was im-
portant to know whether such di-hydroxylated compounds
issued from ALA may exhibit anti-inflammatory proper-
ties. We then investigated the effects of 9,16-di-HOTEs on
COX-2 as well as the 5-LOX pathway.
DISCUSSION
ALA is an essential fatty acid present in vegetable oils,
and as such available in human diet. It is the precursor of
EPA and DHA, but its conversion into EPA and DHA is
rather limiting with around 10% to the former and a fur-
ther 10% to the latter, which means only 1% conversion of
ALA to DHA (47, 48). ALA is also rather well ␤-oxidized
(49, 50), that could partially explain its low accumula-
tion in plasma and tissues. In addition, we describe here a
pathway which could explain some beneficial effects of
ALA in the vascular system at large by reducing both the
Effect of 9,16-di-HOTEs on human recombinant COX-2. The
inhibition of human recombinant COX-2 by 1 ␮M of 9,16-
di-HOTE isomers was assessed by measuring PGD2 and
PGE2 as described above. Results reported in Table 1 show
that 9(S),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic
and 9(R),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic ac-
ids significantly inhibited COX-2 activity by 9% and 21%,
respectively.
Fig. 7. Effect of E,Z,E-9,16-di-HOTEs on platelet
aggregation triggered by collagen. Platelet suspen-
sion (400 ␮l) was preincubated for 1 min at 37°C
and triggered by ف0.05 ng/␮l collagen in the pres-
ence or absence of 1 ␮M 9,16-di-HOTEs. Platelet ag-
gregation was monitored for 4 min. The aggregation
profiles shown are representative of three indepen-
dent experiments. T, transmission.
Di-hydroxylated metabolites from ALA
2091

TABLE 2. Effect of 9,16-di-HOTEs on human leukocyte 5-LOX
in order of increasing retention time, respectively. Such a
configuration is also in agreement with the 5(S),12(R)- and
5(S),12(S)-alltransLTB4 andthe5(S),12(R)-and5(S),12(S)-
all trans LTB5 diastereoisomers already described (43–45). We
conclude that the four di-hydroxylated compounds issued
from ALA treated by sLOX are 9(R),16(S)-dihydroxy-10E,12E,
14E-octadecatrienoicacid,9(S),16(S)-dihydroxy-10E,12E,14E-
octadecatrienoic acid, 9(S),16(S)-dihydroxy-10E,12Z,14E-
octadecatrienoic acid, and 9(R),16(S)-dihydroxy-10E,12Z,
14E-octadecatrienoic acid, in order of increasing retention
time, respectively. Such data suggest that there are two dif-
ferent mechanisms explaining the formation of these 9,16-di-
HOTE isomers. The E,Z,E compounds 9(S),16(S)-di-HOTE
and 9(R),16(S)-di-HOTE would result from a dioxygen-
ation mechanism as previously shown for PDX (18), whereas
the all trans 9,16-di-HOTE isomers might derive from un-
known intermediates.
We also investigated the metabolism of ALA incubated
with human recombinant 15-LOX-2. Interestingly, 9(S)-
HOTE or 9(R)-HOTE were both well converted into the
corresponding 9,16-di-HOTEs whereas ALA was less effi-
ciently metabolized into those di-hydroxylated fatty acids
than by sLOX. Such a result might be biologically relevant
because 9(S)-HOTE can be produced through cyclooxyge-
nases, and 9(R)-HOTE can be produced via aspirinated
COX-2, both in an aborted cyclooxygenation process (54).
The present study also shows some biological effects of the
E,Z,E di-hydroxylated 9,16-di-HOTE isomers. We observed
that 9(S),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic and
9(R),16(S)-dihydroxy-10E,12Z,14E-octadecatrienoic acids
decreased significantly prostaglandin synthesized by recom-
binant COX-1. Moreover, these E,Z,E isomers inhibit platelet
aggregation triggered by collagen both by decreasing the ex-
tent of aggregation and making it partially reversible (Fig. 7).
However, these E,Z,E di-hydroxylated ALA metabolites ap-
pear to be less potent inhibitors than PDX under the same
conditions, although the anti-aggregatory effects observed
are in agreement with our previous data showing that
poxytrins, characterized by an E,Z,E conjugated triene, are
inhibitors of COX-1 (19).
COX-2 is an inducible enzyme becoming abundant at
sites of inflammation, and is upregulated in various carci-
nomas, having a central role in tumorigenesis (55). The
inhibition of COX-2 is a major mode of action to reduce
inflammation (56). In our study, we found that 9(S),16(S)-
dihydroxy-10E,12Z,14E-octadecatrienoic and 9(R),16(S)-
dihydroxy-10E,12Z,14E-octadecatrienoic acids exhibit an
inhibitory effect on the human recombinant COX-2.
Moreover, 9(R),16(S)-dihydroxy-10E,12Z,14E-octadeca-
trienoic acid also decreased significantly the formation of
LTB4 and 5(S)-HETE from arachidonic acid incubated
with human polymorphonuclear leukocytes, indicating
the inhibition of 5-LOX.
It is interesting to note that in all the tests evaluated,
the E,Z,E 9(R),16(S) isomer was a more potent inhibitor
than the E,Z,E 9(S),16(S) isomer. This reinforces the in-
terest of the 15-lipoxygenation of 9(R)-HOTE produced
by aspirinated COX-2 (54) to synergize with aspirin in
atherothrombogenesis.
Control
9(R),16(S)-di-HOTE E,Z,E
LTB4s
9.57 0.66
0.89 0.03
10.46 0.68
8.78 0.78a
0.71 0.05a
9.49 0.81a
5-HETE
5-LOX products
Isolated human leukocytes were incubated in the presence or
absence of 1 ␮M of different 9,16-di-HOTE isomers at 37°C and
triggered with 1 ␮M of ionophore A23187 and 10 ␮M of arachidonic
acid. Oxygenated metabolites were then extracted and quantified by
RP-HPLC. Results expressed in nmol of total LTB4s (LTB4 + LTB4
isomers) and 5-HETE represent the mean SEM of five determinations.
Only the inhibitory effect of 9(R),16(S)-di-HOTE is shown.
aP < 0.05, versus control.
production of pro-inflammatory eicosanoids PGD2/PGE2
and LTB4 (51–53). We recently showed that a new di-hy-
droxylated metabolite from DHA called PDX, which is an
isomer of the anti-inflammatory PD1, exhibits anti-aggre-
gatory properties, both by inhibiting collagen-induced
prostanoid production, including thromboxane A2, and
thromboxane A2-induced aggregation (19). In the present
paper we report the full characterization of the lipoxygen-
ation of ALA, and the anti-inflammatory and anti-aggrega-
tory effects of two LOX end-products. The structure of the
four LOX metabolites that we herein describe have already
been partially reported (23, 24), but the proposed stereo-
chemistry structures of the 9(S),16(R)-di-HOTE do not fit
with our experimental data. On the other hand, their bio-
logical properties have not been investigated.
Our data show that ALA is mainly converted into its 13-
oxygenated derivative 13-HOTE by 15/␻6-LOX. However,
formation of a small amount of the 9-oxygenated product
9-HOTE could also be observed. In contrast to DHA, which
is converted into only one main di-hydroxylated compound
(PDX) via a double lipoxygenation, four di-hydroxylated
compounds were observed from ALA following incubation
with 15-LOX, characterized as 9,16-di-HOTEs. The stereo-
chemistry of these di-hydroxylated compounds was assessed
by incubating 9(S)-HOTE and racemic 9( )-HOTE sepa-
rately with sLOX (see the above results). The UV spectra of
the two main di-hydroxylated products are superimposed
to that of PDX, which possesses a E,Z,E conjugated triene
geometry. The diastereoisomers are likely produced via a
dioxygenation mechanism as previously described for PDX
(18) and these two compounds are likely to have an E,Z,E
conjugated triene geometry. Such a geometry was further
confirmed by high field NMR which allowed us to deter-
1
mine precisely the H-1H J-couplings among the protons
bound to the sp2 carbons in C=C double bonds. The geom-
etry of the conjugated trienes of two additional but less
abundant (around half the amount of the E,Z,E-9(S),16(S)-
di-HOTE and 9(R),16(S)-di-HOTE) di-hydroxylated prod-
ucts was supposed to be E,E,E because their UV spectra were
superimposed to that of all trans LTB4, which has such an
E,E,E conjugated triene geometry. This all trans geometry
was also confirmed by NMR to determine the 1H-1H J-cou-
plings among the protons bound to the sp2 carbons in C=C
double bonds. Based on data from the literature, we may
suggest that the stereochemistry of these two minor products
(Fig. 1B) is 9(R),16(S)- and 9(S),16(S)-all trans-di-HOTE,
2092
Journal of Lipid Research Volume 54, 2013

actions of neuroprotectin D1/protectin D1 and its natural stere-
oisomers: assignments of dihydroxy-containing docosatrienes.
J. Immunol. 176: 1848–1859. [Erratum. 2006. J. Immunol. 176:
3843.]
In conclusion, we observe from the present study that four
di-hydroxylated compounds are produced from ALA treated
by sLOX. They are 9(R),16(S)-dihydroxy-10E,12E,14E-octa-
decatrienoic acid, 9(S),16(S)-dihydroxy-10E,12E,14E-octade-
catrienoic acid, 9(S),16(S)-dihydroxy-10E,12Z,14E-octadeca
trienoic acid, and 9(R),16(S)-dihydroxy-10E,12Z,14E-octa-
decatrienoic acid. Interestingly, 9,16-dihydroxy-10E,12Z,
14E-octadecatrienoic acid isomers exhibit anti-aggregatory
properties as other poxytrins (19). In addition, 9(R),16(S)-
dihydroxy-10E,12Z,14E-octadecatrienoic acid, which can
be produced from 9(R)-HOTE by aspirinated COX-2, inhib-
its both cyclooxygenases and the 5-LOX pathway and appears
to be a potential anti-inflammatory compound in vitro.
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