COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Article abstract of DOI:10.1194/jlr.M017822

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-di-HETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5 S -HETE as a substrate for COX-2 forming 5 S,15 S -diHETE, 5 S,15 R -diHETE, and 5 S,11 R -di-HETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB 4 (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5 S,12 S -diHETE with the unusual 6 E,8 Z,10 E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A 4 -epoxide. Exogenous octadeuterated 5 S -HETE and 15 S -HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes. Copyright

Full text of DOI:10.1194/jlr.M017822

COX-2-dependent and -independent biosynthesis
of dihydroxy-arachidonic acids in activated
human leukocytes
Noemi Tejera, William E. Boeglin, Takashi Suzuki, and Claus Schneider1
Division of Clinical Pharmacology, Department of Pharmacology, and Vanderbilt Institute of Chemical
Biology, Vanderbilt University Medical School, Nashville, TN
Abstract Biosynthesis of 5,15-dihydroxyeicosatetraenoic
acid (5,15-diHETE) in leukocytes involves consecutive oxy-
genation of arachidonic acid by 5-lipoxygenase (LOX) and
15-LOX in either order. Here, we analyzed the contribution
of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-di-
HETE and 5,11-diHETE in isolated human leukocytes acti-
vated with lipopolysaccharide and calcium ionophore
A23187. Transformation of arachidonic acid was initiated
by 5-LOX providing 5S-HETE as a substrate for COX-2
forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-di-
HETE as shown by LC/MS and chiral phase HPLC analyses.
The levels of 5,15-diHETE were 0.45 0.2 ng/106 cells
(mean SEM, n = 6), reaching about half the level of LTB4
(1.3 0.5 ng/106 cells, n = 6). The COX-2 specific inhibitor
NS-398 reduced the levels of 5,15-diHETE to below 0.02
ng/106 cells in four of six samples. Similar reduction was
achieved by MK-886, an inhibitor of 5-LOX activating pro-
tein but the above differences were not statistically signifi-
cant. Aspirin treatment of the activated cells allowed
formation of 5,15-diHETE (0.1 0.05 ng/106 cells, n = 6)
but, as expected, abolished formation of 5,11-diHETE. The
mixture of activated cells also produced 5S,12S-diHETE
with the unusual 6E,8Z,10E double bond configuration, im-
plicating biosynthesis by 5-LOX and 12-LOX activity rather
than by hydrolysis of the leukotriene A4-epoxide. Exogenous
octadeuterated 5S-HETE and 15S-HETE were converted to
5,15-diHETE, implicating that multiple oxygenation path-
ways of arachidonic acid occur in activated leukocytes.
The contribution of COX-2 to the biosynthesis of dihydrox-
ylated derivatives of arachidonic acid provides evidence for
functional coupling with 5-LOX in activated human
leukocytes.—Tejera, N., W. E. Boeglin, T. Suzuki, and C.
Schneider. COX-2-dependent and -independent biosynthe-
sis of dihydroxy-arachidonic acids in activated human leuko-
cytes. J. Lipid Res. 2012. 53: 87–94.
Oxygenation of arachidonic acid at the 5-carbon by
5-lipoxygenase (5-LOX) gives rise to 5S-hydroperoxyei-
cosatetraenoic acid (5S-HPETE). 5S-HPETE undergoes
further transformation by 5-LOX to the leukotriene
epoxide LTA4 or reduction to the hydroxy derivative,
5S-HETE (1). 5-LOX is expressed in neutrophils, eosino-
phils, macrophages, and mast cells, and requires support
by the accessory membrane protein 5-LOX activating
protein (FLAP) for enzymatic activity in intact cells (2).
Whereas 5-LOX is the only enzyme capable of oxygenat-
ing the 5-position of arachidonic acid, several enzymes
can oxygenate the 15-carbon (3): two 15-lipoxygenases,
15-LOX-1 (the 12/15-LOX in the mouse) and 15-LOX-2
(4, 5), COX-1 and COX-2, both forming 15-HETE and
11-HETE as by-products of prostaglandin biosynthesis
(6, 7), and aspirin-acetylated COX-2, a pure 15R-oxyge-
nase (8–11).
Insertion of oxygen at one end of arachidonic acid does
not impinge on the reactive pentadiene system at the other
end, and, therefore, double oxygenation at both the 5-
and 15-carbons of arachidonic acid is readily achievable.
For example, formation of 5,15-diHETE in rat mononu-
clear cells and in human leukocytes is catalyzed by con-
secutive transformation of arachidonic acid by 5-LOX and
15-LOX in either order (12). Consecutive oxygenation of
arachidonic acid by 5-LOX and 15-LOX is also instrumen-
tal in the biosynthesis of lipoxins. Lipoxin formation, how-
ever, additionally requires that one of the enzymes executes
LTA-synthase activity, i.e., the dehydration of a hydroperoxide
to an epoxide. Hydrolysis of the epoxide intermediate
furnishes the final trihydroxylated arachidonic acid de-
rivative (13, 14). The 15R-oxygenating activity of aspirin
acetylated COX-2 can substitute for the 15-LOX activity in
Supplementary key words 5-lipoxygenase • leukotriene • prostaglan-
din • 15-lipoxygenase • aspirin
This work was supported by awards R01GM076592, an ARRA administrative
supplement (3R01GM076592-03S1), and P50GM015431 from the National
Institute of General Medical Sciences. The content is solely the responsibility of
the authors and does not necessarily represent the official views of the National
Institute of General Medical Sciences or the National Institutes of Health.
Abbreviations: APCI, atmospheric pressure chemical ionization;
COX, cyclooxygenase; FLAP, 5-lipoxygenase-activating protein; (di)
H(P)ETE, (di)hydro(pero)xyeicosatetraenoic acid; LOX, lipoxygenase;
LPS, lipopolysaccharide; LT, leukotriene; PFB, pentafluorobenzyl;
SRM, selected reaction monitoring.
Manuscript received 9 June 2011 and in revised form 4 November 2011.
Published, JLR Papers in Press, November 7, 2011
DOI 10.1194/jlr.M017822
1 To whom correspondence should be addressed.
e-mail: claus.schneider@vanderbilt.edu
Copyright © 2012 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 53, 2012
87

d8-5-HETE were prepared by chemical synthesis from arachidonic
acid or d8-arachidonic acid as described (20). 5-HETE was re-
solved into enantiomers using chiral-phase HPLC (21). d8-5-HETE
could not be resolved into the enantiomers on the columns avail-
able (Chiralpak AD, Chiralpak AD-RH) and was used as the race-
mic mixture. [5,6,8,9,11,12,14,15]d8-15S-HETE was prepared using
soybean LOX and reduction of the hydroperoxide with triphe-
nylphosphine (22). 6E,8Z,10E-5S,12S-diHETE was prepared by
reaction of 5S-HETE with recombinant platelet 12-LOX ex-
pressed in the baculovirus/Sf9 insect cell system.
lipoxin biosynthesis, resulting in the formation of aspirin-
triggered lipoxins (15).
Our interest in the biosynthesis of 5,15-diHETE stemmed
from its formation as a byproduct of the oxygenation of 5S-
HETE by COX-2 in vitro (16). This finding implicated cross-
over of the 5-LOX and COX-2 pathways as an alternative
biosynthetic route of 5,15-diHETE in vivo with 5-LOX form-
ing 5S-HETE as the first step, followed by COX-2 catalysis as
the second step. The main product of the reaction is a di-
endoperoxide that is structurally similar to the arachidonic
acid-derived endoperoxide of prostaglandin biosynthesis
(17). The catalytic byproducts include 5,15-diHETE (as a
ف4:1 mixture of the 5S,15S- and 5S,15R-diastereomers) and
5S,11R-diHETE (16). The diHETEs are the 5S-hydroxy ana-
logs of the 15- and 11-HETE byproducts of the COX-1 and
COX-2 reactions with arachidonic acid (18, 19).
Thus, 5,15-diHETE may be biosynthesized through a
multitude of pathways that involve reactions of 5-LOX,
15-LOX, COX-1, and COX-2, or acetylated COX-2 as illus-
trated in Fig. 1. Here, we analyzed the biosynthesis of
5,15-diHETE and 5,11-diHETE in activated human leuko-
cytes. The enzymatic reactions involved were elucidated by
the use of inhibitors of 5-LOX and COX-2 activities, deu-
terated HETE substrates, and stereochemical analysis of
the diHETE products.
Leukocyte preparation and extraction
Leukocytes were isolated from peripheral blood from healthy
human subjects. The study was approved by the Vanderbilt Uni-
versity Medical Center Institutional Review Board (protocol
#091243), and informed consent was obtained from the donors.
Venous blood (45 ml) was collected into a syringe containing
5 ml of citric acid and 10 ml of 6% dextran (250 kDa average mole-
cular weight). The blood was allowed to settle for 1 h and the top
layer was collected and centrifuged. The pellet was washed with
PBS, and remaining red cells were lysed by treatment with a 10-
fold excess of deionized water for 30 s. The leukocytes were cen-
trifuged, washed, and diluted in PBS+ containing 5 mM glucose
to a concentration of 1.5 × 107 cells/ml. 1.0 × 107 cells were di-
luted to 1 ml, LPS (10 ␮g/ml) was added, and the cells were in-
cubated at 37°C for 6 h. After addition of calcium ionophore
A23187 (5 ␮M), the cells were incubated for an additional
15 min before the addition of 10 ng of d4-LTB4 standard, acidifi-
cation to pH 4 using acetic acid, and extraction (Waters HLB
cartridge). Products were eluted from the HLB cartridge with
methanol, evaporated under a stream of nitrogen, and dissolved
in 10 ␮l acetonitrile and 40 ␮l of LC-MS column solvent A.
EXPERIMENTAL PROCEDURES
Materials
Arachidonic acid was purchased from NuChek Prep. d8-Arachi-
donic acid, NS-398, and MK-886 were from Cayman Chemical. Li-
popolysaccharide (LPS) (serotype 0111:B4) was from Calbiochem,
d4-LTB4 was from Biomol/Enzo. 5S-HETE and [5,6,8,9,11,12,14,15]
Leukocyte treatment
The inhibitors NS-398 (10 ␮M), AA-861 (10 ␮M), or MK-886
(5 ␮M) were added to the cells 30 min before the end of LPS
treatment. Aspirin was added at 2 mM final concentration (11).
The deuterated substrates, d8-5S-HETE and d8-15S-HETE, respec-
tively, were added at 50, 100, and 500 ng/106 cells (0.75, 1.5, and
7.5 ␮M, respectively) at the time of addition of A23187 and incu-
bated for 15 min before acidification and extraction.
Resolution of 5,15-diHETE diastereomers
Resolution of the 5S,15S- and 5S,15R-diHETE diastereomers
was achieved after derivatization to the pentafluorobenzyl
(PFB) esters using a Chiralpak AD-RH column (4.6 × 150 mm;
5 ␮m) eluted with a solvent of acetonitrile/ethanol (90/10, by
vol.) at a flow rate of 1 ml/min. The ف77:23 mixture of 5S,15S-
and 5S,15R-diHETE formed by reaction of recombinant COX-2
with 5S-HETE (16) was used to determine the elution order of
the diastereomers. For analysis of 5,15-diHETE formed by leu-
kocytes the cells stimulated with A23187 were extracted using
an HLB cartridge, evaporated, and dissolved in 40 ␮l of penta-
fluorobenzyl bromide (10% in acetonitrile) and 20 ␮l of diiso-
propylethylamine. After 1 h incubation at room temperature,
the samples were evaporated and reconstituted in 50 ␮l of ace-
tonitrile for LC/MS analysis. The samples were analyzed using
LC/MS with atmospheric pressure chemical ionization (APCI)
(23). The same ion transition as in ESI analysis (m/z 335 to 201)
was used. Instrument parameters were optimized by direct liq-
uid infusion of a standard of 15-HETE-PFB ester dissolved in
column solvent.
Fig. 1. Pathways for biosynthesis of 5,15-diHETE. Different com-
binations of 5-LOX, 15-LOX, COX-1, COX-2, or aspirin-acetylated
COX-2 can lead to the formation of 5S,15S- and 5S,15R-diHETE
from arachidonic acid (C20.4).
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Journal of Lipid Research Volume 53, 2012

LC/MS and NMR analysis
Samples were analyzed using a ThermoFinnigan Quantum
Access triple quadrupole mass spectrometer equipped with an
electrospray interface and operated in the negative ion mode.
Instrument specific parameters (sheath and auxiliary gas pres-
sures, temperature, and interface voltage) were optimized using
direct infusion of a solution of PGE2. A Waters Symmetry Shield
C18 column (2.1 × 150 mm; 3 ␮m) was eluted with a linear gradi-
ent of acetonitrile/water, 10 mM NH4OAc from 5/95 (by vol.) to
95/5 (by vol.) at 0.2 ml/min within 10 min. The following transi-
tions were recorded in the selected reaction monitoring (SRM)
mode: 5,15-diHETE: m/z 335 to 201; d8-5,15-diHETE: m/z 343 to
208; 5,11-diHETE: m/z 335 to 183; LTB4: m/z 335 to 195; d4-LTB4:
m/z 339 to 197; 5-HETE: m/z 319 to 115; d8-5-HETE: m/z 327 to
116; 15-HETE: m/z 319 to 175. The analysis was split into two seg-
ments monitoring diHETEs from 0 to 8 min and HETEs from 8
to 15 min. Products were quantified by comparison of their peak
area versus the internal standard d4-LTB4 (6,7,14,15-d4-6Z,8E,10E-
5S,12R-diHETE).
NMR spectra were recorded on a Bruker AV-II 600 MHz spec-
trometer equipped with a cryoprobe. CDCl3 was used as solvent
(␦ 7.25 ppm).
RESULTS
Formation of diHETEs in activated leukocytes
Reversed-phase-HPLC analysis of human leukocytes ac-
tivated with LPS for 6 h followed by A23187 for 15 min
showed formation of five products (I–V) with retention
times and UV spectra that were characteristic of diHETE
derivatives of arachidonic acid (Fig. 2A). Products I and IV
were identified as 5,15-diHETE and 5,11-diHETE, respec-
tively, by comparison of HPLC retention times and
UV spectra with authentic standards (Fig. 2B) prepared
by reaction of recombinant COX-2 with 5S-HETE (16).
Product III was identified as LTB4 by coelution with an
authentic standard and UV analysis (Fig. 2C, D). Product
II was tentatively identified as 6E,8E,10E,14Z-5S,12R,S-
diHETE (i.e., a mixture of 6-trans-LTB4 and 6-trans-12-epi-
LTB4) based on retention time relative to LTB4 and UV
spectrum (Fig. 2A, D) (24). The diastereomers of II are the
major products formed by nonenzymatic hydrolysis of the
LTA4 epoxide.
Product V was identified as 6E,8Z,10E,14Z-5S,12S-
diHETE by comparison of retention time and UV spec-
trum with an authentic standard (Fig. 2C, D). Peak V
coeluted with the 5S,12S-diastereomer while the 5R,12S-
diastereomer was resolved by about 1 min (Fig. 2C).
The configuration of the double bonds of the authentic
standard was confirmed by NMR analysis (J6,7 = 15.2 Hz
Fig. 2. RP-HPLC analysis of diHETEs in activated human leuko-
cytes. A: Leukocytes activated with LPS (6 h) and A23187 (15 min)
form five distinct diHETEs. The absorbance of products I and IV at
235 nm indicates conjugated diene diHETEs, absorbance at 270
nm (II, III, and V) indicates conjugated triene diHETEs. (B) Elu-
tion of the standards 5,15-diHETE and 5,11-diHETE. (C) The
chromatograms of separate analyses of the standards of LTB4 (III),
6E,8Z,10E,14Z-5R,12S-diHETE, and 6E,8Z,10E,14Z-5S,12S-diHETE
(V), respectively, are combined in the same panel. (D) UV spectra
of 5,15-diHETE (I), II (tentatively identified as a mixture of 6-trans-
LTB4 and 6-trans-12-epi-LTB4), LTB4 (III), and 6E,8Z,10E,14Z-
5S,12S-diHETE (V) recorded during the HPLC-diode array analysis
in panel A. A Waters Symmetry C18 column was eluted with ace-
tonitrile/water/acetic acid (37.5/62.5/0.01, by vol.) at 1.0 ml/min
and online diode array detection. The chromatograms shown were
recorded at 235 nm (panels A, B) and 270 nm (panels A, C).
(trans), J8,9 = 10.6 Hz (cis), J10,11 = 15.3 Hz (trans), J14,15
=
9.4 Hz (cis)). The double bond configuration of the
6E,8Z,10E triene implied that V was formed by consecu-
tive oxygenation of arachidonic acid by 5-LOX and
12-LOX in either order, rather than by hydrolysis of
the LTA4 epoxide. The 12-LOX activity was likely due
to platelet 12-LOX. It has been shown that in vitro
mixtures of platelets and neutrophils form 6E,8Z,10E,
14Z-5S,12S-diHETE in response to stimulation with A23187
(25).
Analysis of 5,15-diHETE and 5,11-diHETE formation
We hypothesized that 5,15-diHETE (I) and 5,11-diHETE
(IV) are products of COX-2 catalyzed oxygenation of 5S-
HETE in activated leukocytes (Fig. 1). The role of COX-2
and 5-LOX in the biosynthesis of 5,15-diHETE and 5,11
-diHETE was probed using inhibitors and LC/MS analysis
DiHETE formation in human leukocytes
89

in the SRM mode (Fig. 3A). Treatment of LPS-activated
leukocytes with the COX-2 selective inhibitor NS-398 or
with aspirin 30 min prior to stimulation with A23187 de-
creased 5,15-diHETE and completely inhibited 5,11-di-
HETE (Fig. 3B, C). The lesser effect on 5,15-diHETE was
likely due to 15-LOX activity that would not have been af-
fected by the inhibitors. In addition, aspirin treatment
of COX-2 was expected to induce formation of 5S,15R-
diHETE that coelutes with the 5S,15S-diastereomer (16).
The FLAP inhibitor MK-886 inhibited formation of both
5,15-diHETE and 5,11-diHETE (Fig. 3D).
We used d4-LTB4 as internal standard to quantify
5-HETE, 5,15-diHETE, and LTB4 in activated leukocytes
isolated from six healthy human subjects (Fig. 4). The lev-
els of 5,15-diHETE were 0.45 0.2 ng/106 cells (mean
SEM, n = 6) compared with 5-HETE ranging between 0.4
and 2.4 ng/106 cells (mean 1.0 0.36 ng/106 cells, n = 6)
and LTB4 0.7 – 3.6 ng/106 cells (mean 1.3 0.5 ng/106
cells, n = 6).
MK-886 reduced formation of 5,15-diHETE to below de-
tectable levels in four out of six samples, indicating the
crucial role of 5-LOX in biosynthesis (0.07 0.04 ng/106
cells, n = 6) (Fig. 4). A possible explanation why MK-886
failed to completely inhibit formation of 5,15-diHETE in
two samples is that the cells were partially broken, which
renders the inhibitor ineffective. MK-886 inhibits FLAP
rather than 5-LOX and is only effective in intact cells (26).
Treatment with NS-398 (0.30 0.17 ng/106 cells, n = 6)
reduced the levels of 5,15-diHETE between 5- and 10-fold
in four out of six samples whereas in one sample it had
only a small effect and in the other increased the level of
5,15-diHETE. Inhibition by NS-398 indicates a major al-
though not exclusive role of COX-2 in the biosynthesis of
5,15-diHETE. The portion of 5,15-diHETE that was not in-
hibited by NS-398 was likely formed by 15-LOX expressed
by eosinophils in the leukocyte preparations. Unexpect-
edly, NS-398 also reduced the levels of 5-HETE and LTB4
in some of the samples (not shown). This could have been
due to the ability of NS-398 to inhibit leukocyte lipid body
formation independent of COX-2 inhibition leading to
the suppression of LOX-derived eicosanoids (27). Aspirin
did not completely inhibit formation of 5,15-diHETE
(0.10 0.05 ng/106 cells, n = 6), compatible with the expected
formation 5S,15R-diHETE from 5S-HETE (16). None of
the differences between the means of the treatment groups
were statistically significant using a two-tailed unpaired
t-test.
In addition, formation of 5,11-diHETE is highly indica-
tive of the reaction of COX-2 with 5-HETE. Besides COX-
2, COX-1 is the only other mammalian enzyme capable of
oxygenating the 11-carbon of arachidonic acid, but COX-1
does not react with 5-HETE (17). The inverse reaction (of
5-LOX with 11R-HETE) is very inefficient (16) and thus
unlikely to be relevant in cells. The levels of 5,11-diHETE
were between 0.01 and 0.04 ng/106 cells. 5,11-DiHETE was
reduced below the detectable limit (1 pg/106 cells) upon
treatment with aspirin, MK-886, or NS-398, respectively.
Stereochemical analysis of 5,15-diHETE
The configuration of C-15 of 5,15-diHETE can give an
indication to the enzymes involved in its biosynthesis (cf.
Fig. 1). 5,15-DiHETE formed by consecutive action of
5-LOX and 15-LOX (in either order) is purely 5S,15S (12).
In contrast, oxygenation of 5S-HETE by COX-2 in vitro
leads to formation of a ف1:4 mixture of the 5S,15R- and
5S,15S-diHETE diastereomers, i.e., about 20% of 5,15-di-
HETE has the 5S,15R configuration. Acetylation of recom-
binant COX-2 by aspirin leads to exclusive formation of the
5S,15R diastereomer (16). Analysis of the configuration
of the 5- and 15-carbons in 5,15-diHETE is challenging
Fig. 3. Effect of COX-2 and 5-LOX inhibitors on the formation of 5,15-diHETE, 5,11-diHETE, and 5-HETE by activated leukocytes. Leu-
kocytes were activated with LPS and A23187 (A) without inhibitor, and (B) in the presence of NS-398, (C) aspirin, and (D) MK-886 and ana-
lyzed using LC-ESI-MS. The ion traces for the transitions of 5,15-diHETE (m/z 335 to 201, top), 5,11-diHETE (m/z 335 to 183, middle), and
5-HETE (m/z 319 to 115, bottom) are shown. The signal intensities are given across each row relative to the uninhibited sample in (A) set
to 100%, and corrected using d4-LTB4 as internal standard. The sample in panel B was derived from a different subject and analyzed at a
different time than the samples in panels A, C, and D.
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Journal of Lipid Research Volume 53, 2012

Fig. 4. Quantification of 5,15-diHETE in activated human leuko-
cytes. Samples from 6 healthy volunteers in the absence of or
treated with aspirin, MK-886, or NS-398 30 min prior to 15-min in-
cubation with A23187 were analyzed using LC-ESI-MS in the SRM
mode. The mean of the values is indicated with a horizontal bar.
because the 5S,15S- and 5S,15R-diastereomers do not sepa-
rate using RP-HPLC (12), and only marginally resolve
using straight-phase-HPLC (16). Therefore, we developed
a method for separation of the diastereomers using a Chi-
ralpak AD-RH chiral phase column coupled with LC/MS
detection. 5,15-DiHETE was derivatized to the PFB ester in
order to enhance sensitivity by employing APCI with de-
tection of the [M-PFB]Ϫ molecular ion (23).
Fig. 5. Stereochemical analysis of 5,15-diHETE. LC-MS analysis
of (A) 5,15-diHETE formed by reaction of recombinant COX-2,
(B) 5,15-diHETE formed by activated leukocytes, and (C) 5,15-di-
HETE formed by aspirin-treated activated leukocytes. The samples
were derivatized to the PFB esters and analyzed using a Chiralpak
AD-RH column eluted with acetonitrile/ethanol (90/10, by vol.) at
1 ml/min and detection by LC-APCI-MS operated in the SRM
mode monitoring m/z 335 to 201.
Elution with acetonitrile/ethanol (90/10, by vol.)
achieved baseline resolution of the 5S,15R-diHETE-PFB
and 5S,15S-diHETE-PFB diastereomers. Using recombi-
nant COX-2, the peak areas showed the expected ratio of
20:80 for 5S,15R to 5S,15S (Fig. 5A). Analysis of 5,15-di-
HETE formed by LPS/A23187-activated leukocytes showed
a ratio of ف15:85 of the 5S,15R- to the 5S,15S-diastereomer
(Fig. 5B). Thus, compared with the ratio formed by re-
combinant COX-2 the proportion of the 5S,15R diastere-
omer in the leukocytes was largely retained implying a
major contribution of COX-2 to the synthesis of 5,15-di-
HETE. The slight enrichment (ف10–20%) of the 5S,15S-
diastereomer was likely due to synthesis by a 15-LOX
enzyme. In a second sample, <5% of 5,15-diHETE was the
5S,15R-diastereomer, implicating a lesser contribution of
COX-2 and higher contribution of 15-LOX. Aspirin treat-
ment of activated leukocytes led to a smaller than expected
increase in the proportion of 5S,15R-diHETE, possibly due
to incomplete acetylation of COX-2. A representative sam-
ple (Fig. 5C) showed that about 25% of total 5,15-diHETE
was the 5S,15R-diastereomer.
A23187 and incubated for 15 min. Formation of d8-5,15-
diHETE, d0-5,15-diHETE, and LTB4 was analyzed using
LC/MS in the SRM mode. With increasing concentration
of exogenous d8-5-HETE, the levels of d8-5,15-diHETE in-
creased, and this occurred partly at the expense of forma-
tion of d0-5,15-diHETE (Fig. 6A). This provided evidence
for an activity that catalyzes 15-oxygenation of 5-HETE in
the leukocytes. The 15-oxygenase activity was in part due to
COX-2 because NS-398 reduced the formation of d8-5,
15-diHETE (Fig. 6B). Additional indication that 15-oxygen-
ation of 5-HETE was catalyzed by COX-2 came from the
low levels of 15-LOX activity in the activated leukocytes.
The levels of 15-HETE were about 1-2% of the levels of
5-HETE.
When d8-15S-HETE was added to the LPS-activated leu-
kocytes together with A23187, high incorporation of the
labeled substrate into 5,15-diHETE was observed, even at
the lowest concentration of d8-15S-HETE used (Fig. 6C).
At all three concentrations (50, 100, and 500 ng d8-15S-
HETE /106 cells), the amount of d8-5,15-diHETE was be-
tween 10- and 20-fold higher than d0-5,15-diHETE,
indicating that exogenous 15S-HETE was efficiently con-
verted by the 5-LOX activity in the leukocytes. At the high-
est concentration of d8-15S-HETE added (7.5 ␮M),
formation of endogenous (d0-) 5,15-diHETE was almost
completely suppressed. As expected, the 5-LOX inhibitor
AA-861 reduced the amount of both d8-5,15-diHETE and
d0-5,15-diHETE as well as LTB4 below detectable levels
(Fig. 6D).
Transformation of exogenous 5-HETE and 15-HETE to
5,15-diHETE
We analyzed the ability of activated leukocytes to utilize
either 5-HETE or 15-HETE in the biosynthesis of 5,15-
diHETE by measuring formation of octadeuterated 5,
15-HETE from exogenously added d8-5-HETE and d8-15S-
HETE, respectively. d8-5-HETE was used as a racemic mix-
ture. The enantiomers did not resolve on SP or RP
Chiralpak AD chiral phase HPLC columns although both
give exceptional resolution of unlabeled HETEs (21, 28).
Three different concentrations of d8-5-HETE (50, 100,
and 500 ng/106 cells, equivalent to 0.75, 1.5, and 7.5 ␮M)
were added to LPS-activated leukocytes together with
DiHETE formation in human leukocytes
91

cells is used in the biosynthesis of leukotrienes, lipoxins,
and other lipid autacoids. Thus, analysis of mixtures of
leukocytes can lead to identification of eicosanoids that
are absent or less prominent in monotypic cell popula-
tions. A major goal of this study was to provide evidence
for a functional biosynthetic coupling of 5-LOX expressed
in granulocytes and COX-2 expressed in monocytes.
The main product of the transformation of 5-HETE by
COX-2 in vitro has been identified as an unstable di-
endoperoxide (17); 5,11- and 5,15-diHETE are reaction
by-products (16). The chemical instability makes direct
identification of the di-endoperoxide in vivo a difficult
task. We hypothesized that, instead, formation of 5,15-di-
HETE and 5,11-diHETE could provide evidence for the
oxygenation of 5S-HETE by COX-2 in vivo. While these
studies were in progress, we identified two hemiketal eico-
sanoids as the major transformation products of the un-
stable di-endoperoxide. The hemiketals are present in
activated human leukocytes and induce tubulogenesis of
endothelial cells (32).
Biosynthesis of 5,15-diHETE through crossover of the
5-LOX and 15-LOX pathways has been documented in
leukocytes from normal volunteers (12) and from patients
with eosinophilia (33) and asthma (34). Consecutive oxy-
genation of arachidonic acid by the 5-LOX and 15-LOX
enzymes is illustrated as routes 3 and 6 in Fig. 1. Alterna-
tive pathways could entail the 15-oxygenase activities of
COX-1 and COX-2 (routes 1 and 4 in Fig. 1), and the 15R-
oxygenase activity of aspirin-acetylated COX-2 (routes 2
and 5). Since testing of the alternative pathways required
5-LOX and COX-2 activities we used a crude fraction of
human leukocytes containing neutrophils and eosinophils
in order to provide 5-LOX activity, and monocytes/mac-
rophages for COX-2 activity. COX-2 activity was induced
by LPS (35), followed by stimulation of 5-LOX activity with
calcium ionophore (36). Involvement of COX-2 in the bio-
synthesis of 5,15-diHETE in activated human leukocytes
was evident from three independent findings: 1) signifi-
cant formation of the 5S,15R-diastereomer (about 15% of
total 5,15-diHETE) was indicative of COX-2 activity, re-
sembling the ف20:80 ratio formed by the recombinant en-
zyme (16) [and no 15R-LOX enzyme exists in humans or
any other mammal (37)]; 2) the levels of 5,15-diHETE
were markedly decreased in the presence of the COX-2
specific inhibitor NS-398; and 3) aspirin treatment of the
leukocytes increased the ratio of 5S,15R-diHETE versus
5S,15S-diHETE (while decreasing overall synthesis of
5,15-diHETE) and abolished formation of 5,11-diHETE.
In addition, biosynthesis of 5,11-diHETE was due to COX-2
reaction with 5-HETE because it was blocked by NS-398,
and no 11-LOX activity has been reported in mammals
(3).
Fig. 6. Transformation of exogenous d8-5-HETE and d8-15S-
HETE into d8-5,15-diHETE by activated human leukocytes. (A)
Levels of d8-5,15-diHETE, d0-5,15-diHETE, and LTB4 generated by
incubation of leukocytes activated with LPS and A23187 with 50,
100, or 500 ng of d8-5R,S-HETE. (B) Activated leukocytes were in-
cubated with 100 ng d8-5-HETE in the absence or presence of
10 ␮M NS-398. (C) Formation of d8-5,15-diHETE, d0-5,15-diHETE,
and LTB4 upon incubation of the cells with 50, 100, or 500 ng of
d8-15S-HETE. (D) Activated leukocytes were incubated with 100 ng
d8-15S-HETE in the absence or presence of 10 ␮M AA-861. Data
points are the mean S.E.M. from three different donors.
The levels of LTB4 (which cannot be formed from
5-HETE or 15-HETE) were reduced about 10-fold at the
highest concentration of either of the exogenous HETEs
added (Fig. 6A, C). It is not clear why exogenous 5-HETE
inhibited LTB4 formation but inhibition by 15-HETE
could have been due to outcompeting arachidonic acid as a
substrate for 5-LOX (29). Inhibition of endogenous 5,15-
diHETE by the highest concentration of 15-HETE could par-
tially be due to inhibition of COX-2 by 15S-HETE (30).
An interesting facet of the double oxygenation of arachi-
donic acid at carbons 5 and 15 is that the reactions can
occur in either order. Routes 4, 5, and 6 in Fig. 1 are es-
sentially the inverse of routes 1, 2, and 3, respectively.
What is the order of reaction in the biosynthesis of 5,15-di-
HETE in the activated leukocytes? Because inhibitors are
not a suitable tool to distinguish the order of events, we
DISCUSSION
Eicosanoid biosynthesis in vivo occurs in an environ-
ment that enables transcellular biosynthesis (31). The ex-
change of arachidonic acid substrate and its oxygenated
products between phagocytic, immune, and endothelial
92
Journal of Lipid Research Volume 53, 2012

Fig. 7. Overview of diHETEs formed in activated leukocytes. The transformation of 15-HETE and 12-HETE by 5-LOX and the reaction
of aspirin-acetylated COX-2 have not been included.
added octadeuterated 5-HETE or 15-HETE to the acti-
vated leukocytes and determined their transformation to
5,15-HETE relative to the endogenous pathway. We found
that exogenous 5-HETE was transformed to 5,15-diHETE,
and inhibition by NS-398 proved that the transformation
was catalyzed by COX-2. This confirmed that 5,15-diHETE
in the activated leukocytes was, not to the least part, formed
via route 1 in Fig. 1, i.e., by crossover of the 5-LOX and
COX-2 pathways.
Similar to previous studies (29, 38), we found that addi-
tion of exogenous 15-HETE led to inhibition of the forma-
tion of LTB4 (Fig. 6). This effect has initially been
interpreted as an inhibitory effect of 15-HETE on 5-LOX
activity (38) but was later found to be due to efficient utili-
zation of 15-HETE by 5-LOX at the expense of reaction
with arachidonic acid (29). Two additional lines of evi-
dence support this explanation: 1) studies with 5-LOX pu-
rified from porcine leukocytes showed that 15-HPETE
reacted at about 30% of the maximum velocity obtained
with arachidonic acid (39); and 2) Mancini et al. (40)
showed that recombinant FLAP was able to stimulate oxy-
genation of 15-HETE about twofold (and oxygenation of
12-HETE almost 200-fold). It appears that in activated leu-
kocytes the availability of arachidonic acid or 15-HETE is
limiting the formation of 5,15-diHETE. Taken together,
the abundant formation of 5,15-diHETE from 15-HETE
implies that in the leukocytes 5-LOX can react efficiently
with 15-HETE (route 6 in Fig. 1). This reaction is also a
major pathway of biosynthesis of lipoxins (13, 14).
diHETEs together with 5-HETE accounted for about half
of the observed 5-LOX metabolites, the other half consist-
ing of LTB4 and nonenzymatic hydrolysis products of the
LTA4 epoxide. We did not quantify the levels of cysteinyl
LTs in the samples.
Little is known about the biological role of 5,15-diHETE.
In line with its biosynthesis by leukocytes, 5,15-diHETE
plays a role in the inflammatory response and potentiates
the degranulation of human neutrophils in response to
platelet activating factor, but not f-Met-Leu-Phe, calcium
ionophore A23187, or LTB4 (41). 5S,15S-DiHETE is also a
chemoattractant for eosinophils with an ED50 of 0.3 ␮M
(42)andaprecursortothehighlypotenteosinophilchemoat-
tractant 5-oxo-15-HETE with an ED50 of 5 nM (43).
In summary, formation of 5,11-diHETE and 5,15-di-
HETE in activated human leukocytes is evidence for a
functional biosynthetic cross-over of the 5-LOX and COX-2
pathways. Our studies implicate inhibition of formation of
5,15-diHETE as an additional mechanism of action of
NSAIDs and COX-2 selective inhibitors with as of yet in-
completely understood biological consequences.
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