From the Division of Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206
Received for publication, August 20, 2002, and in revised form, January 22, 2003
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ABSTRACT |
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5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE)
is a metabolite of arachidonic acid shown to possess important
biological activities within different cell types. In the neutrophil, a
specific NADP+-dependent dehydrogenase
utilizes 5-lipoxygenase-derived 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) as the required substrate. In the present
study, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HpETE), rather than 5-HETE, was found to be the biosynthetic precursor of 5-oxo-ETE in
the murine macrophage. The macrophage was not able to convert 5-HETE
into 5-oxo-ETE even when preincubated with phorbol ester or with other
lipid hydroperoxides. The factor responsible for the conversion of
5-HpETE into 5-oxo-ETE was found predominantly in the cytosolic
fraction of the macrophage, with an approximate molecular weight of
50,000-60,000, as assessed by size exclusion chromatography.
Formation of 5-oxo-ETE was rapid and the catalytic protein was found to
have an apparent Km of 5.3 µM for the
eicosanoid. Furthermore, the protein could efficiently utilize 5(R,S)-HpETE as substrate and was heat and
protease labile. This novel pathway of 5-oxo-ETE biosynthesis in the
murine macrophage was consistent with reduction of a 5-hydroperoxy
group to an intermediate alkoxy radical that could be subsequently
oxidized to the 5-oxo product. Such a mechanism would enable racemic
5-HpETE, derived from free radical oxidation of arachidonic acid, to be
efficiently converted into this potent chemotactic eicosanoid.
Arachidonic acid is the precursor of a number of lipid mediators
of diverse activity as well as chemical structure and whose formation
is controlled by the action of several enzymatic systems. One
biosynthetic pathway involves 5-lipoxygenase, which initiates a
cascade of arachidonic acid metabolism leading ultimately to the
formation of a group of biologically active compounds, including the
leukotrienes (1). The molecular events directed by 5-lipoxygenase involve insertion of molecular oxygen at carbon-5 of the arachidonate chain with formation of
5(S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid
(5(S)-HpETE),1
which can be reduced by peroxidases to the hydroxy analog
5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid
(5(S)-HETE) or stereospecifically dehydrated to leukotriene A4 (LTA4) by a second 5-lipoxygenase-catalyzed
step (2). LTA4 can be further converted into
LTB4 by the enzyme LTA4 hydrolase (3) or into
the cysteinyl-leukotrienes LTC4, LTD4, and
LTE4 by the enzyme LTC4 synthase (4).
LTB4 is a potent chemokinetic and chemotactic agent for the
human polymorphonuclear leukocyte (5), whereas LTC4 and
LTD4 are among the most potent mediators of
bronchoconstriction in man (6).
More recently, another 5-lipoxygenase metabolite,
5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), has been identified
as being made within human polymorphonuclear leukocytes (7, 8), eosinophils (9), monocytes, and lymphocytes (10). This eicosanoid elicits a different set of important biological activities as a potent
agonist increasing cytosolic calcium levels, chemotaxis, and
degranulation by a mechanism independent of the LTB4
receptor in the human neutrophil (11, 12). Low concentrations have been
shown to increase the surface expression of the The biosynthesis of 5-oxo-ETE has been extensively examined in human
neutrophils with the identification of a specific microsomal NADP+-dependent dehydrogenase responsible for
the conversion of 5(S)-HETE, but not 5(R)-HETE
(7). In intact cells, significant amounts of this metabolite are
synthesized only when neutrophils are preincubated with phorbol
myristate acetate (PMA), a protein kinase C activator that elevates
NADP+ (22). The same biosynthetic pathway for 5-oxo-ETE has
been also identified in human monocytes and lymphocytes (10).
An alternative pathway for 5-oxo-ETE synthesis could proceed from
5-HpETE, because unsaturated fatty acid hydroperoxides have been shown
to be direct precursors of oxo-fatty acids catalyzed by hematin and
heme-containing proteins such as hemoglobin (23, 24). In a similar
manner, platelet 12-lipoxygenase and soybean 15-lipoxygenase under
anaerobic conditions has been shown to convert 12-HpETE and 15-HpETE,
respectively, into the corresponding 12- and 15-oxo derivatives
(25).
Recently, 5-oxo-ETE was found to be an eicosanoid synthesized within
the elicited murine peritoneal macrophage (26), but the pathway
responsible for the production of 5-oxo-ETE was not investigated. The
objective of the present study was to define the biosynthetic pathway
that leads to the synthesis of 5-oxo-ETE in the macrophage and
elucidate the mechanism responsible for the formation of this keto
eicosanoid in this cell type.
Materials--
5-Oxo-ETE, 5(S)-HETE,
5(S)-HpETE, 5(R,S)-HpETE,
15(S)-hydroperoxyeicosatetraenoic acid
(15(S)-HpETE), 13(S)-hydroperoxyoctadecadienoic acid (13(S)-HpODE), d8-5-HETE,
and d7-5-oxo-ETE were purchased from Cayman
Chemical Company (Ann Arbor, MI). All solvents were HPLC grade and
obtained from Fisher. Type I "plus" water was obtained using a
MilliQ water system (Millipore Corp., Bedford, MA) fed with deionized
water. CompleteTM protease inhibitor mixture tablets were
obtained from Roche Molecular Diagnostics. Dulbecco's modified
Eagle's medium and Hanks' balanced salt solution were purchased from
Cellgro by Mediatech Inc. (Herndon, VA). Stannous chloride anhydrous
(SnCl2), PMA, trypsin, hematin, and phosphate-buffered
saline were purchased from Sigma. Thioglycollate was purchased from BD
Difco (Franklin Lakes, NJ).
Preparation of Murine Elicited Peritoneal
Macrophages--
Elicited macrophages were obtained by injecting 1 ml
of 4% thioglycollate into the peritoneum of ICR mice. After 3 days,
the mice were euthanized in a CO2 atmosphere. For the
experiments with intact cells, the peritoneum was lavaged once with 10 ml of Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin, and peritoneal macrophages were plated onto polystyrene 6-well cell culture dishes (Corning Inc., Corning, NY) at a concentration of 3 × 106 cells/well. For some experiments, the peritoneum was
lavaged once with 10 ml of cold Hanks' balanced salt solution and
cells were kept in suspension at the concentration of 20-30 × 106 cells/ml.
Preparation of Cytosolic and Microsomal
Fractions--
Peritoneal macrophages were washed once with lysis
buffer (NaCl, 0.4 M; phosphate buffer, 0.2 M,
pH 7.4; and one CompleteTM protease inhibitor mixture
tablet for each 20 ml of buffer) and then resuspended in lysis buffer
at the concentration of 100 × 106 cells/ml. Cells
were disrupted by sonication and then centrifuged at 12,000 × g for 10 min at 4 °C; the resulting supernatant was subjected to centrifugation at 100,000 × g for 60 min
at 4 °C. The cytosolic fraction (supernatant) and the pellet
(microsomal fraction), resuspended in lysis buffer, were kept on ice
until used. For some experiments the cytosol fraction (100 µl) was
resuspended in a boiling water bath (94 °C) for 20 min prior to
testing for enzymatic activity.
Cell and Cellular Fraction Incubations--
Plated macrophages
were washed twice with 1 ml of KRPD buffer (KCl, 4.8 mM;
CaCl2, 0.97 mM; MgSO4, 1.2 mM; NaH2PO4, 3.1 mM; Na2HPO4, 12.5 mM; and dextrose,
0.2%) and incubations were performed in 1 ml of KRPD. After 5 min of
thermal equilibration at 37 °C, macrophages were incubated with
5(S)-HpETE or 5(R,S)-HpETE (1 µM) for 30 min at 37 °C; supernatants were then
collected in 1 volume of ice-cold methanol containing 5 ng of
d8-5-HETE and 10 ng of
d7-5-oxo-ETE as internal standards. Identical
incubations were conducted using human neutrophils (3 × 106/ml) prepared according to the method of Haslett
et al. (27).
Additional experiments were performed in macrophages, where
5(S)-HETE (1 µM) was coincubated with
15(S)-HpETE or 13(S)-HpODE (1 µM)
and 5(S)-HpETE (1 µM) with
d8-5(S)-HETE (2 µM) for
30 min at 37 °C. Human neutrophils and murine macrophages (5 × 106 cells/ml in Hanks' balanced salt solution) were
preincubated with either PMA (final concentration 30 nM) or
vehicle (Me2SO) for 6 min at 37 °C and then
5(S)-HETE (final concentration, 1 µM) was
added and incubated for an additional 20 min. Cytosolic and
microsomal fractions from 5 × 106 macrophages
(diluted with lysis buffer to 1 ml) were incubated with
5(S)-HpETE (1 µM) or
5(R,S)-HpETE (1 µM) for 10 min at
37 °C, after 5 min of thermal equilibration; incubations were
terminated with 1 volume of ice-cold methanol containing the internal standards.
Cytosolic fractions were also incubated with 5(S)-HpETE or
5(S)-HETE (1 µM for 10 min at 37 °C) after
pretreatment for 5 min at 37 °C with different cofactors
(NADP+, NADPH, NADH, all at the concentration of 1 mM), and further experiments were conducted by boiling the
cytosol for 20 min or adding trypsin (2.5 mg/ml) to the cytosol
preparation for 10 min at 37 °C and incubating it with
5(S)-HpETE (1 µM) for 10 min at 37 °C. Time
course experiments were performed incubating cytosolic fractions from
5 × 106 macrophages with 5(S)-HpETE (1 µM) at the times indicated at 37 °C, after a 5-min
thermal equilibration. The effect of 5-HpETE concentration on 5-oxo-ETE
synthesis was studied with increasing concentrations of
5(S)-HpETE (0.1-100 µM) for 1 min at
37 °C. In separate experiments, 13-HpODE and 15-HpETE (1 µM) were incubated with the cytosol fraction under
identical conditions. Protein content in the cytosol aliquots was
determined by the microbicinchoninic acid assay (Pierce) protocol using
bovine serum albumin as standard.
Metabolite Separation by RP-HPLC and Analysis by Electrospray
Ionization-Mass Spectrometry--
The quantitative analysis of
5-oxo-ETE production in each incubation was carried out by stable
isotope dilution mass spectrometry. Because the conversion from 5-HpETE
to 5-oxo-ETE has been reported as an artifact of HPLC analysis (25),
SnCl2 (100 mM ethanol stock solution) was added
to each sample (0.5 mM final concentration) for 30 min at
room temperature before solid phase extraction. This chemically reduced
any remaining 5-HpETE into 5-HETE prior to HPLC separation.
Supernatants from different experiments were diluted with water to a
final concentration of less than 20% methanol and solid phase
extraction was performed using Bond-Elut C18 cartridges (Varian Inc.,
Harbor City, CA), preconditioned with 1 ml of methanol and washed with
1 ml of water; methanol eluates (1 ml) were taken to dryness using a
SpeedVac evaporating centrifuge (Savant Instruments, Farmingdale, NY),
reconstituted in 40 µl of HPLC mobile phase A (8.3 mM
acetic acid buffered to pH 5.7 with NH4OH) + 20 µl of methanol, and injected into an HPLC gradient pump system directly interfaced into the electrospray source of a triple quadrupole mass
spectrometer (Sciex API 3000, PerkinElmer Life Sciences, Thornhill,
Ontario, Canada). A linear gradient from 15% mobile phase B
(acetonitrile/methanol, 65/35, v/v) to 100% B was used to elute a
150 × 1-mm Columbus 5-µm C18 reversed phase column (Phenomenex,
Rancho Palos Verde, CA), at the flow rate of 50 µl/min. Mobile phase
B was increased from 15 to 55% in 10 min, to 80% in 25 min, to 100%
in 30 min and held at 100% B for a further 5 min. Mass spectrometric
analyses were performed in the negative ion mode using multiple
reaction monitoring of the specific transitions m/z 317 Size Exclusion Chromatography--
Aliquots of 10 µl of
macrophage cytosolic fractions and boiled cytosol fractions were loaded
on a hydrophilic bonded silica size exclusion column (BioSep-SEC-S2000
Peek 300 × 7.50 mm, Phenomenex) eluted with phosphate buffer, 200 mM, pH 7.3, at a flow rate of 1 ml/min; 1-min fractions
were collected from the column for 14 min. Aliquots of 10 µl from
each fraction were then tested for activity by incubating them with
5(S)-HpETE (1 µM) for 10 min at 37 °C and
further analyzed by RP-HPLC followed by electrospray ionization-mass spectrometry. Identical experiments were conducted using cytosolic and boiled cytosolic fractions pretreated for 5 min at
room temperature with hematin (0.5 and 5 µM) and with cytosolic fractions pretreated with trypsin (2.5 mg/ml) for 10 min at
37 °C. A standard curve to assess the approximate molecular weight
corresponding to each fraction collected was built by injecting on the
same column 50 µg of each of the following proteins: IgG (150 kDa),
bovine serum albumin (66 kDa), glutathione S-transferase (25 kDa), insulin (5800 Da), and vitamin B12 (1355 kDa); UV
absorbance was monitored at 214 and 280 nm.
Intact Cell Incubations--
Elicited peritoneal macrophages
(3 × 106 cells/ml) were incubated for 30 min at
37 °C with 5(S)-HETE (1 µM) and the
formation of specific products was analyzed by combined liquid
chromatography-mass spectrometry. Specific ion transitions formed by
collisional activation were monitored to detect the elution of
5-oxo-ETE (m/z 317
When elicited macrophages were incubated under identical conditions
with 5(S)-HpETE (1 µM) followed by
purification and analysis by mass spectrometry, a significant
production of 5-oxo-ETE (7.13 ± 0.56 ng/106 cells)
was observed (Fig. 2). Furthermore,
incubation of racemic 5(R,S)-HpETE (1 µM) resulted in an almost identical level of 5-oxo-ETE production (6.79 ± 0.61 ng/106 cells). However, the
incubation of the human neutrophil with either 5(S)-HpETE or
5(R,S)-HpETE) did not lead to any significant biosynthesis of 5-oxo-ETE (0.13 ± 0.05 and 0.17 ± 0.04, respectively).
These results suggested that a hydroperoxide may be required for
biosynthesis of 5-oxo-ETE, but did not unambiguously define the
conversion of the 5-HpETE directly into 5-oxo-ETE. An alternative possibility would be a dual role for the hydroperoxide to stimulate a
secondary biochemical pathway, adding a co-factor, which could then
activate the biosynthetic pathway of 5-HETE to 5-oxo-ETE in a manner
quite analogous to that previously described for the NADPH oxidase
pathway in neutrophils. To investigate whether or not other
hydroperoxides coincubated with 5(S)-HETE could stimulate 5-oxo-ETE formation from the hydroxyeicosanoid, cells were incubated with 15(S)-HpETE or 13(S)-HpODE (1 µM each) in the presence of 5(S)-HETE (1 µM) at 37 °C for 30 min. Neither hydroperoxide caused a significant enhancement of the production of 5-oxo-ETE (Fig. 3) in the LC/MS/MS analysis. The specific
transition for 5-oxo-ETE (m/z 317 Subcellular Localization--
Cytosolic and microsomal fractions
from murine peritoneal macrophages were prepared after sonication and
successive centrifugations at 12,000 and 100,000 × g.
Each of these subcellular fractions were incubated with
5(S)-HpETE (1 µM) for 10 min at 37 °C. The biosynthetic activity for 5-oxo-ETE was found predominantly within the
cytosolic fractions (Table I) with
17.2 ± 14 and 6.5 ± 0.5 pmol/106 cells of
5-oxo-ETE formed in the cytosolic and microsomal fractions, respectively. Various cofactors were also added to each of these subcellular fractions, including NADP+, NADPH, and NADH
(each at 1 mM) to examine whether or not they had any
effect on 5-oxo-ETE biosynthesis from either 5(S)-HpETE or
5(S)-HETE (1 µM). After pretreatment of each
of the subcellular fractions with the cofactors for 5 min at 37 °C,
the production of 5-oxo-ETE was assessed by LC/MS/MS. There was no
significant increase in 5-oxo-ETE biosynthesis when NADP+
and NADH were added; however, there was a significant decrease in
5-oxo-ETE production (p < 0.01) when NADPH was added.
The production of 5-oxo-ETE was 10-15 times higher when 5-HpETE was
used as substrate relative to 5-HETE (Table
II).
The process involved in the conversion of 5(S)-HpETE into
5-oxo-ETE was found not to be greatly altered by boiling for 20 min,
because the rate of 5-oxo-ETE production dropped only 20% after
heating. Furthermore, this treatment caused the formation of
substantial denatured proteins observed as an abundant precipitate that
had no biochemical activity (data not shown).
Incubation of racemic 5-HpETE with the cytosolic fraction prepared from
separate macrophages was found to yield substantial 5-oxo-ETE
(16.2 ± 2.2 pmol/106 macrophages), virtually
identical to that observed when the same preparation of macrophage
cytosol was incubated with the same concentration of
5(S)-HpETE (17.2 ± 1.4 pmol/106
macrophages) (Table II). Thus, the cytosolic fraction displayed the
same lack of stereospecificity observed with intact cell incubations in
the formation of 5-oxo-ETE. The cytosolic fraction also catalyzed conversion of 13-HpODE and 15-HpETE into their respective oxo-lipids, but to a reduced extent with 6.6 pmol/106 cells of
13-oxo-ODE and 6.0 pmol/106 cells of 15-oxo-ETE under
conditions identical to those used for Table I.
The effect of 5-HpETE substrate concentration on the total rate of
5-oxo-ETE revealed saturation behavior with approximate Vmax of 92.2 pmol/mg of protein/min and apparent
Km of 5.3 µM 5-HpETE (Fig.
5B). Furthermore, the
formation of 5-oxo-ETE from 5-HpETE was relatively rapid and
incubations longer than 10 min led to a diminution of the quantity of
5-oxo-ETE present in these cells. Because it is known that 5-oxo-ETE
can be rapidly metabolized to several metabolites, including a
glutathione adduct termed FOG7, it is likely that metabolic
reactions decreased the apparent level of 5-oxo-ETE at these longer
incubation times (Fig. 5A).
Size Exclusion Chromatography--
An evaluation of the molecular
size of the factor present in the cytosol responsible for the
conversion of 5-HpETE into 5-oxo-ETE was evaluated using size exclusion
chromatography. Aliquots (10 µl) of macrophage cytosol and boiled
cytosol (20 min) were injected onto an HPLC size exclusion
chromatographic column and 1-min fractions collected for 14 min.
Fractions were then tested for the presence of substances that would
catalyze the conversion of 5-HpETE to 5-oxo-ETE under the standard
conditions of 1 µM substrate concentration for 10 min at
37 °C. Only fractions eluting at 7 and 8 min had the capacity to
convert 5-HpETE to 5-oxo-ETE (Fig. 6).
Calibration of the HPLC size exclusion column with a number of proteins
of different molecular weight (Fig. 6, inset) suggested that
the factor(s) present in the cytosol had an apparent molecular weight between the 60,000 and 25,000 markers, calculated to be 55,000. This
molecular weight suggested that a reasonably large protein was
responsible for the catalytic activity rather than a low molecular weight substance such as inorganic iron or a small prosthetic group
such as hematin. After boiling the cytosol (Fig. 6B) there was a substantial loss of catalytic activity in the size exclusion fractions (7-8 min), in sharp contrast to the results obtained from
the analysis of the crude cytosol and boiled cytosol (Table I), where
only a slight drop in catalytic activity was observed. This suggested
that perhaps the high molecular weight protein released a factor such
as hematin, which could carry out this reaction of 5-HpETE to 5-oxo-ETE
as had been previously described (23). Attempts to chromatograph
hematin either with the indicated mobile phase or at pH 9 to increase
hematin solubility were unsuccessful with this column. Even injecting
larger amounts of hematin (8 nmol) did not result in elution from the
column of detectable quantities of this porphyrin.
Hematin was also added to the cytosol before as well as after boiling
to final concentrations of 0.5 and 5 µM. With untreated cytosol, 2.0 to 2.5 ng of 5-oxo-ETE was formed at both concentrations of added hematin, identical to the results reported in Fig. 6. After
boiling the cytosol and adding hematin (0.5 and 5 µM),
the catalytic activity of size exclusion fractions eluting between 7 and 8 min (Fig. 6B) were also not increased (data not shown).
Additional experiments were also carried out with treatment of the
cytosol preparation with the serine protease trypsin (2.5 mg/ml final
concentration). Measurement of the catalytic activity of the crude
cytosol after trypsin treatment revealed no loss of activity (Table
II). However, size exclusion separation of the trypsinized cytosol
resulted in substantial loss of catalytic activity in the components
eluting between 7 and 8 min (Fig. 6C).
The murine peritoneal macrophage has been known for sometime to be
an active cell in eicosanoid biosynthesis. It expresses cyclooxygenase,
both COX-1 and COX-2 (29), as well as 5-lipoxygenase, which can
metabolize arachidonic acid to 5(S)-HpETE and leukotrienes. As found in the studies reported here, the macrophage is also capable
of efficient conversion of 5-HpETE into 5-oxo-ETE, another biologically
active eicosanoid widely thought to be a product only of the
5-lipoxygenase pathway. Within the macrophage 5-oxo-ETE is conjugated
with glutathione to afford the adduct FOG7, which is
chemotactic for the eosinophil as well as neutrophil, but does not
elevate intracellular calcium in the latter cell type (30). The
formation of FOG7 has now been shown to be catalyzed by
LTC4 synthase (31), which is also expressed in the murine
peritoneal macrophage. Thus, a host of lipid mediators can result from
the oxidation of arachidonic acid within the peritoneal macrophage. However, the biosynthesis of 5-oxo-ETE from 5-HpETE shown in this report is not critically dependent upon the stereochemistry at carbon-5, in that the racemic 5(R,S)-HpETE was
fully capable of being converted to 5-oxo-ETE. Because the chiral
center in 5-HpETE is lost in this conversion to the
sp2 carbonyl carbon of 5-oxo-ETE, it is
impossible to ascertain whether or not an enzymatically derived, chiral
hydroperoxide or free radical-derived hydroperoxide (racemic) is the
precursor of this conjugated dienone eicosanoid. Nonetheless, this
pathway describes an efficient way to utilize free radical-derived HpETE.
5-Oxo-ETE biosynthesis has been extensively studied in the human
polymorphonuclear leukocyte, where it is specifically derived from
5(S)-HETE by a NADP+-dependent
eicosanoid dehydrogenase (7). In the neutrophil, 5-oxo-ETE is produced
only after stimulation with phorbol ester that activates NADPH oxidase
with consequent elevation of NADP+. The microsomal
NADP+-dependent dehydrogenase does not convert
5(R)-HETE into 5-oxo-ETE (7). In the peritoneal macrophage,
there is no evidence to suggest that this
NADP+-dependent eicosanoid dehydrogenase is
expressed even though NADPH oxidase is present (32). 5-HpETE was found
to be a precursor of 5-oxo-ETE when either 5(S)- or
5(R,S)-HpETE were incubated with the macrophage.
Furthermore, the addition of various cofactors as well as other lipid
hydroperoxides did not enhance production of 5-oxo-ETE from 5-HpETE.
The only effect observed by cofactors was a reduction in the formation
of 5-oxo-ETE when NADPH was added to cytosol preparations. This
undoubtedly was because of an increased conversion of 5-HpETE to 5-HETE
by peroxidases dependent on NADPH (33). When macrophages were incubated
with a mixture of stable isotope-labeled 5-HETE and unlabeled 5-HpETE,
the resulting 5-oxo-ETE was unlabeled, clearly supporting the
hypothesis that 5-HETE was not a precursor in 5-oxo-ETE biosynthesis in
the macrophage, but rather 5-HpETE.
A further difference in the biosynthetic pathway of 5-oxo-ETE in the
neutrophil versus the macrophage was the primary location of
the factors responsible for the synthesis of 5-oxo-ETE. In the
peritoneal macrophage the cytosolic fraction retains the major biosynthetic activity, whereas in the neutrophil the microsomal fraction has been described as the locus of 5-hydroxyeicosanoid dehydrogenase. Furthermore, the molecular weight of the macrophage catalytic factor appeared to be ~55,000. The catalytic activity of
the cytosolic fraction was observed to be only slightly diminished by
boiling when the crude cytosol was used for testing and unaffected by
trypsin pretreatment. However, much different results were obtained
when attempting to partially purify the component in cytosol
responsible for this biochemical conversion in that a substantial loss
of activity in the 50-60-kDa region was observed. One possible
explanation for the failure to detect the active component after size
exclusion chromatography would be formation of hematin or a similar
heme prosthetic group released from a heme-containing protein during
boiling and trypsin treatment, but this low molecular weight porphyrin
was tightly bound by the size exclusion column packing material.
Separate experiments clearly showed that hematin did not traverse the
HPLC column and became irreversibly absorbed. However, the extensive
loss of the high molecular weight factor (Fig. 6, B and
C) that catalyzed conversion of 5-HpETE to 5-oxo-ETE was
consistent with the presence of a protein catalyzing this conversion
process in macrophage cytosol.
The formation of various conjugated dienone eicosanoids has been
studied extensively, particularly as products of the reaction of
hydroperoxides with iron-containing metalloproteins and heme derivatives. Dix and Marnett (23) found that
13-hydroperoxy-9-cis-11-trans-octadecadienoic acid was converted into 13-keto-9,11-octadecadienoic acid as a major
product in the presence of hematin, the oxidized form of heme. Other
epoxyhydroxy and trihydroxy fatty acid metabolites were also formed by
this reaction, which was suggested to proceed through formation of an
alkoxy radical lipid hydroperoxide in a one-electron reduction and the
concomitant formation of a ferryl-hydroxyl complex of hematin. Whereas
the stereochemistry of this initial reduction step was not investigated
in these studies, the hypothesis that the initial reduction involves
the hydroperoxy group rather than attack at the hydroperoxy carbon atom
suggests that this reaction would be insensitive to the chirality of
the hydroperoxide. Hematin was also found to effectively convert
10-hydroperoxyoctadec-8-enoic acid into 10-oxo-octadec-8-enoic acid in
almost 80% yield. The formation of this latter The conversion of hydroperoxides to oxo derivatives has also been
reported to be catalyzed by nonheme iron-containing enzymes including
lipoxygenase, such as 12-lipoxygenase and soybean 15-lipoxygenase (25).
The 12-lipoxygenase was found to readily convert
12-hydroperoxy-5,8,10,15-eicosatetraenoic acid into
12-oxo-5,8,10,15-eicosatetraenoic acid. Hematin was also found to be
effective in catalyzing conversion of either 12-HpETE into 12-oxo-ETE
or 15-HpETE into 15-oxo-ETE. Clearly, various iron-containing proteins
are capable of converting hydroperoxides into the corresponding oxo
derivatives, including metalloproteins not containing heme, but iron
chelated by histidine residues. There have also been reports that
cytochrome c, horseradish peroxidase (36), and
heat-denatured platelet preparations (37) can convert 12-hydroperoxyeicosatetraenoic acid into 12-oxo-ETE. These observations are consistent with the hypothesis that the conversion of 5-HpETE to
5-oxo-ETE in the macrophage may involve an iron-containing protein and
intermediate formation of a 5-alkoxyl radical (Fig. 7).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
integrin CD11b, actin polymerization and adherence (13). It is the most active lipid-derived chemoattractant factor for human eosinophils (14,
15), with a potency in the range of the CC chemokines, eotaxin and
RANTES (regulated on activation normal T cell expressed and secreted),
both of which also enhance 5-oxo-ETE-induced chemotaxis (16). This keto
eicosanoid also causes L-selectin shedding, surface expression of
CD11b, calcium mobilization, and actin polymerization (17). It can also
activate directional migration and actin polymerization within the
human monocyte (18), and lead to volume reduction of guinea pig
intestinal epithelial cells (19). Furthermore, 5-oxo-ETE has been shown
to promote eosinophil transmigration through basement membranes (20),
suggesting an important role for this eicosanoid in the recruitment of
these potent inflammatory cells in pathologic conditions such as asthma
and allergy. Recently, a specific G-protein-linked receptor with high
affinity for 5-oxo-ETE has been reported (21).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
203 for 5-oxo-ETE,
m/z 319
115 for 5-HETE,
m/z 324
210 for
d7-5-oxo-ETE, and m/z 327
116 for d8-5-HETE eluting from the RP-HPLC
column. Quantitation of 5-oxo-ETE and 5-HETE in different samples was
performed using a standard isotope dilution curve as previously
described (28). The quantitation of 13-oxo-octadecadienoic (m/z 253
113) and 15-oxo-eicosatetraenoic
acids (m/z 317
113) were carried out in a
similar fashion.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
203) and 5-HETE
(m/z 319
115) as well as internal standards
added in this experiment for quantitative analysis,
d8-5-HETE (m/z 327
116) and d7-5-oxo-ETE (m/z
324
210). There was no significant production of 5-oxo-ETE derived
from the exogenously added 5-HETE in the murine macrophage (Fig.
1); in contrast, the incubation of 5-HETE
with human polymorphonuclear leukocytes resulted in the formation of a
small, but clearly measurable 5-oxo-ETE (0.58 ± 0.02 ng/106 cells) as previously reported (22). When PMA was
added to the intact cells to stimulate the NADPH oxidase (22), there
was still no conversion of 5-HETE to 5-oxo-ETE by the macrophage, but a
significant stimulation of polymorphonuclear leukocyte production of
5-oxo-ETE (3.95 ± 0.14 ng/106 cells) was observed.
These results suggested a substantial difference in the biosynthetic
pathway leading to 5-oxo-ETE in the murine macrophage compared with the
previously described pathway from 5-HETE in the neutrophil.
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Fig. 1.
Effect of pretreatment with PMA on 5-oxo-ETE
synthesis in murine peritoneal macrophages and human neutrophils after
incubation with 5(S)-HETE. Murine peritoneal
macrophages (3 × 106/ml) and human neutrophils
(5 × 106/ml) were incubated with either vehicle
(Me2SO) or PMA (30 nM) for 6 min at 37 °C
and then incubated for a further 20 min with 5(S)-HETE (1 µM). 5-Oxo-ETE was analyzed by RP-HPLC followed by
electrospray ionization-mass spectrometry. Relative abundance of the
specific ion transition relative to deuterium-labeled internal standard
were used to calculate absolute quantities of the metabolite. Mean ± S.E. are of three different cell preparations, with each sample run
in duplicate. The production of 5-oxo-ETE from 5-HETE was significantly
different in the neutrophil relative to the macrophage (**,
p < 0.01).
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Fig. 2.
5-Oxo-ETE synthesis in murine peritoneal
macrophages and human neutrophils after incubation with 5-HpETE.
Murine peritoneal macrophages (3 × 106/ml) and human
neutrophils (5 × 106/ml) were incubated for 30 min at
37 °C with 5(S)-HpETE and
5(R,S)-HpETE (1 µM). 5-Oxo-ETE was
analyzed by RP-HPLC followed by electrospray ionization-mass
spectrometry. Mean ± S.E. are of four different cell
preparations, with each sample run in duplicate. The production of
5-oxo-ETE from 5-HpETE was significantly different in the neutrophil
relative to the macrophage (**, p < 0.01).
203)
indicated the elution of 5-oxo-ETE for which there was only a small
quantity observed at this retention time. Additional experiments were
carried out by incubating 5(S)-HpETE (1 µM) in the presence of d8-5(S)-HETE (2 µM) to assess whether or not there was any direct
precursor role for 5-HpETE. In this experiment the formation of
5-oxo-ETE from the d8-5(S)-HETE would
be revealed by a product eluting at the expected retention time at 27.5 min only having a molecular anion at m/z 324 (d7-product). Collision-induced decomposition of
this molecular anion would result in a product ion at
m/z 210 and this transition could be used to
detect the formation of any d7-5-oxo-ETE from
d8-5-HETE. In this experiment there was a robust
formation of unlabeled 5-oxo-ETE as indicated by the component eluting
at 27.5 min, having the transition m/z 317
203 for unlabeled 5-oxo-ETE, and yet very little conversion of
d8-5-HETE into
d7-5-oxo-ETE (Fig.
4). These studies further supported a
direct conversion of 5-HpETE into 5-oxo-ETE without intermediate
formation of 5-HETE.
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Fig. 3.
Mass spectrometric analysis by multiple
reaction monitoring of supernatants from peritoneal macrophages
incubated with 5(S)-HETE in the presence of
hydroperoxides. Mass spectrometric analysis of extracts from the
supernatant of murine peritoneal macrophages (3 × 106/ml) incubated for 30 min at 37 °C with
5(S)-HETE (1 µM) in the presence of
(A) 15(S)-HpETE (1 µM) or
(B) 13(S)-HpODE (1 µM). Elution of
5-oxo-ETE was detected by monitoring the specific transition
m/z 317 203.
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Fig. 4.
Mass spectrometric analysis by multiple
reaction monitoring of supernatants from peritoneal macrophages
incubated with
d8-5(S)-HETE and
5(S)-HpETE. Mass spectrometric analysis of
extracts from the supernatant of murine peritoneal macrophages (3 × 106/ml) incubated for 30 min at 37 °C with
5(S)-HpETE (1 µM) in the presence of
d8-5(S)-HETE (2 µM).
A, elution of 5-oxo-ETE was detected by the ion transition
m/z 317 203. B, elution of
d7-5-oxo-ETE was detected by the ion transition
m/z 324
210.
5-Oxo-ETE synthesis by intact cells and subcellular fractions from
murine macrophages
5-Oxo-ETE synthesis by peritoneal macrophage cytosolic fractions in the
presence of different cofactors and by boiled cytosol
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Fig. 5.
Time course of 5-oxo-ETE production by
peritoneal macrophage cytosolic fractions incubated with
5(S)-HpETE. Murine peritoneal macrophage
cytosolic fractions (from 5 × 106 cells) were
incubated for 1, 3, 10, and 30 min at 37 °C with
5(S)-HpETE (1 µM). A, 5-oxo-ETE was
analyzed by RP-HPLC followed by electrospray ionization-mass
spectrometry. Mean ± S.E. are of three different cell
preparations, with each sample run in duplicate. B, an
aliquot of macrophage cytosol (equivalent to 5 × 106
cells) was incubated with 5(S)-HpETE (0.1, 0.3, 1, 3, 10, 30, and 100 µM) for 1 min at 37 °C. The apparent
kinetic parameters were calculated after curve fitting to the
Michaelis-Menten equation.
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Fig. 6.
5-Oxo-ETE synthesis in fractions eluted from
size exclusion column. Aliquots (10 µl) of (A)
cytosolic fractions (from 5 × 106 cells),
(B) boiled cytosol fractions, and (C) from
cytosolic fractions pretreated with trypsin (2.5 mg/ml) were loaded on
a size exclusion column and 1-min fractions (1 ml) were collected for
14 min. From each fraction, 10 µl was incubated for 10 min at
37 °C with 5(S)-HpETE (1 µM). 5-Oxo-ETE was
analyzed by RP-HPLC followed by electrospray ionization-mass
spectrometry. Inset, standard curve obtained running on the
size exclusion column (50 µg) of (a) IgG, (b)
bovine serum albumin, (c) glutathione
S-transferase, (d) insulin, and (e)
vitamin B12. For these measurements UV absorbance was
monitored at 214 and 280 nm.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
-unsaturated
ketone was thought to be a result of a one-electron oxidation of the
alkoxide radical by Fe4+ = O, which is the oxidized heme
formed during the initial reduction of the hydroperoxide to the alkoxyl
radical (34). The conversion of lipid hydroperoxides and in particular
13-hydroperoxy-9,11-octadecadienoic acid into the corresponding
conjugated dienone, 13-keto-9,11-octadecadenoic acid, was also found to
be catalyzed by hemoglobin in a rather efficient manner (24). Whereas
the mechanism responsible for formation of this dienone was not
investigated, its formation would be consistent with an intermediate
alkoxy radical by a one-electron reduction reaction, followed by a
one-electron oxidation to the corresponding ketone after removal of a
hydrogen atom from the chiral center. While the conversion of a
hydroperoxide to a keto moiety would formally be a dehydration
reaction, the involvement of heme-containing proteins (hemoglobin) as
well as hematin clearly suggested involvement of redox chemistry of the
chelated iron. Even Fe(III) and Fe(II), as cysteine chelates, were
found to be capable of converting the linoleic hydroperoxide to
13-oxo-9,11-octadecadienoic acid in high yields (35).
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Fig. 7.
Proposed mechanism for the conversion of
racemic 5-HpETE to 5-oxo-ETE through initial one-electron reduction to
the 5-alkoxy radical intermediate catalyzed by an iron-containing
factor followed by one-electron oxidation by removal of the C-5
hydrogen atom.
The facile formation of 5-oxo-ETE from 5-HpETE in the macrophage, but
not the neutrophil, the metabolism of 5-oxo-ETE to FOG7, and the lack of a stereochemical requirement for lipoxygenase-derived 5(S)-HpETE as precursor, suggest a unique pathway to signal
free radical-based lipid peroxidation in the macrophage. We have
previously found that specific phospholipid molecular species, namely
plasmalogen phospholipids containing arachidonate esterified at the
sn-2 position, can be efficiently oxidized at carbon-5 of
arachidonate by radical reactions while still in the ordered membrane
bilayer (38). Thus, initiation of peroxidation at lipid membranes could
result in an elevated production of racemic 5-HpETE esterified to the phospholipid backbone. Subsequent action of phospholipase
A2 would release racemic 5-HpETE. Both enantiomers of this
hydroperoxide could then be converted into a single product, namely
5-oxo-ETE, by the mechanisms suggested above (Fig. 7). Whereas
5-oxo-ETE is known to exert potent biological activities, it is highly
lipophilic and likely would not leave the biosynthetic cell because of
membrane association and affinity to fatty acid-binding proteins.
However, it is known that the macrophage can efficiently convert
5-oxo-ETE into FOG7, which is substantially less lipophilic
and is readily released from cells. Furthermore, FOG7
retains considerable biological activity, being a potent chemotactic
factor for eosinophils and neutrophils (31). Thus, it is possible that
an amplification of cellular events mediated by the
5-oxo-ETE/FOG7 pathway could result from free radical
oxidation of arachidonic acid. The conversion of 5-HpETE to
5-oxo-ETE/FOG7 could also serve as a unique signal following exposure to reactive oxygen species. Whereas it is unknown at
the present time whether one or several iron-containing metalloproteins are involved in the process of reduction and oxidation of 5-HpETE into
5-oxo-ETE, it is clear that this alternative pathway of 5-oxo-ETE biosynthesis can operate in relevant cells involved in the inflammatory response.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL36577.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 303-398-1849;
Fax: 303-398-1694; E-mail: murphyr@njc.org.
Published, JBC Papers in Press, January 23, 2003, DOI 10.1074/jbc.M208496200
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ABBREVIATIONS |
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The abbreviations used are: 5(S)-HpETE, 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid; 5(S)-HETE, 5-hydroxy-6,8,11,14-eicosatetraenoic acid; LTA4, leukotriene A4; 5-oxo-ETE, 5-oxo-6,8,11,14-eicosatetraenoic acid; PMA, phorbol myristate acetate; RP-HPLC, reverse phase-high performance liquid chromatography.
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