(Received for publication, March 3, 1995; and in revised form, June 23, 1995)
From the
The intermediate metabolic events which degrade hydroxy
polyunsaturated fatty acids is largely unknown. Such molecules are
common products of lipid peroxidation and lipoxygenase catalyzed
oxidation of arachidonic acid. Metabolism of two
5,12-dihydroxyeicosatetraenoic acids, 6-trans-LTB (leukotriene B
), and
6-trans-12-epi-LTB
was studied in HepG2
cells (a human-derived hepatoma cell line). Extensive metabolism was
observed with a major metabolite identified as 4-hydroxy-6-dodecenoic
acid for both epimers. Incubation of 6-trans-LTB
epimers at shorter times revealed the formation of intermediate
metabolites, including 6-hydroxy-4,8-tetradecadienoic acid and
8-hydroxy-4,6,10-hexadecatrienoic acid suggesting
-oxidation as
the major pathway leading to the formation of the common terminal
metabolite. Two additional metabolites were structurally elucidated as
5-oxo-6,7-dihydro-LTB
and 6,7-dihydro-LTB
which
have not been previously described. Formation of
5-oxo-6,7-dihydro-LTB
and 6,7-dihydro-LTB
were
also observed during metabolism of
6-trans-12-epi-LTB
in human
polymorphonuclear leukocytes. Of particular interest is the metabolism
of these compounds by
-oxidation from the carboxyl terminus, a
process which is not observed with leukotriene B
or
leukotriene C
. Identification of these metabolites
suggested the operation of the 5-hydroxyeicosanoid dehydrogenase
pathway followed by a
-reductase metabolic pathway
which has not been previously described. This pathway of
-oxidation may limit the activity of various 5,12-diHETEs
including nonenzymatic hydrolysis products of LTA
and also
the recently described B
-isoleukotrienes.
Various isomeric 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acids
(5,12-diHETEs) ()can be formed within mammalian cells both
by enzymatic and nonenzymatic processes. Several isomers of this family
of arachidonic acid metabolites have potent biological activities and
are thought to play significant roles as lipid mediators. The first
committed step in leukotriene biosynthesis is the formation of
leukotriene A
(LTA
) by
5-lipoxygenase(1) . Leukotriene A
hydrolase
enzymatically hydrates this triene epoxide to leukotriene B
(LTB
) (2) which is thought to play an
important role as a chemotactic factor for the human polymorphonuclear
leukocyte(3) . In addition, there is a competitive nonenzymatic
hydrolysis of LTA
leading to the formation of two epimeric
dihydroxyeicosatetraenoic acids, 6-trans-LTB
and
6-trans-12-epi-LTB
. These two
leukotrienes are substantially less potent compared to the enzymatic
product LTB
, but there is evidence to suggest they retain
significant biological activity(4) . Recently, we have
identified the formation of a family of free radical-derived
5,12-diHETEs which have substantial biological activity(5) .
These compounds, which have been termed B
-isoleukotrienes,
are not enzymatic products of arachidonic acid metabolism, but rather
are a result of reactive oxygen species attack on arachidonic acid
while esterified to phospholipids.
Termination of the biological
activity of 5,12-diHETE species is thought to be a result of metabolic
inactivation. The most widely studied is LTB for which a
specific cytochrome P-450
(6, 7, 8) catalyzes the formation of
20-hydroxy-LTB
and 20-carboxy-LTB
in the human
polymorphonuclear leukocyte. LTB
is also rapidly taken up
by the hepatocyte and metabolized by cytochrome P-450
-oxidation
and by subsequent
-oxidation in both mitochondria and
peroxisomes(9, 10, 11) . Recently, it was
found that for those cells which do not express the high affinity
P-450
, an alternative pathway of LTB
metabolism exists. A 12-hydroxyeicosanoid
dehydrogenase/
-reductase pathway leads to the
conversion of the conjugated triene in LTB
to a conjugated
diene and numerous subsequent
-oxidation metabolites and
glutathione conjugates result(12, 13) .
Considerably less is known about the metabolism of 5,12-diHETE
isomers that are not substrates for the cytochrome
P-450. Neither 6-trans-LTB
epimer is efficiently metabolized by the specific cytochrome
P-450
(14) . This observation reveals the
importance of the double bond geometry at carbon 6 when comparing the
metabolic fate of LTB
with these isomeric 5,12-diHETEs.
Furthermore, it is interesting to note that the metabolism of
prostaglandins proceeds by
-oxidation from the carboxyl
terminus(15) . Such C-1
-oxidation has not been observed
as a major pathway for LTB
metabolism. The only exception
was the identification of a
-oxidation intermediate,
3-hydroxy-LTB
, formed when LTB
was metabolized
in rat hepatocytes in the presence of
ethanol(16, 17) .
The 5,12-diHETEs are hydroxy,
unsaturated fatty acids that could be substrates for -oxidation
from the carboxyl terminus. Studies of the metabolism of structurally
related monohydroxyeicosatetraenoic acids, 12-HETE and 5-HETE, revealed
-oxidation from the C-1 terminus to be a major pathway of
metabolism. Metabolism of 12-HETE by
-oxidation in mouse
peritoneal macrophages led to the formation of both
8-hydroxy-hexadecatrienoic acid and 4-hydroxy-dodecenoic acid as major
metabolites(18) . Recently, a microsomal 5-hydroxyeicosanoid
dehydrogenase activity was reported to exist within human
polymorphonuclear leukocytes that reversibly converted
5(S)-HETE to 5-oxo-ETE and also converts
6-trans-12-epi-LTB
to a 5-oxo
metabolite(19) . This 5-oxo metabolite was further converted to
dihydro products identified as 5-oxo-6,11-dihydro-LTB
and
6,11-dihydro-LTB
(19) .
In the present work,
metabolism of 6-trans-12-epi-LTB and
6-trans-LTB
was examined in a human-derived
hepatoma cell line, HepG2 cells. These cells metabolize LTB
by a unique
-oxidation process from C-1 with the formation
of 10-hydroxy-octadecatetraenoic acid(20) . However, the
metabolism of the 6-trans epimers of LTB
was
completely different from that previously described for
LTB
. The results reported here reveal that
-oxidation
for these 5,12-diHETEs is dependent on the geometry of the first double
bond (
) in the conjugated triene. Such metabolic
processes could play an important role in limiting the activity of free
radical-derived B
-isoleukotrienes generated in
vivo.
Catalytic hydrogenation of metabolites was performed as described earlier (23) prior to PFB/TMS derivatization. Oxidative ozonolysis of 200-400 ng of sample was performed without solvent as described earlier (13) prior to PFB/TMS derivatization as described above.
Figure 1:
Reverse phase HPLC analysis with
radioactivity monitoring of HepG2 metabolites released from the cells
following: (A) 24 h incubation with
6-trans-12-epi-LTB and (B) 5 h
incubation with
6-trans-LTB
.
The time course for the formation of the major metabolite of
6-trans-12-epi-LTB (Metabolite 1) is
shown in Fig. 2A and the time courses for the formation
of the remaining intermediate metabolites are shown in Fig. 2B. At all time points, metabolite 1 was the major
radioactive metabolite. Metabolites 2, 3, and 4 reached a maximum
concentration of approximately 5% of the total radioactivity between 3
and 9 h of incubation and declined to less than 1% by 24 h. Metabolite
5 reached a maximum concentration of approximately 15% of the total
radioactivity between 3 and 9 h and thereafter declined and was not
detected at 24 h. Similar time courses were obtained when HepG2 cells
were incubated with 6-trans-LTB
. Radioactive
metabolites were analyzed by GC/MS analysis in both the negative ion
mode and positive ion mode following derivatization to the
pentafluorobenzyl ester/trimethylsilyl ether (PFB/TMS) compounds.
Figure 2:
Formation of HepG2 extracellular
metabolites during incubation with
6-trans-12-epi-LTB from 3 to 24 h
expressed as a percent of the total recovered radioactivity for each
metabolite identified in Fig. 1. A, time course for
formation of the major metabolite, metabolite 1. B, time
course for the formation of the remaining metabolites, metabolites 2,
3, 4, and 5.
Figure 3: Positive ion electron ionization (70 eV) mass spectra of the PFB/TMS derivatives of: A, 4-hydroxy-6-dodecenoic acid (metabolite 1); B, 6-hydroxy-4,8-tetradecadienoic acid (metabolite 2); and C, 8-hydroxy-4,6,10-hexadecatrienoic acid (metabolite 3) with UV spectrum shown in inset.
Figure 4:
Mass spectral analysis of the PFB/TMS
derivative of 5-oxo-6,7-dihydro LTB (metabolite 4). A, negative ion chemical ionization mass spectrum with UV
spectrum shown in the inset. B, positive ion electron
ionization (70 eV) mass spectrum.
Figure 5:
Proposed mechanism leading to formation of
the ion observed at m/z 167 in the electron ionization mass
spectrum of the PFB/TMS derivative of
5-oxo-6,7-dihydro-LTB.
Figure 6:
Electron ionization (70 eV) mass spectra
of: (A) the PFB/TMS derivative of 6,7-dihydro-LTB (metabolite 5) with UV spectrum shown in the inset and (B) the PFB/[
H
] TMS
derivative of 6,7-dihydro-LTB
.
Figure 7:
Proposed mechanism leading to formation
of the ions observed at m/z 395 and 279 in the electron
ionization mass spectrum of the PFB/TMS derivative of
6,7-dihydro-LTB.
The observed ion at m/z 435 also
contained one OTMS group as evidenced by a shift of 9 atomic mass units
to m/z 444 in the analysis of the
PFB/[H
]TMS derivative. This ion may
be due to loss of acetylene (26 atomic mass units) from the base peak
at m/z 461. This would be a favorable process with
loss of a small neutral molecule possibly involving loss of carbons 11
and 12 with OTMS transfer to C-10. The corresponding ion in the methyl
ester/TMS derivative, m/z 269, may likewise be due to
loss of 26 atomic mass units from m/z 295 rather than
an unlikely vinylic fragmentation of the C-10,C-11 bond with the
conjugated diene at C-7 and C-9 as was previously suggested (19, 25) . A C-7,C-9 position for the conjugated diene
moiety would also be expected to result in an abundant fragment ion due
to fragmentation of the C-11,C-12 bond with loss of a neutral allylic
stabilized radical (C-1 though C-11) and formation of an observed ion
at m/z 213
(TMSO
=CHCH
CH =
CHC
C
). Lack of an observed ion at m/z 213 then implies in the spectra reported here as
well as in the spectra of the previously identified
6,11-dihydro-LTB
metabolite derivatized as the methyl
ester/TMS compound (19, 25) that location of the
conjugated diene is best supported by positive ion GC/MS data as a
6,7-dihydro-LTB
structure.
In order to unambiguously
assign double bond location, metabolite 5 was subjected to oxidative
ozonolysis and negative ion GC/MS analysis of the products following
PFB/TMS derivatization. With double bonds located at C-8 and C-10, the
expected dicarboxylic acids were a 4-carbon monohydroxy fragment
corresponding to C-11 through C-14 and an 8-carbon monohydroxy fragment
corresponding to C-1 through C-8. Following PFB/TMS derivatization,
these fragments were observed at m/z 385 and 441 with
EC values of 13.4 and 17.9, respectively (Fig. 8). The spectrum
of the derivatized 4-carbon fragment is identical to that obtained from
oxidative ozonolysis followed by PFB/TMS derivatization of LTB with an observed base peak at m/z 385 (loss of
one PFB group) and minor ions observed at m/z 341
(loss of 44 atomic mass units) and at m/z 295 (loss
of TMSOH from m/z 385) (Fig. 8A). The
spectrum of the PFB/TMS derivatized 8-carbon fragment is also dominated
by an observed ion resulting from loss of one PFB group at m/z 441 (Fig. 8B). Expected fragments
resulting from oxidative ozonolysis of a dihydro metabolite containing
double bonds at positions C-7 and C-9, a 5-carbon monohydroxy diacid
and a 7-carbon monohydroxy diacid resulting in expected PFB/TMS
derivatized fragments observed at m/z 399 and 427,
respectively, were not observed. The above data for metabolite 5 is
then consistent with the structure
5,12-dihydroxy-8,10,14-eicosatrienoic acid
(6,7-dihydro-LTB
).
Figure 8:
Negative ion chemical ionization GC/MS
analysis of the PFB/TMS derivatives of ozonolysis products of
6,7-dihydro-LTB. A, ion profile at m/z 385 with the inset showing negative ion mass spectrum of
fragment containing C-11 through C-14 observed at scan 153 with EC
value = 13.4, and B, ion profile at m/z 441
with the inset showing negative ion mass spectrum of fragment
containing C-1 through C-8 observed at scan 297 with EC value =
17.9.
It was previously observed that oxo
compounds elute during RP HPLC analysis after the corresponding hydroxy
compound when acetonitrile is present in the organic
phase(19, 26) , but in the present system using only
methanol in the organic phase, the 5-oxo compound eluted slightly
before the corresponding hydroxy compound. The reverse order of elution
depending on the presence or absence of acetonitrile was also observed
using synthetic 12-oxo-LTB which eluted from RP HPLC
slightly before LTB
when only methanol was used in the
organic phase but eluted after LTB
when the organic phase
consisted of 1:1 methanol:acetonitrile.
Figure 9:
Reverse phase HPLC analysis with
radioactivity monitoring of extracellular metabolites produced by human
polymorphonuclear leukocytes following 50 min incubation with
6-trans-12-epi-LTB. Inset shows
the UV monitor trace at 230 nm revealing the conjugated
diene-containing metabolites, 5-oxo-6,7-dihydro-LTB
(4) and 6,7-dihydro-LTB
(5) , with
end absorption of 6-trans-12-epi-LTB
(
= 269 nm). Resolution at the UV
spectrophotometer is better than observed at the radioactivity monitor
due to on-line mixing of the HPLC effluent with scintillation mixture
prior to radioactivity detection.
The metabolism of 6-trans-LTB and
6-trans-12-epi-LTB
by HepG2 cells was
quite similar both qualitatively and quantitatively. Based on the
chemical structures of the identified metabolites, the sequence of
metabolic events is proposed in Fig. 10. Initial metabolism
likely involved oxidation of the 5-hydroxy group to form
5-oxo-LTB
which has been previously described(19) .
This ketone could be reduced to 5-oxo-6,7-dihydro-LTB
by an
enzymatic activity which converts an
,
-unsaturated keto
moiety to the corresponding saturated ketone. We have termed this
activity
-reductase. This same enzymatic pathway was
found in human polymorphonuclear leukocytes when metabolism of
6-trans-12-epi-LTB
was examined and exact
double bond location in the observed metabolites determined by
oxidative ozonolysis. It is likely that the initially described
reductase pathways suggested to lead to the formation of
6,11-dihydro-LTB
(19, 27) actually
resulted in the formation of 6,7-dihydro-LTB
.
Figure 10:
Proposed metabolism of
6-trans-LTB and
6-trans-12-epi-LTB
in HepG2 cells
involving 5-hydroxydehydrogenase/
-reductase and
-oxidation.
The
reduction of ,
-unsaturated ketones, which are metabolites of
various eicosanoids, is a major metabolic process. The metabolism of
LTB
by 12-hydroxyeicosanoid dehydrogenase leads to the
formation of an
,
-unsaturated ketone, 12-oxo-LTB
.
Subsequent metabolism involving
-reductase results in
the formation of 10,11-dihydro metabolites which have been identified
in several human cell
types(13, 28, 29, 30) . A major
route of metabolism of many prostaglandins involves initial oxidation
of the 15-hydroxy group with formation of 15-oxo prostanoids which are
,
-unsaturated ketones. This process is catalyzed by a widely
distributed enzyme, 15-hydroxyprostaglandin dehydrogenase ( (31) and references therein). Further metabolism by
15-keto-
-reductase results in formation of
13,14-dihydro-15-oxo metabolites(32, 33) .
The
chain shortened metabolites identified during metabolism of both
6-trans-LTB and
6-trans-12-epi-LTB
by HepG2 cells
suggested that initial metabolism by the 5-hydroxyeicosanoid
dehydrogenase/
-reductase pathway was followed by
-oxidation as outlined in Fig. 10. It was previously
demonstrated that metabolism of
6-trans-12-epi-LTB
by human
polymorphonuclear leukocytes occurred with formation of
dihydro-LTB
with loss of the proton label at the C-5 carbon
atom suggesting an intermediate formation of
5-oxo-dihydro-LTB
(27) . Further
-oxidation
could occur from either dihydro intermediate; however, if
-oxidation occurred primarily from the 6,7-dihydro-LTB
intermediate, identification of an 18-carbon dihydroxy
intermediate might be expected. As no chain shortened 18-carbon
metabolites were observed,
-oxidation is suggested to occur
primarily from 5-oxo-6,7-dihydro-LTB
. The probable CoA
ester-dependent initial steps in peroxisomal
-oxidation of
5-oxo-6,7-dihydro-LTB
involving acyl-CoA oxidase, enoyl-CoA
hydratase, 3-hydroxyacyl-CoA dehydrogenase, and
-ketothiolase
would lead to formation of an 18-carbon intermediate containing a keto
group 3 carbon atoms removed from the CoA ester moiety. This could be
rapidly metabolized by a second
-ketothiolase step to produce the
first chain shortened intermediate metabolite that is released to the
medium, 8-hydroxy-4,6,10-hexadecatrienoic acid. Such a pathway would
also suggest that the formation of 6,7-dihydro-LTB
from
5-oxo-6,7-dihydro-LTB
must be reversible. The reversibility
of a similar step has been suggested in the metabolism of LTB
by human keratinocytes where 12-hydroxyeicosanoid
dehydrogenase/
-reductase activity resulted in the
formation of C-12 epimeric isomers of
10,11-dihydro-LTB
(13) . Metabolism of
6,7-dihydro-LTB
to 5-oxo-6,7-dihydro-LTB
would
involve 5-hydroxyeicosanoid dehydrogenase activity. It cannot be
determined from the results reported here if this is the same
5-hydroxyeicosanoid dehydrogenase activity that is responsible for
metabolism of 6-trans-LTB
to
5-oxo-6-trans-LTB
.
Conversion of
8-hydroxy-4(Z),6(E),10(Z)-hexadecatrienoic
acid to 6-hydroxy-4,8-tetradecadienoic acid and 4-hydroxy-6-dodecenoic
acid has been reported in metabolism of 12-HETE and was suggested to
result from peroxisomal -oxidation(18) . The monohydroxy
hexadecatrienoic acid identified in the present study may differ in the
stereochemistry of the conjugated diene with a
4(E),6(E) stereochemistry unchanged from the trans,trans stereochemistry of the C-8 and C-10
double bonds of 6-trans-LTB
and
6-trans-12-epi-LTB
, but normal pathways
of
-oxidation from this metabolite would also result in formation
of the chain shortened metabolites. Subcellular localization of this
metabolic activity has not been determined for HepG2 metabolism.
Little is known about the metabolic processes that are involved in
degradation of 5,12-diHETEs or other hydroxy polyunsaturated fatty
acids that may be formed by lipid peroxidation. An exception is the
metabolism of LTB for which there are specific enzymes
responsible for metabolic inactivation. Detailed studies of the
individual steps in
-oxidation of polyunsaturated fatty acids is
known to be complex as exemplified by the discovery of 2,4-dienoyl-CoA
reductase (34) as well as specific enzymes that conjugate
double bonds up to 5-carbon atoms removed from the CoA ester
moiety(35) . The presence of a hydroxyl group in the fatty acid
backbone further alters the normal steps of
-oxidation. The
conjugated diene alcohol structural motif is common to a wide variety
of products that have undergone either lipoxygenase-catalyzed
oxygenation, autoxidation, or free radical catalyzed mechanisms of
lipid peroxidation to form conjugated diene alcohols(36) . The
metabolic reaction observed for the 5,12-diHETEs studied here suggests
a common reaction pathway for such molecules, that being oxidation of
the conjugated diene alcohol to the conjugated dienone followed by
reduction to a nonconjugated ketone.
-Oxidation can then proceed
through this functionalized carbon atom.