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A previously unidentified leukotriene produced by human polymorphonuclear leukocytes
Vol. 120, No. 2, 1984
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
April 30, 1984
Pages 548-552
A PREVIOUSLY UNIDENTIFIED LEUKOTRIENE PRODUCED BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES William
George
Nolan
Veterans Administration Medical of Utah School of Medicine, Salt
University Received
Jubiz,
March
20,
Center and Lake City,
Utah 84148
1984
unidentified leukotriene was isolated from incubaSUMMARY: A previously tions of human polymorphonuclear leukocytes with ionophore A23187. The compound eluted and 20-carboxy LTB4 on SP-HPLC and between LTB4 between the two 6-trans isomers of Ultraviolet LTB4 on RP-HPLC. spectroscopy revealed three absorption bands at 258 nm, 268 nm and 278 nm. 1802 was incorporated by the OH at both C5 and Cl2. Oxidative ozonolysis indicated the presence of 5s. 12s configuration. The structure of the newly identified leukotriene is 5S, 12S-dihydroxy-icosatetraenoic acid. Stereochemistry of the double bonds and biologic activity were not investigated.
Arachidonic
acid
is
the
enzymatic
pathways
catalyzed
dependent
oxidizing
system
include
a series
diverse
biologic
trienes,
are
bonds 8,
or
are
While
working
consistent phase
peak high
spectroscopy at
by
to
of
B4
between
a typical
(LTB4)
12-dihydroxy
5,
both
groups
hydroxy
new leukotriene 0006-291X/84 Copyright All rights
0
is
leukotriene
5S, 12S-dihydroxy-icosatetraenoic
Inc. reserved.
548
system exhibiting
the
conjugated acid
several
we have
leukodouble
at carbons
acids
LTB4
a small on
(SP-HPLC).
spectrum
with is
Thus.
the acid.
but
straight-
Ultraviolet absorption
bands
characteristic
ozonolysis
5.
intermediates.
observed
20-carboxy
Oxidative
$1.50
acids
three
mass spectrum
acid.
lipoxygenase
arachidonic
and
had an S configuration.
1984 by Academic Press. of reproduction in any form
of
three
and an NADPH-
compounds,
chromatography
258 nm, 268 nm and 278 nm and the
dioxygenated
these
through
LTB4
liquid
of the
via
hydroperoxi-icosatetraenoic
leukotrienes
leukotriene
performance
of
formation
into
of
presence
oxidation
the
eluting
revealed
the
compounds
and trihydroxy
A group
Initial
metabolized with
dihydroxy
of
lipoxygenase
The products
(1).
characterized
15 leads
which
(l-3).
activity
(4).
of a variety
by cyclooxygenase,
of monohydroxy.
triene
12 and
precursor
of a
revealed structure
that of
this
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 120, No. 2, 1984
MATERIALS
AND METHODS
Procedures for isolation and stimulation of polymorphonuclear leukocytes (PMNL) and for extraction of the leukotrienes released into the medium have been described previously (5). In short, PMNL obtained from human peripheral blood were sedimented with dextran, purified by suspended in Hank's buffer pH 7.4. Ficoll-Hypaque gradient centrifugation. incubated at 37O C and stimulated with 5 uM ionophore A23187 for five minutes. Reaction was stopped with 1.5 volumes of methanol and the Following treatment leukotrienes were extracted with ether at pH 3.0. samples were injected into a Waters HPLC system with diazomethane, equipped with a U6K injector, a model M-45 solvent delivery system, a model 450 variable wavelength detector and an Omni Scribe B-500 recorder (Houston Instruments, Austin, Texas 78753). Leukotrienes were eluted from a straight-phase Radial-PAK cartridge (SI, 8 mm x 10 cm, 5u) in a Z-module with hexane/isopropanol/acetic acid 97/3/0.01 V/V/V at 4 ml/min. Peaks at 270 nm were collected for scanning ultraviolet (UV) spectroscopy and gas chromatography-mass spectrometry (GC-MS). In addition, the peaks obtained from the SP-HPLC were injected into a reverse-phase Radial-PAK cartridge with methanol/water/acetic acid (Cl89 8 mm ID x 10 cm. 5 u) and eluted 75/25/0.01 V/V/V at 2 ml/min. This procedure allowed to establish a relationship between compound A and the other 5,12-dihydroxy compounds. Incubations were carried out in the presence or absence of 18 02. of the products for ultraviolet Oxidative ozonolysis and preparation spectroscopy and GC-MS were performed by published methods (5-7). RESULTS A SP-HPLC incubating
PMNL with
eluting
at
exhibited
18.4
278
accounting
presence
mass
205, units
(Figure
28).
graphed
with
2% of
first
10,
those
of
derivative
to
dihydroxy-icosatetraenoic
to
Products the
in
a chain both
387
498
length
As it
cis. the
trans.
SP-HPLC
were
in
are
metabolite 258 nm, 268
extremely
small,
(c-value)
acid.
Shifting
molecular
of
018
at
compound
acid in
Figure
23.6
2A) and of
ion
by four
C5 and
Cl2
A cochromato-
of dimethyl-L-malate
GC-MS of 5s.
shown
ion
the the
of
(Figure
the
of
A revealed
identical
cis)-icosatetraenoic
549
A,
at
compound
derivatives
chromatogram
bands
of
ozonolysis
happens,
Compound
ZO-carboxy
A
incorporation
(-)-menthoxycarbonyl
by
1.
equivalent
and an increase
oxidative
Figure
obtained
Gas chromatography of
compounds
indicates of
its
LTB4.
of
494
and
compound
spectra
14 (trans, peak
of
Mass
383 to
in
esters)
absorption
with
ion
shown
with
isomers
5.12
(methyl
LTB4
quantities
dimethyl-2-L-hydroxyadipate. 8,
between
UV pattern
than
is
two
corresponded to
products
A23187
trimethylsilylether
of
24.9.
203
and
The
less
ester
the
ionophore
minutes
nm. for
methyl
it
of
a characteristic
nm and
and
chromatogram
and
12S-dihydroxy-6. (5,12-diHETE), 1 revealed
the the
Vol.
120,
No.
2, 1984
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
i.lZ-diHETE
0
4
B
12
16
20
, 24
Minutes
FIGURE
presence and eluted
1.
of
SP-HPLC chromatogram of an etner extract from an incubation of human PMNL (60 ml of 100 x 106 cells/ml) with 5 uM ionophore A-23187 for 5 min at 370 C. Z-module system, Radial-PAK cartridge SI, 8 rmn ID x 10 cm, 5 u. System: hexane/isopropanol/acetic acid 97/3/0.01 V/V/V. Pump rate 4 ml/min. Ultraviolet absorption was monitored at 270 nm. Sample was treated with diazomethatie prior to injection. 25X indicates where the detector sensitivity was increased 25 times.
an
isomer,
incorporation between
not of
the
two
previously
018
at
6-trans
identified, CS
isomers
and
C,2.
of
LTB4.
with On
a
c-value
RP-HPLC
of
24.9
compound
A
_DISCUSSION A previous of LTB4
human and
unidentified PMNL
20-carboxy
with
leukotriene ionophore LT84
on
has
A-23187. SP-HPLC
The and
550
been
between
isolated compound the
from migrates 6-trans
incubations between isomers
of
Vol.
0
70
120,
90
No.
110
BIOCHEMICAL
2, 1984
130
150
170
190
no
230
AND
250
BIOPHYSICAL
270
ml
310
RESEARCH
330
350
370
COMMUNICATIONS
390
410
430
450
470
m/e
B FIGURE
LTB4 was
on
2.
Mass spectra of compound A, C value = 23.6. A = 016, 8 = 018. Shift on ion 203 to 205. ion 383 to 387 and increase in the molecular ion by four mass units indicate incorporation of and The structure O18 at % c12. corresponds to 55. 12S-dihydroxy-icosatetraenoic acid.
RP-HPLC.
observed.
The
compound
and
hydroxyl
groups.
tion
isomer
like
trans,
small
that
the
In
excluded.
addition,
suggests
compound. traenoic
The acid
material
the
double
the
we
have
cis.
trans-trans-trans-cis
biologic
effects
other an
compound
remains
to
be
and
for did
bonds.
It
trans, have
isomer
elusive
5S,
nm
both
not is
cis the
allow
possible configura-
trans.
trans. cannot
of
(5.12-diHETE).
278
5.12-dihydroxy
configuration
the
be
dioxygenated The
configuration this
nm
possibilities
identified
of
268
obtained
may
compound a
S
trans.,
isomer
However,
5,12-dihydroxy-trans-cis-trans-cis 24.9
of
C=24.9
configuration.
cis
an
have
nm,
a dioxygenated
revealed
of may
258
of
quantities
C=23.6
and
at
that
stereochemistry
with
LTB4
is
ozonolysis
The the
the
absorption
mass-spectrum
oxidative
investigating that
Ultraviolet
for
C value this
of new
12S-dihydroxy-icosate-
determined.
fiCKNOWLEDGMENTS
authors
Supported wish
by the to thank
Research Service Dr. Dana Wilson
of for 551
the Veterans allowing
us
Administration. to use his
The equipment
.#I
Vol. 120, No. 2, 1984 for performing help.
BIOCHEMICAL ozonolysis
and
to
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Gwenevere
Shaw
for
expert
secretarial
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Samuelsson, B. (1983) Science 200, 568-575. Morrison, A.R., and Pascoe, N. (1981) Proc. Natl. Acad. Sci. 78, 1375-7378. Capdevila, J., Marnetta, L.J., Chacos, N., Prough, R.A.. and Estrabrook, R.W. (1982) Proc. Natl. Acad. Sci. 79, 767-770. Borgeat, P., and Samuelsson, B. (1979) J. Biol. Chem. 254, 2643-2646. Jubiz, W. (1983) Biochem. Biophys. Res. Commun. 110, 842-850. Jubiz, W., Radmark, 0.. Lindgren, J.A., Malmsten, C., and Samuelsson, 8. (1981).Biochem. Biophys. Res. Commun. 99, 976-986. Jubiz, W., Radmark, O., Malsten, C., Hansson, G., Lindgren, J.A.. Pamblad, J., Uden. A., and Samuelsson, B. (1982) J. Biol. Chem. 257, 6106-6110.
552
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
April 30, 1984
Pages 548-552
A PREVIOUSLY UNIDENTIFIED LEUKOTRIENE PRODUCED BY HUMAN POLYMORPHONUCLEAR LEUKOCYTES William
George
Nolan
Veterans Administration Medical of Utah School of Medicine, Salt
University Received
Jubiz,
March
20,
Center and Lake City,
Utah 84148
1984
unidentified leukotriene was isolated from incubaSUMMARY: A previously tions of human polymorphonuclear leukocytes with ionophore A23187. The compound eluted and 20-carboxy LTB4 on SP-HPLC and between LTB4 between the two 6-trans isomers of Ultraviolet LTB4 on RP-HPLC. spectroscopy revealed three absorption bands at 258 nm, 268 nm and 278 nm. 1802 was incorporated by the OH at both C5 and Cl2. Oxidative ozonolysis indicated the presence of 5s. 12s configuration. The structure of the newly identified leukotriene is 5S, 12S-dihydroxy-icosatetraenoic acid. Stereochemistry of the double bonds and biologic activity were not investigated.
Arachidonic
acid
is
the
enzymatic
pathways
catalyzed
dependent
oxidizing
system
include
a series
diverse
biologic
trienes,
are
bonds 8,
or
are
While
working
consistent phase
peak high
spectroscopy at
by
to
of
B4
between
a typical
(LTB4)
12-dihydroxy
5,
both
groups
hydroxy
new leukotriene 0006-291X/84 Copyright All rights
0
is
leukotriene
5S, 12S-dihydroxy-icosatetraenoic
Inc. reserved.
548
system exhibiting
the
conjugated acid
several
we have
leukodouble
at carbons
acids
LTB4
a small on
(SP-HPLC).
spectrum
with is
Thus.
the acid.
but
straight-
Ultraviolet absorption
bands
characteristic
ozonolysis
5.
intermediates.
observed
20-carboxy
Oxidative
$1.50
acids
three
mass spectrum
acid.
lipoxygenase
arachidonic
and
had an S configuration.
1984 by Academic Press. of reproduction in any form
of
three
and an NADPH-
compounds,
chromatography
258 nm, 268 nm and 278 nm and the
dioxygenated
these
through
LTB4
liquid
of the
via
hydroperoxi-icosatetraenoic
leukotrienes
leukotriene
performance
of
formation
into
of
presence
oxidation
the
eluting
revealed
the
compounds
and trihydroxy
A group
Initial
metabolized with
dihydroxy
of
lipoxygenase
The products
(1).
characterized
15 leads
which
(l-3).
activity
(4).
of a variety
by cyclooxygenase,
of monohydroxy.
triene
12 and
precursor
of a
revealed structure
that of
this
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 120, No. 2, 1984
MATERIALS
AND METHODS
Procedures for isolation and stimulation of polymorphonuclear leukocytes (PMNL) and for extraction of the leukotrienes released into the medium have been described previously (5). In short, PMNL obtained from human peripheral blood were sedimented with dextran, purified by suspended in Hank's buffer pH 7.4. Ficoll-Hypaque gradient centrifugation. incubated at 37O C and stimulated with 5 uM ionophore A23187 for five minutes. Reaction was stopped with 1.5 volumes of methanol and the Following treatment leukotrienes were extracted with ether at pH 3.0. samples were injected into a Waters HPLC system with diazomethane, equipped with a U6K injector, a model M-45 solvent delivery system, a model 450 variable wavelength detector and an Omni Scribe B-500 recorder (Houston Instruments, Austin, Texas 78753). Leukotrienes were eluted from a straight-phase Radial-PAK cartridge (SI, 8 mm x 10 cm, 5u) in a Z-module with hexane/isopropanol/acetic acid 97/3/0.01 V/V/V at 4 ml/min. Peaks at 270 nm were collected for scanning ultraviolet (UV) spectroscopy and gas chromatography-mass spectrometry (GC-MS). In addition, the peaks obtained from the SP-HPLC were injected into a reverse-phase Radial-PAK cartridge with methanol/water/acetic acid (Cl89 8 mm ID x 10 cm. 5 u) and eluted 75/25/0.01 V/V/V at 2 ml/min. This procedure allowed to establish a relationship between compound A and the other 5,12-dihydroxy compounds. Incubations were carried out in the presence or absence of 18 02. of the products for ultraviolet Oxidative ozonolysis and preparation spectroscopy and GC-MS were performed by published methods (5-7). RESULTS A SP-HPLC incubating
PMNL with
eluting
at
exhibited
18.4
278
accounting
presence
mass
205, units
(Figure
28).
graphed
with
2% of
first
10,
those
of
derivative
to
dihydroxy-icosatetraenoic
to
Products the
in
a chain both
387
498
length
As it
cis. the
trans.
SP-HPLC
were
in
are
metabolite 258 nm, 268
extremely
small,
(c-value)
acid.
Shifting
molecular
of
018
at
compound
acid in
Figure
23.6
2A) and of
ion
by four
C5 and
Cl2
A cochromato-
of dimethyl-L-malate
GC-MS of 5s.
shown
ion
the the
of
(Figure
the
of
A revealed
identical
cis)-icosatetraenoic
549
A,
at
compound
derivatives
chromatogram
bands
of
ozonolysis
happens,
Compound
ZO-carboxy
A
incorporation
(-)-menthoxycarbonyl
by
1.
equivalent
and an increase
oxidative
Figure
obtained
Gas chromatography of
compounds
indicates of
its
LTB4.
of
494
and
compound
spectra
14 (trans, peak
of
Mass
383 to
in
esters)
absorption
with
ion
shown
with
isomers
5.12
(methyl
LTB4
quantities
dimethyl-2-L-hydroxyadipate. 8,
between
UV pattern
than
is
two
corresponded to
products
A23187
trimethylsilylether
of
24.9.
203
and
The
less
ester
the
ionophore
minutes
nm. for
methyl
it
of
a characteristic
nm and
and
chromatogram
and
12S-dihydroxy-6. (5,12-diHETE), 1 revealed
the the
Vol.
120,
No.
2, 1984
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
i.lZ-diHETE
0
4
B
12
16
20
, 24
Minutes
FIGURE
presence and eluted
1.
of
SP-HPLC chromatogram of an etner extract from an incubation of human PMNL (60 ml of 100 x 106 cells/ml) with 5 uM ionophore A-23187 for 5 min at 370 C. Z-module system, Radial-PAK cartridge SI, 8 rmn ID x 10 cm, 5 u. System: hexane/isopropanol/acetic acid 97/3/0.01 V/V/V. Pump rate 4 ml/min. Ultraviolet absorption was monitored at 270 nm. Sample was treated with diazomethatie prior to injection. 25X indicates where the detector sensitivity was increased 25 times.
an
isomer,
incorporation between
not of
the
two
previously
018
at
6-trans
identified, CS
isomers
and
C,2.
of
LTB4.
with On
a
c-value
RP-HPLC
of
24.9
compound
A
_DISCUSSION A previous of LTB4
human and
unidentified PMNL
20-carboxy
with
leukotriene ionophore LT84
on
has
A-23187. SP-HPLC
The and
550
been
between
isolated compound the
from migrates 6-trans
incubations between isomers
of
Vol.
0
70
120,
90
No.
110
BIOCHEMICAL
2, 1984
130
150
170
190
no
230
AND
250
BIOPHYSICAL
270
ml
310
RESEARCH
330
350
370
COMMUNICATIONS
390
410
430
450
470
m/e
B FIGURE
LTB4 was
on
2.
Mass spectra of compound A, C value = 23.6. A = 016, 8 = 018. Shift on ion 203 to 205. ion 383 to 387 and increase in the molecular ion by four mass units indicate incorporation of and The structure O18 at % c12. corresponds to 55. 12S-dihydroxy-icosatetraenoic acid.
RP-HPLC.
observed.
The
compound
and
hydroxyl
groups.
tion
isomer
like
trans,
small
that
the
In
excluded.
addition,
suggests
compound. traenoic
The acid
material
the
double
the
we
have
cis.
trans-trans-trans-cis
biologic
effects
other an
compound
remains
to
be
and
for did
bonds.
It
trans, have
isomer
elusive
5S,
nm
both
not is
cis the
allow
possible configura-
trans.
trans. cannot
of
(5.12-diHETE).
278
5.12-dihydroxy
configuration
the
be
dioxygenated The
configuration this
nm
possibilities
identified
of
268
obtained
may
compound a
S
trans.,
isomer
However,
5,12-dihydroxy-trans-cis-trans-cis 24.9
of
C=24.9
configuration.
cis
an
have
nm,
a dioxygenated
revealed
of may
258
of
quantities
C=23.6
and
at
that
stereochemistry
with
LTB4
is
ozonolysis
The the
the
absorption
mass-spectrum
oxidative
investigating that
Ultraviolet
for
C value this
of new
12S-dihydroxy-icosate-
determined.
fiCKNOWLEDGMENTS
authors
Supported wish
by the to thank
Research Service Dr. Dana Wilson
of for 551
the Veterans allowing
us
Administration. to use his
The equipment
.#I
Vol. 120, No. 2, 1984 for performing help.
BIOCHEMICAL ozonolysis
and
to
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Gwenevere
Shaw
for
expert
secretarial
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Samuelsson, B. (1983) Science 200, 568-575. Morrison, A.R., and Pascoe, N. (1981) Proc. Natl. Acad. Sci. 78, 1375-7378. Capdevila, J., Marnetta, L.J., Chacos, N., Prough, R.A.. and Estrabrook, R.W. (1982) Proc. Natl. Acad. Sci. 79, 767-770. Borgeat, P., and Samuelsson, B. (1979) J. Biol. Chem. 254, 2643-2646. Jubiz, W. (1983) Biochem. Biophys. Res. Commun. 110, 842-850. Jubiz, W., Radmark, 0.. Lindgren, J.A., Malmsten, C., and Samuelsson, 8. (1981).Biochem. Biophys. Res. Commun. 99, 976-986. Jubiz, W., Radmark, O., Malsten, C., Hansson, G., Lindgren, J.A.. Pamblad, J., Uden. A., and Samuelsson, B. (1982) J. Biol. Chem. 257, 6106-6110.
552