From the
Isoprostanes are a family of prostaglandin (PG) isomers formed
in an enzyme-independent manner. They circulate in plasma and are
excreted in urine. One of them, 8-epi PGF
Activation of
platelets by threshold concentrations of collagen, thrombin, and
arachidonic acid resulted in formation of 8-epi PGF
Confirmation
of the nature of the material formed by platelet COX as 8-epi
PGF
In addition to its generation by free radical
catalyzed mechanisms, 8-epi PGF
The isoprostanes are families of prostaglandin isomers formed in
a free radical catalyzed manner from arachidonic acid
(1, 2) . They are produced via peroxyl radical isomers
which undergo endocylcization and subsequent reduction; isoprostanes of
the E and F series have been reported. Given the manner of their
formation, it has been proposed that measurement of isoprostanes might
offer a quantitative index of free radical generation in vivo.
Existing indices of this process are of controversial validity when
applied to clinical studies. Consequently, our understanding of the
role of free radicals in human disease has been confused
(3, 4) , and there is a paucity of data underlying the
selection and usage of antioxidant drugs in vivo. Particular
attention has focused on one of the isoprostanes, 8-epi
PGF
Human Subjects The study was scrutinized and approved by the Ethics Committee of the
Mater Hospital, Dublin, Ireland. All participants signed an informed
consent document prior to participation in the study. The volunteers
were Caucasian males, aged 21-49 years. All were nonsmokers and
abstained from all medication for at least 14 days prior to
participation in the study. No abnormality was revealed on clinical
examination or on full blood count, platelet count, or routine
biochemical screening. Blood was collected, without stasis, into a
plastic syringe, using a 21-gauge needle from an antecubital vein after
the subject had been sitting for at least 5 min. Samples were drawn
into 3.8% sodium citrate in a ratio of 9:1. The samples were taken
between 9:00 a.m. and 11:00 a.m. at least 12 h after the last meal.
The role of COX was assessed by preincubating PRP or WP
for 5 min at 37 °C with aspirin (100 µM; Aspidol,
Maggioni, Milan, Italy) or indomethacin (10 µM; Sigma) and
inhibition of activity confirmed by the absence of an aggregation
response to arachidonic acid (1 mM; Cayman Chemical Co., Ann
Arbor, MI). The mechanism of 8-epi PGF
For electron impact-mass
spectrometry (EI/MS) studies, the methyl ester was formed by dissolving
the sample in 100 µl of methanol and 500 µl of a dilute
solution of diazomethane in ether and allowing it to stand at room
temperature for 30 min. GC-MS All GC-MS studies were performed on a Delsi-Nermag Automass 150
(Delsi-Nermag, Argenteuil, France) equipped with a Varian 1077
split/splitless injector operated in the splitless mode, at 280 °C.
Helium was used as the carrier gas. The interface was maintained at 280
°C, the ion source at 250 °C. The MS was operated in the
negative ion, chemical ionization mode, utilizing methane as the
reagent gas. The ion source was maintained at 250 °C. For electron
impact studies, the ionization energy was 70 eV, and the source
temperature was 190 °C.
Threshold concentrations of collagen
(0.5-2 µg/ml) induced irreversible aggregation in PRP
after a lag phase of 30 s. Three min after addition of agonist, the
levels of TxB
We studied WP to extend these observations and
incorporated measurement of 12-HETE as a non-COX, enzymatic product of
arachidonic acid in platelets. Collagen increased all three products in
WP coincident with aggregation. Aspirin pretreatment abolished
formation of TxB
Second,
[
F
Following
hepatic injury with carbon tetrachloride, formation in the membrane
precedes their release into the effluent when the liver is perfused
in situ. Similarly, oxidation of low density lipoprotein,
either by Cu
Attention has particularly focused upon one of these compounds,
8-epi PGF
To elucidate the mechanism of formation of
F
The
possibility existed that the peak in the endogenous material
corresponding to the internal standard for 8-epi PGF
More evidence was acquired by
obtaining an electron impact mass spectrum of a mixture of authentic
8-epi PGF
Third, human platelets made
octadeuterated 8-epi PGF
Data consistent with this observation have been
previously reported by others. Corey et al. (32) proposed a schema by which 8-epi PGF
The formation of 8-epi PGF
Formation of the compound as a
prostaglandin may provide intuitive support for the concept of distinct
receptors for 8-epi PGF
The
biological importance of this compound as an autacoid remains to be
established. It is noteworthy in this regard, that 8-epi PGF
The antioxidants and aspirin were incubated for 5 min
at 37 °C before adding the stimuli.
is a
vasoconstrictor and mitogen, effects which are prevented by thromboxane
antagonists. Given that 8-epi PGF
may be formed by
cyclooxygenase (COX) (Corey, E. J., Shih, C., Shig, N-Y., and Shimoji,
K. (1984) Tetrahedron Letts. 44, 5013-5016; Hecker, M.,
Ullrich, V., Fischer, C., and Meese, C. O. (1987) Eur J. Biochem. 169, 113-123) and that this might confound its use
as an index of free radical generation, we sought to characterize the
mechanism of its formation by human platelets.
coincident with that of the COX product, thromboxane, and the 12
lipoxygenase product, 12-hydroxyeicosatetraenoic acid, as detected by
selected ion monitoring assays using gas chromatography-mass
spectrometry. The effect appeared selective for 8-epi PGF
among the F
isoprostanes. Pretreatment of platelets
with aspirin or indomethacin abolished 8-epi PGF
formation. COX-independent activation of platelets by high doses
of collagen or thrombin, by the phorbol ester, phorbol 12-myristate
13-acetate, or the prostaglandin endoperoxide analog, U 46619 was not
associated with 8-epi PGF
formation.
included its cochromatography over three highly
resolving high performance liquid chromatography systems,
identification by electron impact mass spectrometry, and its formation
by partially purified COX. Inhibition of platelet thromboxane formation
was associated with augmented 8-epi PGF
formation.A major component of 8-epi PGF
formed in serum by
healthy volunteers was shown to be sensitive to inhibition by aspirin
ex vivo.
may be formed as a PG
by human platelets. Given that activation of platelet COX characterizes
many of the human syndromes which are putatively associated with free
radical generation, assessment of the contribution of this pathway is
relevant to the use of 8-epi PGF
as an index of lipid
peroxidation in vivo.
.
(
)
This compound is among
the most abundant of the F
isoprostanes formed under
physiological conditions in humans
(5) and induces
vasoconstriction in the renal and pulmonary circulations
(6, 7) , mitogenesis in NIH 3T3 cells
(8) , and
platelet shape change, but not aggregation and the release reaction
(9, 10) . These effects are prevented by thromboxane
receptor antagonists. We have developed a sensitive and specific assay
for this compound and have shown its excretion in human urine to be
increased during coronary reperfusion and in chronic cigarette smokers,
both settings putatively associated with increased free radical
generation
(11, 12) . However, activation of platelet
cyclooxygenase (COX), as reflected by increased excretion of
thromboxane metabolites, also characterizes these conditions
(13, 14) . COX-dependent formation of 8-epi
PGF
might confound its usefulness as an analytical
target reflective of free radical formation in vivo,
particularly in the setting of coincident platelet activation. Thus, we
addressed the hypothesis that this compound may be a product of COX
metabolism of endogenous arachidonic acid in human platelets.
Studies of Platelet Function
Platelets were
harvested as described previously (15). Briefly, platelet-rich
plasma (PRP) was prepared by centrifugation of the blood sample at 160
g for 10 min and platelet-poor plasma (PPP) by
centrifugation of PRP at 900
g for 10 min at room
temperature. Washed platelets (WP) were isolated from PRP after
centrifugation and resuspended in calcium and magnesium-free
Hank's balanced salt solution at pH 7.4, containing 10%
autologous PPP. Platelet number was adjusted to 3
10
platelets/ml with PPP or Hank's balanced salt solution.
Platelet aggregation was studied either in PRP or in WP by using a
PAP-4 model BIO-DATA aggregometer (BIO-DATA Corporation, Hatboro, PA),
at 37 °C, in siliconized cuvettes with continuous stirring.
Platelet aggregation was performed using threshold concentration (TC)
of agonists, defined as the lowest concentration that gave an
irreversible aggregation tracing with an amplitude between 65 and 85%
of maximal.
formation was
also explored under similar conditions using vitamin E (500
µM),mannitol (5 mM), deoxyribose (5
mM; all from Sigma). We also used the thromboxane synthase
inhibitors
(16, 17) ,1(7-carboxyheptyl)imidazole-HCI (10
µM) and sodium furegrelate (U-63557A, 50 µM)
(Cascade Biochem., Berkshire, United Kingdom). Solid-phase Extraction (SPE), Thin Layer Chromatography (TLC), and
Derivatization Briefly, product formation was stopped at fixed times after addition of
the platelet agonists by adding glacial acetic acid and lowering the pH
to 3-3.5. Tetra and hepta deuterated internal standards of
TxB
and 12-HETE (Cayman Chemical Co., Ann Arbor, MI),
respectively, were then added to the platelet preparation and the
samples applied to a 100-mg octadecylsilyl (ODS) solid-phase extraction
column (Alltech Associates Inc., Deerfield MI) that had been prepared
as per the manufacturer's instructions. The column was then
washed with 25% methanol, 75% water and 100% hexane, dried, then eluted
with 100% ethyl acetate. The samples were initially derivatized as
pentafluorobenzyl (PFB) esters by adding 10 µl of
diisopropylethylamine and 20 µl of 10% PFB Br in acetonitrile and
allowing the reaction to proceed for at least 10 min at room
temperature. This reaction mixture was dried under nitrogen and applied
to a TLC plate (LK6D, 60A Silica Gel Plates Whatman Inc., Clifton NJ).
The mobile phase was 100% ethyl acetate. The extractions were then
dried under a stream of nitrogen, the trimethylsilyl ether derivative
was formed by adding 10 µl of BSTFA Supelco Inc., Bellafonte, PA)
and 10 µl of pyridine and allowing the reaction to proceed for 10
min at room temperature. The reaction mixture was then dried under
nitrogen, the sample redissolved in 20 µl of dodecane, and analyzed
by GC-MS. 8-Epi PGF
was measured using an
O
-labeled internal standard derivatized as the
PFB ester as described above. The reaction mixture was dried under
nitrogen and applied to a TLC plate (LK6D, 60A Silica Gel Plates). The
mobile phase was 80% ethyl acetate and 20% heptane. The extractions
were dried under a stream of nitrogen and the
tert-butyldimethylsilyl ether derivative formed by adding 10
µl of MTBSTFA (Sigma) and 10 µl of pyridine, and allowing the
reaction to proceed for 24 h at room temperature. The reaction mixture
was then dried under nitrogen, the sample redissolved in 20 µl of
dodecane, and analyzed by GC-MS.
8-Epi PGF
A 30-µm DB-1
capillary column of 0.25 mm inner diameter with 0.25 µm of coating
was utilized. The temperature program was 190-320° at 20
°C/min. The retention time was approximately 17 min. A 40-m DB-1
0.18 mm inner diameter, 0.4-µm coating was used for studies aimed
at examining the purity of the 8-epi PGF, using the
same temperature program. The retention time on this system was
approximately 1 h. Integration times for selected ion monitoring
studies were 500 ms for each of the two ions monitored; m/z 695 for 8-epi PGF
and m/z 699 for
[
O
]8-epi PGF
.
TxB
Platelet TXAwas
measured as its hydrolysis product, TxB
. A 15-m DB-1
capillary column of 0.25 mm inner diameter with 0.25 µm of coating
was used; the temperature program was as above described for 8-epi
PGF
. The retention time was approximately 7 min, and
ions monitored were m/z 614 for TxB
and m/z 618 for [
H
]TxB
.
12-HETE
A 10-m DB-1 column of 0.25 mm inner
diameter with 0.25 µm of coating was used with a temperature
program as described above. The retention time was approximately 6 min,
and the ions monitored were m/z 391 for 12-HETE and m/z 399 for [H
]12-HETE. High Performance Liquid Chromatography (HPLC) A Hewlett-Packard (HP)1050 HPLC in line with an HP 1050 UV detector and
a Flo-One Radioactivity detector (Radiomatic Instruments, Meriden, CT)
was used for all HPLC experiments. Straight phase (SP) chromatography
was performed on an Ultrasphere Si 5-µm column, 4.6 mm inner
diameter
25 cm (Beckman Instruments, Fullerton, CA). Reverse
phase (RP) chromatography utilized an Ultrasphere ODS 5-µm column,
4.6 mm
25 cm. The flow rate was 1 ml/min in all experiments. Incubation of Arachidonic Acid (AA) with Partially Purified COX-1 AA (10 µg) or [
H
]AA (10 µCi)
in 20 µl of methanol was added to 1 ml of Tris buffer, pH 8,
containing 2 nM phenol, 1 mM EDTA, and 500 units of
COX-1. The reaction was allowed to proceed for 1 min at 37 °C, at
which time 150 µl of glacial acetic acid and 3 ml of ether
containing 1 mg of triphenyl phosphine was added. After vortexing and
centrifugation, the ether was removed, dried under N
, and
applied to a TLC plate which was then developed with a mobile phase of
10% methanol, 90% ethyl acetate, 0.1% glacial acetic acid. A 1-cm zone
centered on 8-epi PGF
was scraped, extracted, and
further purified by HPLC on an ODS column utilizing a mobile phase of
25% acetonitrile, 75% water, 0.1% glacial acetic acid. The elution
volume was 24-26 ml. Ex Vivo Study Four healthy volunteers (age 30 ± 8 years), who had not taken any
medication during the previous 2 weeks, were given 1 g of aspirin. All
were nonsmokers. Blood samples were taken before aspirin (base line),
at 3 h and at 24 h after the ingestion and the serum was analyzed for
TxB
and 8-epi PGF
. Statistical Analysis Data were initially analyzed using analysis of variance. Pairwise
comparisons were made using the Student's t test, where
appropriate. Data are displayed as mean ± standard deviation.
Formation of Arachidonic Acid Products by Human
Platelets
All three products, TxB, 12-HETE, and
8-epi PGF
were below the detection levels of their
corresponding assays (2 ng/ml, 2 ng/ml, and 2 pg/ml, respectively) in
unstimulated PRP and WP.
(130 ± 20 ng/ml) and 8-epi
PGF
(80 ± 15 pg/ml) had risen dramatically
( n = 5; p < 0.00 1). Addition of aspirin or
indomethacin 5 min prior to agonist completely prevented formation of
both TxB
and 8-epi PGF
. Consistant with
this observation, threshold concentrations of peroxide-free arachidonic
acid (20-50 µM) also increased TxB
(400
± 25 ng/ml) and 8-epi PGF
(350 ± 30
pg/ml) which were both inhibited by the COX inhibitors ( n = 4).
and 8-epi PGF
; it also
caused 12-HETE formation to fall from 320 to 140 ng/ml ( p <
0.001). Formation of the three products appeared related in time; an
initial lag phase in the first minute preceded initiation of
aggregation, which was maximal 3 min after addition of agonist when
product formation had reached a plateau (Fig. 1). Interestingly,
the conditions of the assay allowed chromatographic separation of an
endogenous peak corresponding to the retention time of authentic
O
-labeled 8-epi PGF
from
peaks which probably correspond to other F
isoprostanes
(Fig. 2, center panel). Although internal
standards for the other compounds were not included in the assay, the
agonist-induced increments appeared selective for the peak comigrating
with the 8-epi PGF
internal standard irrespective of
the platelet agonist (Fig. 2, lower panel). A similar,
apparently selective increase in 8-epi PGF
formation
was observed when aggregation was induced by threshold concentration of
thrombin (0.1-0.3 unit/ml). The pattern was similar to that
observed with collagen, although the rise in 8-epi PGF
appeared to precede that of the other two products
(Fig. 3 A). Again, aspirin completely suppressed
TxB
and 8-epi PGF
and partially suppressed
12-HETE formation. Similar results were obtained with threshold
concentrations (1-3 µM) of the calcium ionophore
A23187 (Fig. 3 B).
Figure 1:
Time course of eicosanoid formation in
collagen-stimulated washed human platelets. Significant ( p < 0.001) increases occurred coincident with aggregation in the
predominant COX product TxB(
), the predominant
12-lipoxygenase product, 12-HETE (
), and 8-epi PGF
(
). Pretreatment of platelets with aspirin (asa 100
µM) prevented aggregation and completely inhibited
TxB
and 8-epi PGF
formation. Production of
12-HETE was significantly ( p < 0.00 1) reduced ( n = 6). A representative tracing of platelet aggregation is
depicted in the lower panel: addition of collagen is indicated
by the arrow (
) and aggregation is reflected by a change
of light transmission ( LT) from base
line.
Figure 2:
Selected ion monitoring of 8-epi
PGF. The upper trace shows a peak at m/z 699 corresponding to authentic
O
-labeled
internal standard. The center trace ( m/z 695) shows the signal before the stimulus, and the small peaks are
likely to be isoprostanes. The lower trace shows a peak
( m/z 695) corresponding to the retention time of the authentic
8-epi PGF
. Other isoprostanes are present, but
obscured by the intense 8-epi PGF
signal. The
inset shows the trace magnified four times to reveal the other
isoprostanes.
Figure 3:
Time course of eicosanoid formation in
thrombin stimulated washed platelets (0.1-0.3 unit/ml)
( a), and in A23187-stimulated washed platelets (1-3
µM) ( b). Aspirin (asa) 100 µM was
incubated for 5 min before the stimulus ( n =
5).
Formation of 8-epi PGFreflected COX activation rather than platelet aggregation.
Induction of platelet aggregation in a COX-independent manner by using
high doses (10 µg/ml) of collagen in the presence of aspirin was
unaccompanied by an increase in 8-epi PGF
or TxB
(Fig. 4). Similar results were obtained with high dose
thrombin. While COX inhibitors suppressed the increment in 8-epi
PGF
in platelets activated with threshold
concentrations of thrombin and collagen, three structurally distinct
free radical scavengers, vitamin E, mannitol, and deoxyribose all
failed to inhibit its formation (). Aggregation of
platelets with threshold concentrations of the thromboxane receptor
agonist U46619 (1-3 µM) or of phorbol myristate
acetate (100-300 nM) was unassociated with a detectable
increment in either TxB
or 8-epi PGF
.
Figure 4:
Aggregation of washed human platelets
stimulated with collagen (2 µg/ml) ( A) is associated with
8-epi PGF and TxB
formation. Pretreatment
with aspirin (100 µM) prevents aggregation ( B)
and formation of both. Increasing the concentration of collagen (10
µg/ml) ( C) allows aggregation to occur despite
pretreatment with aspirin, but the formation of 8-epi PGF
along with TxB
remains inhibited ( n.d., not
detectable).
Several lines of evidence are consistent with formation of 8-epi
PGFin a COX-dependent manner by human platelets.
These include (i) coincident kinetics of formation with the COX
product, TxA
, in activated platelets; (ii) inhibition of
formation coincident with that of TxA
by two structurally
distinct COX inhibitors; (iii) dissociation of aggregation and 8-epi
PGF
formation when aggregation occurs in a
COX-independent manner; (iv) selective elevation of 8-epi
PGF
as compared to other isoprostanes.
Effect of Thromboxane Synthase Inhibition on Platelet
8-epi PGF
Washed platelets were
incubated with the selective thromboxane synthase inhibitors
1-(7-carboxyheptyl)imidazole hydrochloride and sodium furegrelate
(U-63557A) for 5 min at 37 °C, then stimulated with collagen (1
µg/ml) or AA (30 µM). The samples were analyzed for
8-epi PGFFormation
and TxB
formation. The
inhibitors completely prevented TxB
production, but did not
prevent the formation of 8-epi PGF
which was elevated
an average 6-8-fold for both collagen and AA (Fig. 5, A and B)
(18) .
Figure 5:
Effect of two thromboxane synthase
inhibitors, U6557A (50 µM) and carboxyheptyl-imidazole (10
µM), on 8-epi PGF and TxB
formation in arachidonic acid ( AA) (30 µM) and
collagen ( CL) (1 µg/ml) stimulated washed platelets. The
inhibitors were incubated 5 min at 37 °C before adding the stimuli.
Pretreatment with 100 µM aspirin ( ASA) abolished
aggregation and prevented formation of both TxB
and 8-epi
PGF
. The synthase inhibitors prevented formation of
TxB
but an increased production of 8-epi PGF
from pretreatment values was observed ( n =
5).
Confirmation of 8-Epi PGF
To verify the authenticity of the endogenous
peak comigrating with the internal standard in the quantitative GC-MS
assay as 8-epi PGFas a Product
of COX-1 Activity
, we performed several further
experiments. First, AA was incubated with partially purified ram
seminal vesicle COX-I using the conditions described above. An internal
standard, consisting of [
O
]8-epi
PGF
was added to the ether, and the mixture was
derivatized as the PFB ester, subjected to TLC, and the tBDMS
derivative was formed. When subjected to negative ion chemical
ionization GC-MS, a product with the molecular weight and retention
time of 8-epi PGF
was observed (not shown).
H
]arachidonic acid was incubated
with COX-I and the product extracted by SPE, partially purified by TLC,
mixed with 10 µg of authentic 8-epi PGF
, and
subjected to HPLC purification using the technique of Morrow et al. (5) . 8-Epi PGF
was then monitored by UV
absorption at 210 nm; tritiated products were monitored with a
Flo-One/Beta radiodetector. Briefly, coelution was demonstrated in
three systems: (i) SP HPLC of the underivatized compound, mobile phase:
12% isopropanol, 88% hexane, 0.1% glacial acetic acid, retention time
24.6 min for the unlabeled, and 26 min for the tritiated product; (ii)
RP HPLC of the underivatized compound, mobile phase: 28% acetonitrile,
72% water, 0.1% glacial acetic acid, retention time 23.6 min for the
unlabeled and 22 min for the tritiated product; and (iii) RP HPLC of
the PFB ester, mobile phase: 50% acetonitrile, 50% water, retention
time 22.3 min for the unlabeled and 21.9 min for the tritiated product.
Retention time differences between the unlabeled material and the
products possessing the hydrogen/tritium substitution in eight
locations were significant (up to 1.4 min). To account for this
variation, the PGF
was purified from the incubation
and chromatographed with authentic PGF
in the same
three systems. The relative retention times enabled compensation for
the isotope effect (Fig. 6). The isotope effect was larger than
seen by Morrow et al. (5) due to the fact that their
standard was labeled with a single tritium.
Figure 6:
A)
Coelution of a COX-1 product of
[H
]-arachidonic acid (
-
) with the UV absorption profile of authentic 8-epi
PGF
(-) after reverse phase HPLC of the
underivatized compound (mobile phase: 28% acetonitrile, 72% water, 0.1%
glacial acetic acid). The slight difference in retention time reflects
the isotope effect (see text). B, coelution of
[
H
]PGF
(
-
) from the same incubation and authentic PGF
(-) using the same HPLC conditions as in
A.
Third, 80
µM AA was incubated with washed human platelets and after
addition of O
internal standard, SPE, TLC, and
PFB, tBDMS derivatization, the zone cochromatographing with 8-epi
PGF
was injected onto a 40 m DB-1 capillary column
with a 0.18-mm inner diameter, 0.4-µm coating. Complete GC-MS
conditions are described above. This system yielded a retention time of
approximately 1 h; no difference in retention time was observed between
the [
O
]8-epi PGF
internal standard and the product of the incubation, other than
the expected isotope effect (the internal standard eluted 15 s earlier
than both the platelet product and authentic 8-epi
PGF
).
Gas Chromatography-Electron Impact Mass Spectrometry
(GC-EI-MS)
We applied GC-EI-MS to provide structural elucidation
of the compound detected in the negative ion chemical ionization GC-MS
assay. Platelets were incubated with
[5,6,8,9,11,12,14,15-d]arachidonic acid for 3 min
at 37 °C. The product of this incubation was subjected to
extraction and purification as described above, except that the final
derivative was the methyl ester TMS ether and mixed with authentic
8-epi PGF
. This technique is useful for demonstrating
identity between two compounds because the mass spectrum will be
present in two forms that are identical except for the presence of
deuterium in one (Fig. 7). For assignment of structure to the
ions, we have referred to the study of Middleditch and Desiderio
(19) on the mass spectrum of the TMS ether-TMS ester derivative
of PGF
. Although we used the TMS ether-methyl ester,
the spectra are analogous in the ions that contain the ester and, since
the ester group plays little role in the fragmentation of most ions,
most fragments are identical. The presence of eight deuterons alters
the GC retention in a predictable fashion, causing the deuterated
analog to elute slightly before the natural product. This difference,
although only approximately 1 s, allowed the use of reconstructed ion
chromatograms (RIC) to ascertain the origin of each individual ion (see
Fig. 8
). RICs were also useful for demonstrating that several
ions present in the spectrum ( m/z 361, 437, and 451) had
different GC elution profiles, and therefore did not originate from the
compounds of interest.
Figure 7:
An electron impact mass spectrum of the
product of the incubation of human platelet with
[5,6,8,9,11,12,14,15-d]arachidonic acid
coinjected with authentic 8-epi PGF
derivatized as the
methyl ester TMS ether. The lower panel has been magnified by
a factor of 3. The inset shows 8-epi PGF
methyl ester TMS ether, the single asterisks (*) denote
the sites of deuterium labeling. Ions at m/z 361, 437, and 451
originate from a coeluting impurity (**) (see
text).
Figure 8:
Reconstructed ion chromatograms showing
the slight isotope effect on the GC retention times of the two
compounds. m/z 521 and 592 originate from the
H
-labeled product, as indicated by their
slightly earlier (approximately 1 s) retention
time.
We interpret the spectrum in the following
manner: m/z 584 is the molecular ion of the unlabeled product;
its [H
] analog is m/z 592.
m/z 569 represents the loss of 15 (CH
; from TMS);
its
H
analog is m/z 577. m/z 513 is the loss of 71 (C16-20); since this portion of the
molecule is not labeled, the analog m/z 521 retains all eight
deuterons. m/z 494 represents the loss of 90
(trimethylsilanol, TMSOH); the analog can result from a loss of 90
( m/z 502) or 91 ( m/z 501) depending on whether the
loss of TMSOH involves a proton or a deuteron. The ion current is
divided between these two ions. m/z 479 is the loss of 105
(90+15; TMSOH+CH
). The analog is seen as a loss
of 105 ( m/z 487) or 106 ( m/z 486), again depending on
whether a proton or deuteron is abstracted. m/z 423 (M-161)
originates from the loss of TMSOH
(90) and C16-20
(71) . Its analog can occur at M-161 ( m/z 431) or M-162
( m/z 430) for reasons stated above. m/z 404 reflects
the loss of two TMSOH groups (2
90). Since each TMSOH can
abstract either a proton or a deuteron, its analog appears at m/z 410, 411, and 412. m/z 397 originates from the exclusion
of TMSOC
H
(116) from M-71. This loss
involves C9, with its -OTMS group and C10. Its analog retains seven
deuterons and is obscured by the larger m/z 404. An RIC of
m/z 404 yields a peak profile consistent with having two
components, one originating from the unlabeled standard, the other from
the deuterated compound. m/z 333 reflects the loss of
90+90+71. Since its analog can lose zero, one, or two
deuterons, it appears at m/z 339, 340, and 341. m/z 307 originates from the expulsion of TMSOC
H
(116) from M-(90+71). Since C9 with its deuteron is
lost and the TMSOH can remove a proton or a deuteron, its analog
appears at m/z 313/314. Middleditch and Desiderio
(19) described an ion at m/z 313 which could account
for the fact that m/z 313 in this spectrum is larger than
would be predicted. Two fragments of m/z 243 are reported by
Middleditch; the first consists of the intact five-membered ring, the
second is C11-15. The former would be expected to retain three
deuterons, the latter four. The small ions at m/z 246 and 247
have RICs consistent with their origin from the deuterated product.
m/z MDRV 217 originates from a rearrangement of C9-11.
It should retain two deuterons, placing its analog at m/z 219.
This ion is, in fact, elevated above its expected size, as predicted by
other second isotope peaks in the spectrum.
Ex Vivo Study
Basal serum levels for
TxB, and 8-epi PGF
were 250 ± 20
ng/ml and 231 ± l5 pg/ml, respectively. Three h after aspirin
administration the levels dropped to 4 ± 1 ng/ml and 40 ±
10 pg/ml, respectively, reflecting average reductions of 98% for
TxB
and 83% for 8-epi PGF
. Suppression of
serum 8-epi PGF
ex vivo persisted 24 h after
aspirin administration (Fig. 9).
Figure 9:
Serum
levels of TxB2 () and 8-epi PGF
(
)
before (base) and at 3 and 24 h after administration of aspirin to
healthy volunteers ( n = 4).
isoprostanes are a family of PGF
isomers, reportedly formed by free radical catalyzed peroxidation
of arachidonic acid, independent of the action of COX
(1) .
Evidence for this pathway of formation includes enhanced formation
in vivo in animal models of enhanced oxidative stress, such as
poisoning with carbon tetrachloride
(20) , or depletion of
endogenous antioxidants, such as by iron overload
(21) .
Isoprostanes are formed initially from arachidonic acid in situ in phospholipids and are putatively cleaved from the membrane by
phospholipases A
(22, 23) .
, a metal-independent source of peroxyl
radicals, or by coincubation with endothelial cells
(24, 25, 26) results in initial formation in
the phospholipid, followed by release of the isoprostanes. Although the
precise chemistry of the reactions which lead to their formation
remains to be elucidated, the discovery of isoprostanes affords a
potential index of free radical catalyzed events in vivo, as
they can be measured in plasma and urine
(12, 27) .
, which has been shown to possess biological
activity as a vasoconstrictor
(6, 7) . This effect is
prevented by pharmacological antagonists of the thromboxane receptor.
It is unclear whether 8-epi PGF
incidentally exerts
its effects via this receptor or whether it acts via a distinct, but
related receptor
(8) . Pharmacological assessment of the effects
of 8-epi PGF
suggests that it acts as a partial
agonist at the thromboxane receptor that transduces the aggregation
response
(10, 12, 28) . Interestingly, despite
its lack of potency as a stimulant of aggregation, 8-epi PGF
can readily elicit the platelet shape change that normally
precedes the aggregation response, and this phenomenon is accompanied
by an increase in [Ca
i]
(12, 28) . We have previously shown that the thromboxane
antagonist, GR 32191, segregates forms of the thromboxane receptor
which mediate aggregation and the release reaction from those which
transduce the platelet shape change and vasoconstriction
(29, 30) .
isoprostanes in human platelets, we initially studied the
time course of appearance of 8-epi PGF
in response to
activation by the physiological agonists collagen and thrombin. We
developed a stable isotope dilution assay, using an
O
-labeled internal standard and gas
chromatography-mass spectrometry
(11) . To our surprise, there
was a marked increase in the peak comigrating with the internal
standard for 8-epi PGF
, coincident with platelet
aggregation. Importantly, closely migrating peaks, presumed to reflect
other F
isoprostanes, did not increase correspondingly.
Pretreatment with the COX inhibitors aspirin and indomethacin
completely abolished this increment in 8-epi PGF
,
together with the major COX product in platelets, thromboxane
A
, as reflected by its hydrolysis product, thromboxane
B
. Furthermore, induction of aggregation in a
COX-independent manner by a variety of approaches (the phorbol ester
phorbol 12-myristate 13-acetate, the endoperoxide analog U46619, or by
high doses of collagen or thrombin in the presence of aspirin) was
unassociated with 8-epi PGF
formation.
actually represented several unresolved species. To address this
possibility, we obtained several lines of evidence that the COX
metabolite was actually 8-epi PGF
. First, we added
radiolabeled arachidonic acid to the semipurified COX. The tritiated
product of this reaction was then mixed with authentic 8-epi
PGF
and passed over three highly resolving
chromatographic systems; the product of the reaction coelutes with
authentic 8-epi PGF
.
and the product isolated from an incubation
of octadeuterated AA with platelets. The mass spectrum shows a series
of ions corresponding to those of 8-epi PGF
and
another series corresponding to a deuterated analog. While the EI mass
spectrum of PGF
would be similar, this compound is
clearly chromatographically resolved from 8-epi PGF
under the conditions of the assay.
from octadeuterated
arachidonic acid in an aspirin-sensitive manner, as detected in a
selected ion monitoring assay. If the 8-epi PGF
had
been formed from either PGD
or PGE
, this would
have involved loss of one of the ring deuteriums, resulting in a
heptadeuterated product
(31) . Thus, human platelets and perhaps
other cells, possess the capacity to form 8-epi PGF
as
a prostaglandin.
might
be formed via the corresponding endoperoxide by a biomimetic
cyclization and Hecker et al. (9) demonstrated that
8-epi PGF
is a product of the ram seminal vesicle COX.
Additional support for a COX-dependent source of 8-epi PGF
formation in human platelets is provided by two experiments with
structurally distinct inhibitors of thromboxane synthase. These
compounds increase 8-epi PGF
formation coincident with
inhibition of TxA
(18) . Finally, we have shown
coelution of the putative COX-1 product with authentic 8-epi
PGF
by GC-MS as the PFB tBDMS derivative and as the
ME-TMS derivative. We have also shown HPLC coelution of the
underivatized compounds by straight phase chromatography and of the PFB
ester derivative by straight phase and reverse phase chromatography.
in a COX-dependent
manner does not exclude the possibility that this reflects generation
of free radicals associated with enzyme turnover. However, formation
via peroxyl radical isomers of arachidonate is unlikely. We found that
three structurally distinct, free radical scavengers failed to prevent
8-epi PGF
formation by stimulated human platelets
under conditions where aspirin was effective. Furthermore, its
production was not accompanied by the appearance of the array of
isomers typical of isoprostane production. Finally, the biomimetic
schema outlined by Corey et al. (32) renders such an
explanation unnecessary.
, as has been suggested
(8) . Although its biological effects are prevented by
thromboxane receptor antagonists, it displaces ligand weakly
(8, 12) from the recombinant thromboxane receptor
cloned from human placenta
(33) . The recombinant PGF
receptor is not activated by 8-epi
PGF
.
(
)
A single thromboxane
receptor gene has been cloned
(34) . However, subtle changes in
primary sequence may result in discriminant affinity for ligands
(35) , and the recent discovery of tissue specific expression of
splice variants
(36) raises the possibility that 8-epi
PGF
might exhibit greater affinity for a variant other
than the placental isoform. Alternatively, post-translational
modifications of the receptor, perhaps initiated by oxidizing
conditions, may favor 8-epi PGF
as a ligand.
is a considerably less abundant COX product than thromboxane in
human platelets. However, the demonstration that 8-epi PGF
is formed in serum and that it is substantially depressed ex
vivo in volunteers administered aspirin indicates the potential
relevance of these observations to its formation in vivo.
Several of the clinical situations putatively associated with free
radical generation, in which we have found elevated urinary 8-epi
PGF
, including syndromes of vascular reperfusion and
chronic cigarette smoking, are associated with platelet COX activation
(13, 14) . Increased excretion of 8-epi PGF
may reflect the generation of free radicals by the vasculature
(37) in these clinical settings. If so, utilization of such an
index would be pertinent to the rational development of antioxidant
drugs in humans. Clearly, delineation of the extent to which a
COX-dependent pathway might confound interpretation of urinary 8-epi
PGF
is critical to its use as a quantitative index of
free radical generation in vivo.
Table:
Effect of aspirin (ASA) and the
antioxidants, vitamin E (vit E, 500 µM), mannitol (MN, 5
mM) and deoxyribose (DR, 5 mM) on eicosanoid
formation in collagen and thrombin-stimulated washed platelets ( n
= 5).
;
RIC, reconstructed ion chromatogram; BSTFA,
bis-[trimethylsilyl]trifluoroacetamide; MTBSTFA,
N-[ tert-butyldimethylsilyl]- N-methyltrifluoroacetamide;
ODS, octadecylsilyl; TMS, trimethylsilyl.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.