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INTRODUCTION |
Isoprostanes (IsoPs)1
are prostaglandin (PG)-like compounds that are formed nonenzymatically
in vivo by free radical-induced peroxidation of arachidonic
acid (AA). Their formation proceeds through bicyclic endoperoxide
PGH2-like intermediates. The endoperoxide intermediates are
reduced to form PGF2-like compounds (F2-IsoPs) (1) or undergo rearrangement to form E-ring and D-ring compounds (E2/D2-IsoPs) (2) and thromboxane-like
compounds (isothromboxanes) (3). A novel aspect of the formation of
IsoPs is that, unlike cyclooxygenase-derived prostaglandins, IsoPs are
formed in situ esterified to phospholipids and subsequently
released (4). Quantification of F2-IsoPs has emerged as one
of the most accurate approaches to assess oxidant injury in
vivo (5, 6). Furthermore, IsoPs are capable of exerting potent
biological activity (5, 6).
Docosahexaenoic acid (C22:6
3) (DHA) has been the subject of
considerable interest owing to the fact that it is highly enriched in
the brain, particularly in gray matter, where it comprises approximately 25-35% of the total fatty acids in aminophospholipids (7, 8). Although DHA is present in high concentrations in neurons,
neurons are incapable of elongating and desaturating essential fatty
acids to form DHA. Rather, DHA is synthesized primarily by astrocytes
after which it is secreted and taken up by neurons (9). Although the
precise function of DHA in the brain is not well understood, deficiency
of DHA is associated with abnormalities in brain function (10). We
considered the possibility that IsoP-like compounds could be formed by
free radical-induced peroxidation of DHA. Because such compounds would
be two carbons longer in length than IsoPs, it would be inappropriate
to term these compounds IsoPs. Since DHA is highly enriched in neurons in the brain, we therefore propose to term these compounds
"neuroprostanes" (NPs).
One of our interests in the possibility that IsoP-like compounds could
be formed from DHA derives from the fact that a role for free radicals
in the pathogenesis of a number of neurodegenerative diseases,
e.g. Alzheimer's diesease, Parkinson's disease,
Huntington's disease, and amyotrophic lateral sclerosis, has been
suggested (11-13). Thus, quantification of such compounds might
provide a unique marker of oxidative injury in the brain. Furthermore,
these compounds, like IsoPs, could potentially exert biological
activity. This possibility is supported by the finding that
PGF4
, the four series F-prostaglandin corresponding to
the structure expected from cyclooxygenase action on C22:6, is
approximately equipotent with cyclooxygenase-derived
PGF2
in contracting gerbil colonic smooth muscle strips
(13). In addition, the formation of NPs esterified in lipids might be
expected to have significant effects on the biophysical properties of
neuronal membranes, which might impair normal neuronal function. This
may be particularly relevant, since it has been suggested that one of
the physiological functions of DHA may be to maintain a certain state
of membrane fluidity and promote interactions with membrane proteins
that are optimum for neuronal function (14, 15).
The mechanism by which F4-NPs could be formed is outlined
in Fig. 1, A-C. As noted,
five DHA radicals are initially generated, which following addition of
molecular oxygen, results in the formation of eight peroxyl radicals.
These peroxyl radicals then undergo endocyclization followed by further
addition of molecular oxygen to form eight bicylic endoperoxide
intermediate regioisomers (not shown), which are then reduced to form
eight F-ring NP regioisomers. Each regioisomer is theoretically
comprised of eight racemic diastereomers for a total of 128 compounds.
A nomenclature system for the IsoPs has been established and approved
by the Eicosanoid Nomenclature Committee in which the different
regioisomer classes are designated by the carbon number on which the
side chain hydroxyl is located with the carboxyl carbon designated as
C-1 (16). Thus, in accordance with this nomenclature system, the F-ring
NP regioisomers are similarly designated as 4-series
F4-NPs, 7-series F4-NPs, etc.
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EXPERIMENTAL PROCEDURES |
Materials--
Docosahexaenoic acid, pentafluorobenzyl bromide,
and diisopropylethylamine were purchased from Sigma; dimethylformamide,
undecane, and 1-butaneboronic acid from Aldrich;
N,O-bis(trimethylsilyl)trifluoroacetamide from Supelco
(Bellefonte, PA);
[2H9]N,O-bis(trimethysilyl)trifluoroacetamide
from Regis Chemical (Morton Grove, Il); organic solvents from Baxter
Healthcare (Burdick and Jackson Brand, McGaw Park, Il); C-18 Sep-Paks
from Waters Associates (Milford, MA); 60ALK6D TLC plates from Whatman
(Maidstone, UK); and [2H4]PGF2
from Cayman Chemical (Ann Arbor, MI).
Oxidation of DHA--
DHA and AA were oxidized in
vitro using iron/ADP/ascorbate as described (17).
Purification and Analysis of F4-NPs--
Free and
esterified F4-NPs were extracted using a C-18 Sep-Pak
cartridge, converted to a pentafluorobenzyl ester, purified by TLC,
converted to a trimethylsilyl ether derivative, and quantified by
stable isotope dilution negative ion chemical ionization gas chromatography mass spectrometry using
[2H4]PGF2
as an internal
standard using a modification of the method described for the
quantification of F2-IsoPs (18). Instead of scraping 1 cm
below to 1 cm above where PGF2
methyl ester migrates on
TLC for analysis of F2-IsoPs, the area scraped was extended
to 3 cm above where PGF2
methyl ester migrates. This
extended area of the TLC plate was determined to contain F4-NPs by analyzing small 5-mm cuts using approaches for
their identification described below. The
M-·CH2C6F5 ions were
monitored for quantification (m/z 593 for F4-NPs and m/z 573 for
[2H4]PGF2
). Quantification of
the total amount of F4-NPs and F2-IsoPs was
determined by integrating peak areas. Formation of cyclic boronate
derivatives and hydrogenation were performed as described (19).
Electron ionization mass spectra were obtained using a Finnigan Incos
50B quadropole instrument as described (19).
Analysis of F4-NPs in Human Cerebrospinal
Fluid--
Cerebrospinal fluid was obtained from seven subjects
following informed consent. Subjects with Alzheimer's disease
(n = 4) had been diagnosed with probable Alzheimer's
disease during life. Control subjects (n = 3) were
age-matched individuals without clinical evidence of dementia or other
neurological disease; each had annual neuropsychological testing with
all test scores within the normal range. Ventricular cerebrospinal
fluid was collected as part of a rapid autopsy protocol. Mean
post-mortem intervals were 2.9 ± 0.3 h in control subjects
and 2.7 ± 0.2 h in Alzheimer's patients. Brains were
evaluated using standard criteria for Alzheimer's disease (20, 21).
Patients with brainstem or cortical Lewy body formation, or significant
cerebrovascular disease, were excluded. Control subjects demonstrated
only age-associated alterations. Statistical analysis of data was
performed using the unpaired t test.
Molecular Modeling of NP-containing
Phosphatidylserine--
Molecular modeling was performed with
Macspartan computer software.
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RESULTS |
A representative selected ion current chromatogram obtained from
the analysis for F4-NPs following oxidation of DHA in
vitro with iron/ADP/ascorbate is shown in Fig.
2. A series of m/z 593 peaks
eluted over approximately a 90-s period beginning approximately 30 s after the elution of the
[2H4]PGF2
internal standard.
F4-NPs would be expected to have a longer GC retention time
than PGF2
because their C-value is two units higher. It
should be pointed out to avoid confusion that the time scales of some
of the chromatograms obtained from the analysis of F4-NPs
shown in subsequent figures are compressed or expanded compared with
that in Fig. 2; this may give the impression that the relative
abundances/pattern of the different isomers detected differs.
Furthermore, the retention times over which the F4-NPs
elute may differ somewhat in the different figures, because these
analyses were performed on different days using different columns that
vary somewhat in length.

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Fig. 2.
Selected ion current chromatograms obtained
from the analysis of F4-NPs generated during
iron/ADP/ascorbate-induced oxidation of DHA in vitro.
The series of peaks in the m/z 593 ion current chromatogram
represent putative F4-NPs, and the single peak in the
m/z 573 ion current chromatogram represents the
[2H4]PGF2 internal
standard.
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Analysis of these compounds as a
[2H9]trimethylsilyl ether derivative resulted
in a shift in the m/z 593 peaks to m/z 620, indicating the presence of three hydroxyl groups (not shown). Analysis
following catalytic hydrogenation is shown in Fig.
3. Prior to hydrogenation, no peaks were
present 8 Da above m/z 593 at m/z 601. However,
following hydrogenation, intense peaks appear at m/z 601, indicating the presence of four double bonds. The pattern of the
hydrogenated compounds differs significantly from that of the
nonhydrogenated compounds, because hydrogenation converts the compounds
into new compounds that are resolved differently than the
nonhydrogenated compounds.

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Fig. 3.
Analysis of putative F4-NPs
before and after catalytic hydrogenation. In the absence of
hydrogenation, intense peaks are present in the m/z 593 ion
current chromatogram representing F4-NPs and absent are
peaks of significant intensity 8 atomic mass units higher at
m/z 601. Following catalytic hydrogenation, intense peaks
appear at m/z 601, indicating that the m/z 593 compounds have four double bonds.
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F4-NPs are formed by reduction of endoperoxide
intermediates (Fig. 1). Thus, the cyclopentane ring hydroxyls must be
oriented cis, but they can be either
,
or
,
.
Evidence that these compounds contained a cyclopentane (prostane) ring
with cis-oriented hydroxyls was obtained by analyzing the
compounds as a cyclic boronate derivative (Fig.
4). PGF2 compounds with
cis-oriented prostane ring hydroxyls will form a cyclic
boronate derivative bridging the ring hydroxyls (22). The
M-·CH2C6F5 ion for the
cyclic boronate derivative is m/z 515. When the compounds
were analyzed as a pentafluorobenzyl ester, trimethysilyl ether
derivative, no intense peaks were present at m/z 515. However, when the pentafluorobenzyl ester derivatives were treated with 1-butaneboronic acid and then converted to a trimethylsilyl ether derivative, the intense peaks at m/z 593 were no longer
present and intense peaks appeared at m/z 515. Again, the
pattern of the m/z 515 peaks differs from that of compounds
that were not treated with 1-butaneboronic acid because of differences
in resolution of the individual compounds as a cyclic boronate
derivative.

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Fig. 4.
Formation of a cyclic butylboronate
derivative of putative F4-NPs. The
M-·CH2C6F5 ion for the
pentafluorobenzyl ester, cyclic butylboronate, trimethylsilyl ether
derivative is m/z 515. In the absence of treatment of the
compounds with 1-butaneboronic acid, the peaks representing the
putative F4-NPs are present in the m/z 593 ion
current chromatogram, and no peaks of significant intensity are present
in the m/z 515 ion current chromatogram. However, analysis
of compounds treated with 1-butaneboronic acid revealed a disappearance
of the m/z 593 peaks and the appearance of intense peaks at
m/z 515.
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Finally, these compounds were subjected to analysis by electron
ionization mass spectrometry as a methyl ester, trimethylsilyl ether
derivative. Multiple mass spectra consistent with compounds representing the different regioisomers of F4-NPs eluted
from the GC column over approximately 45 s. This elution time
differs from that of the pentafluorobenzyl ester derivatives used for negative ion chemical ionization, because methyl esters elute from the
GC column much earlier and thus the duration over which they elute is
compressed. When analyzed by electron impact mass spectrometry, the
different F4-NP regioisomers would be expected to give
characteristic
-cleavage ions of the trimethylsiloxy substituents on
the side chains (Fig. 5). One of the mass
spectra obtained is shown in Fig. 6. The
ions designated with "A" are ions that would be generated from all
of the different regioisomers. These include, in addition to the
molecular ion at m/z 608, m/z 593 (M
15, loss of ·CH3), m/z 539 (M - 90, loss of
Me3SiOH), m/z 518 (M
2 × 90)), m/z 501 (M
(90 + 15)), m/z 487 (M
121, loss of ·OCH3 + 90), m/z 217 (Me3SiO-CH=CH=O+SiMe3), a
characteristic ion of F-ring prostanoids (23), and m/z 191 (Me3SiO+=CH-OSiMe3), a
rearrangement ion characteristic of F-ring prostanoids (23). The ions
designated (17-S), (4-S), etc. indicate ions generated specifically
from 17-series, 4-series, etc. regioisomers. These include the
following: (a) 10-series regioisomer ions m/z 539, (M
69, loss
·CH2(CH2)2CH3),
m/z 449 (M
(69 + 90), m/z 359 (M
(69 + 2 × 90)), (b) 17-series regioisomer ion
m/z 437 (M
171, loss of ·CH(Me3
SiOH)CH2CH=CHCH2CH3),
(c) 7-series regioisomer ion m/z 409 (M
(109 + 90), loss of
·CH2CH=CHCH2CH=CHCH2CH3 + 90), (d) 13-series regioisomer ions m/z 401 (M
207, loss of
·CH2CH=CHCH2CH=CHCH2CH=CH(CH2)2COOCH3),
m/z 311, (M
(207 + 90)), m/z 219 (M
(309 + 90), loss of
·CH(Me3SiOH)CH2CH=CHCH2CH=CHCH2CH=CH(CH2)2CO OCH3 + 90), and (e) 4-series regioisomer ion m/z 279 [M
(149 + 2 × 90), loss of ·CH2CH=CHCH2CH=CHCH2CH=
CHCH2CH3 + 2 × 90). The six ions further designated with an asterisk represent specific
-cleavage ions of the
trimethylsiloxy substituents of different regioisomers as shown in Fig.
5. These data indicated that this was a mass spectrum of a mixture of
six of the eight regioisomers co-eluting simultaneously from the GC
column. This evidence for the presence of predicted six out of eight
regioisomers supports the proposed mechanism of formation of these
compounds outlined in Fig. 1.

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Fig. 5.
Predicted specific -cleavage ions of the
trimethylsiloxy substituents on the side chains of the different
F4-NP regioisomer series. The -cleavage ions for
the regioisomer series designated by asterisks were
prominent ions in the mass spectrum shown in Fig. 6.
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Fig. 6.
Electron ionization mass spectrum obtained of
putative F4-NPs as a methyl ester, trimethylsilyl ether
derivative. An intense molecular ion is present at m/z
608. The ions designated with an "A" are common ions
generated from all regioisomers (see text for explanation). The
designations (17-S), (4-S), etc. indicate ions specifically generated
by compounds in the 17-series, 4-series regioisomers, etc. (see text
for explanation). Ions further designated with an asterisk
are specific -cleavage ions of the trimethylsiloxy substituents for
the different regioisomer classes as indicated in Fig. 5.
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The time course of formation of F4-NPs during oxidation of
DHA using iron/ADP/ascorbate was rapid, reaching a maximum level of
approximately 5 µg/mg DHA at 50 min (Fig.
7). We then compared the amounts of
F2-IsoPs formed from oxidation of AA with the amounts of
F4-NPs formed from DHA. In these experiments, equal molar
amounts of AA and DHA were co-oxidized with Fe/ADP/ascorbate and the
total amounts of F2-IsoPs and F4-NPs generated
quantified. Interestingly, the relative amounts of F4-NPs
formed exceeded that of F2-IsoPs by a mean of 3.4-fold
(Fig. 8).

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Fig. 8.
Relative amounts of F4-NPs and
F2-IsoPs formed during co-oxidation of equal amounts of DHA
and AA in vitro.
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We then undertook experiments to determine whether F4-NPs
are present esterified in brain lipids in vivo (Table
I). Both F2-IsoPs and
F4-NPs were present at readily detectable levels esterified
in lipids of normal whole rat brain at levels of 10.3 ± 3.1 and
7.0 ± 1.4 ng/g, respectively (n = 4). A selected
ion current chromatogram obtained from one of these analyses is shown in Fig. 9. Although the levels of
F2-IsoPs were slightly higher than the levels of
F4-NPs, these differences were not significant (p > 0.05). However, levels of F4-NPs
esterified in the cortex of newborn pig brain (13.1 ± 0.8 ng/g)
greatly exceeded levels of F2-IsoPs (2.9 ± 0.4 ng/g)
by a mean of 4.5-fold (n = 3) (p < 0.0001). Note that the pattern of F4-NP peaks detected
esterified in brain differs somewhat than that of compounds formed by
oxidation of DHA in vitro. We have also observed slight
differences in the pattern of F2-IsoPs formed from
oxidation of arachidonic acid in vitro compared with that of
compounds present esterified in tissue lipids. Although the reason for
these differences has not been firmly established, a reasonable
explanation for this is that there may be steric influences of
phospholipids on the formation of different isomers from esterified
substrate.
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Table I
Levels of F2-IsoPs and F4-NPs measured esterified to
lipids in whole normal rat brain (n = 4) and in brain cortex
from newborn pig (n = 3)
F2-IsoPs and F4-NPs were measured as free compounds
following base hydrolysis of a Folch lipid extract of brain tissue as
described under "Experimental Procedures." The data are expressed
as nanograms of F2-IsoPs and F4-NPs measured per g of
wet weight of tissue.
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Fig. 9.
Selected ion current chromatogram obtained
from the analysis for F2-IsoPs and F4-NPs
esterified in whole rat brain. The peaks in the m/z 569 ion current chromatogram represent F2-IsoPs; the peak in
the m/z 573 ion current chromatogram is
[2H4]PGF2 ; the peaks in the
m/z 593 ion current chromatogram represent
F4-NPs. The total amounts of F2-IsoPs and
F4-NPs present were 7.9 and 6.3 ng/g brain tissue,
respectively.
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As a measure of specificity of the assay to detect esterified
F4-NPs in tissues, we analyzed for F4-NPs
esterified in lipids in 1 ml of human plasma, which contains only very
small amounts of DHA (Fig. 10) (7).
Intense peaks were present in the m/z 569 ion current
chromatogram representing F2-IsoPs but absent were peaks of
significant intensity in the m/z 593 ion current chromatogram that would indicate the presence of F4-NPs at
levels above the lower limits of detection (~5 pg/ml).

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Fig. 10.
Selected ion current chromatogram obtained
from the analysis for F2-IsoPs and F4-NPs
esterified in lipids in 1 ml of plasma. The intense peaks present
in the m/z 569 ion current chromatogram represent
F2-IsoPs. The peak in the m/z 573 ion current
chromatogram represents the
[2H4]PGF2 internal standard.
Absence are peaks in the m/z 593 ion current chromatogram
representing F4-NPs at a level above the lower limit of
detection (~5 pg/ml).
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Although F4-NPs can be readily detected esterified in the
brain, the utility of such measurements to assess oxidative injury would primarily be restricted to animal models of neurological disorders or brain samples obtained post-mortem from humans. We therefore examined whether F4-NPs could be detected in
cerebrospinal fluid obtained from four patients with Alzheimer's
disease and three age-matched control subjects. F4-NPs were
detected in 1-2 ml of cerebrospinal fluid from the control subjects at
a level of 64 ± 8 pg/ml. Of considerable interest was the finding
that the concentrations measured in the patients with Alzheimer's
disease were significantly higher (110 ± 12 pg/ml)
(p < 0.05). A selected ion current chromatogram
obtained from the analysis of F4-NPs in cerebrospinal fluid
from a patient with Alzheimer's disease is shown in Fig.
11. The pattern of F4-NP
peaks detected in free form in cerebrospinal fluid differs somewhat
from the pattern peaks detected esterified in tissue phospholipids
(Fig. 9). Similar differences have been observed for the pattern of
F2-IsoP peaks detected in free form in plasma and urine
compared with the pattern of peaks detected esterified in tissue
phospholipids as free compounds following base hydrolysis of a tissue
lipid extract. Although the reason for these differences has not been
established, this may be explained by differences in the efficacy of
phospholipases to hydrolyze different isomers from phospholipids.
Cerebrospinal fluid concentrations of F2-IsoPs were
similarly increased in patients with Alzheimer's disease but were
lower than the levels of F4-NPs in both control subjects
and Alzheimer's patients (46 ± 4 and 72 ± 7 pg/ml,
respectively).

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Fig. 11.
Selected ion current chromatogram obtained
from the analysis for F4-NPs in cerebrospinal fluid from a
patient with Alzheimer's disease.
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DISCUSSION |
These studies have elucidated a new class of
F2-IsoP-like compounds formed in vivo by free
radical-induced peroxidation of DHA. Free radical-induced peroxidation
of AA results not only in the formation of F-ring IsoPs but also D-ring
and E-ring IsoPs and thromboxane-like compounds (isothromboxanes) (2,
3). Thus, although it remains the subject of future studies, it is likely that analogous compounds in addition to F-ring NPs are also
formed as products of nonenzymatic peroxidation of DHA.
One of our motivations for determining whether IsoP-like compounds
could be formed as peroxidation products of DHA involves the
possibility that quantification of these compounds might provide a
unique marker of oxidative injury in the brain that could be exploited
to investigate the role of free radicals in the pathogenesis of
neurological disease. The ultimate hope would be that quantification of
F4-NPs could be utilized in studies of neurological disease in humans during life. Our findings indicate that these compounds are
readily detected esterified in lipids in the brain. However, the
application of such measurements in humans would be limited to samples
of brain removed surgically or post-mortem samples of human brain.
Measurements of F4-NPs made in human brain samples obtained
after death could be quite problematic because of the possibility of
artifactual generation of NPs by autoxidation of DHA during the time
interval between death and sample procurement. This is not a unfounded
concern as we previously demonstrated the occurrence of the generation
of significant quantities of IsoPs by autoxidation of arachidonic acid
in plasma, even when stored at
20 °C (19).
Although invasive, cerebrospinal fluid is frequently obtained for
diagnostic purposes in patients with suspected neurological disorders.
Thus, the availability of a marker of oxidative injury in the brain
that could be measured in cerebrospinal fluid intra vitam
would be an important advance. Thus, the finding that
F4-NPs could be detected in human cerebrospinal fluid
clearly has potentially important clinical applications. Recently we
have shown that markers of lipid peroxidation are increased in
cerebrospinal fluid of patients with Alzheimer's disease (24, 25).
However, these assays have shortcomings related to measurement of
reactive molecules, i.e. 4-hydroxynonenal, and require large
volumes of fluid. However, F4-NPs were detectable using
negative ion chemical ionization mass spectrometry in 1-2 ml of
cerebrospinal fluid from normal subjects, an amount that can usually be
obtained safely from patients for diagnostic purposes. Although it was
a limited study, the finding that F4-NP concentrations in
cerebrospinal fluid from patients with Alzheimer's disease were
significantly higher than levels in age-matched control subjects
highlights the potential of this approach to provide insights into the
role of free radicals in the pathogenesis of neurological disorders.
Another potentially important aspect of this finding is that serial
measurements of F4-NPs in cerebrospinal fluid might provide
a biochemical assessment of disease progression as well as a means to
monitor efficacy of therapeutic intervention, e.g. with
antioxidants, during life. To our knowledge, no other method has proven
to be reliable to obtain such information.
One question that arises is whether there is a distinct advantage of
measuring either IsoPs or NPs to assess oxidative injury in the brain.
The answer to this question is not forthcoming from the results of the
studies reported herein. However, it is of interest that the relative
amounts of F4-NPs formed during oxidation of DHA in
vitro exceeded the amounts of F2-IsoPs generated from an equivalent amount of AA by as much as 3.4-fold (Fig. 8). This is
consistent with the fact that of the naturally occurring fatty acids,
DHA is the most easily oxidizable (15). This suggests that measurement
of F4-NPs in some situations may provide a more sensitive
index of oxidative injury in the brain than measurement of
F2-IsoPs. The ratio of levels of AA and DHA, and thus the
capacity to form IsoPs and NPs, respectively, varies significantly
between different regions of the brain (white matter, gray matter),
different cell types (neurons, astrocytes, oligodendrocytes), and
subcellular fractions (myelin, synaptosomes) (7, 8, 26). In this
regard, we found that levels of F4-NPs and
F2-IsoPs esterified in whole rat brain were similar,
whereas levels of F4-NPs were higher than levels of
F2-IsoPs in the cortex of newborn pigs and in human cerebrospinal fluid. Therefore, there may be distinct advantages associated with measuring either IsoPs or NPs to assess oxidant injury
in the brain depending on the site of oxidant injury and the
predominant cell types involved. Thus, the best approach at this time,
which will provide valuable insight into this question, would be to
quantify both IsoPs and NPs in a variety of situations involving
different types of oxidative insults to the brain both in experimental
animals and in human neurological disorders. The practicality of this
approach will be facilitated by the fact that the method of assay we
developed allows simultaneous measurement of both F4-NPs
and F2-IsoPs in the same sample.
There are additional ramifications that are potentially relevant to
neuropathobiology that emerge from this discovery. Two of the IsoPs,
previously referred to as 8-iso-PGF2
and
8-iso-PGE2, now termed 15-F2t-IsoP and
15-E2t-IsoP according to the approved nomenclature for
IsoPs (16), have been found to possess potent biological activity
ranging from effects on vascular and bronchial smooth muscle,
endothelin release, platelet function, to cellular proliferation (5,
6). Of interest has been the evidence obtained which suggests that
these IsoPs may exert their vascular effects by interacting with a
unique receptor (5, 6). Thus, the possibility exists that NPs might
also be found to possess important biological actions that may be
relevant to the pathophysiology of oxidant injury to the brain. As
mentioned, this possibility is greatly supported by the finding that
C22-PGF4
is bioactive (7). This compound is one of the
F4-NPs that would be formed, although, analogous to IsoPs,
compounds in which the side chains are oriented cis likely
predominate over compounds in which the side chains are oriented
trans in relation to the cyclopentane ring (19). However, in
the case of the IsoPs, inversion of the stereochemistry of the upper
side chain of PGF2
and PGE2 affords
different and/or more potent biological actions (5, 6).
In addition, phospholipids containing esterified NPs are very unnatural
and unusual molecules. Shown in Fig. 12
is a molecular model of phosphatidylserine with palmitate esterified at
the sn-1 position and a 13-series NP (13-F4t-NP)
esterified at the sn-2 position. Mass spectral evidence for
the formation of 13-series F4-NPs during oxidation of DHA
was presented in Fig. 6. Trailing downward on the right from the polar
head group above is palmitic acid. Trailing downward and then curving
sharply upward on the left is the NP molecule in which the cyclopentane
ring is seen at the top. Unmistakably, this is a remarkably distorted
molecule. Thus, enhanced formation of these unusual phospholipids in
neuronal membranes in settings of oxidant injury to the brain might
lead to profound alterations in the biophysical properties of the
membrane, e.g. degree of fluidity, which in turn might
greatly impair normal neuronal function. Future studies using synthetic
NP-containing aminophospholipids in model membranes to assess the
extent to which these unique phospholipids alter membrane properties
should provide valuable insight into the potential relevance of the
formation of these phospholipids in settings of oxidative neuronal
injury.

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Fig. 12.
Ball and wire molecular model of
phosphatidylserine containing palmitate esterified in the
sn-1 position and a 13-series NP (13-F4t-NP)
esterified in the sn-2 position. Trailing downward on
the right from the polar head group above is palmitic acid.
Trailing downward and then curving sharply upward on the
left is the NP molecule in which the cyclopentane ring is
seen at the top.
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