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INTRODUCTION |
A role for free radicals has been implicated in the pathogenesis
of a wide variety of human diseases including atherosclerosis, cancer,
and neurodegenerative diseases (1). Unsaturated fatty acids are a major
target of free radical attack leading to lipid peroxidation. A variety
of compounds is generated as products of free radical-induced lipid
peroxidation including reactive aldehyde species, such as
4-hydroxynonenal (4-HNE)1 and
malondialdehyde (MDA). These reactive aldehydes are considered important mediators of oxidant injury due to their ability to covalently modify proteins and DNA, which can disrupt important cellular functions and can cause mutations (2). Furthermore, adduction
of aldehydes to apolipoprotein B in LDL has been strongly implicated in
the mechanism by which LDL is converted to an atherogenic form that is
taken up by macrophages, eventuating in the formation of foam cells
(3). Previously we reported the formation of prostaglandin (PG)-like
compounds in vivo, termed isoprostanes (IsoPs), by free
radical-induced peroxidation of arachidonic acid (4). Analogous to the
cyclooxygenase enzymatic pathway, intermediates in the IsoP pathway are
PGH2-like bicyclic endoperoxides. In aqueous media,
PGH2 is unstable and undergoes rearrangement with a
t1/2 of approximately 5 min at 37 °C (5).
Originally, it was shown PGH2 undergoes rearrangement to
form PGE2 and PGD2 (6). More recently, Salomon
and colleagues (5) demonstrated that PGH2 also rearranges
to form
-ketoaldehyde secoprostanoids. These compounds have been
termed levuglandin E2 and D2 because of their structural similarities with levulinaldehyde. Levuglandins comprise approximately 20% of the total rearrangement products of
PGH2 (5).
Initially we reported the formation of F2-IsoPs, which are
produced by reduction of the IsoP endoperoxide intermediates (4). We
recently discovered a key role for glutathione in effecting the
reduction of IsoP endoperoxides to F2-IsoPs (7). However, the reduction of IsoP endoperoxides is not completely efficient. In
this regard, we have shown that the IsoP endoperoxides undergo rearrangement in vivo to form E2-IsoPs,
D2-IsoPs, and isothromboxanes (8, 9).
Based on the observation that the IsoP endoperoxides undergo
rearrangement in vivo, we explored whether such
rearrangement also results in the formation of levuglandin-like
compounds, for which we propose the term isolevuglandins (IsoLGs). Our
interest in this possibility stems from the fact that the
-ketoaldehyde moiety confers remarkable reactivity to these
compounds. In this regard, Salomon and colleagues (10, 11) have shown
that LGE2 rapidly adducts to amines and, in addition,
readily undergoes further reaction to form extensive protein-protein
and protein-DNA cross-links. Thus, if formed, IsoLGs might participate
as important mediators of oxidant injury. Therefore, we undertook
studies to explore whether IsoLGs are formed during oxidation of
arachidonic acid and studies to elucidate the nature of the IsoLG
adduct that would be formed with lysine residues on proteins. The
mechanism by which IsoLGs are generated by free radical-induced
oxidation of arachidonic acid is shown in Fig.
1. Four regioisomers of both D2-IsoLGs and E2-IsoLGs are formed, each of
which is theoretically comprised of four racemic diastereomers, for a
total of 32 IsoLGE2 and 32 IsoLGD2 compounds.
The designation "D" and "E" is the same as for the
cyclooxygenase-derived LGs and refers to the location of the keto
group. If the keto group is located at C-9, the molecule is designated
an E2-IsoLG, and if it is located at C-11, the molecule is
designated as a D2-IsoLG. Salomon and co-workers (12) had previously used a carbon numbering system from 1 to 17 beginning with
the carboxyl carbon, and the chains containing the carbonyl groups were
considered as substituents. However, to retain consistency with the
official nomenclature for isoprostanes that has been approved by the
Eicosanoid Nomenclature Committee, sanctioned by JCBN of IUPAC (13),
the original 20 carbon numbering system used for the IsoP endoperoxide
precursors will be retained. In accordance with the IsoP nomenclature,
the different regioisomers are designated by the carbon number on which
the side chain hydroxyl is located, as indicated in Fig. 1.

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Fig. 1.
Predicted mechanism of formation of IsoLGs
via the IsoP pathway. Four IsoP bicyclic endoperoxide regioisomers
are formed which then undergo rearrangement to form four
E2-IsoLG and four D2-IsoLG regioisomers. Each
E2-IsoLG and D2-IsoLG regioisomer is
theoretically comprised of four racemic diastereomers.
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EXPERIMENTAL PROCEDURES |
Oxidation of Arachidonic Acid--
Five mg of arachidonic acid
(Nu Chek Prep, Elysian, MN) or 1-palmitoyl,
2-arachidonoyl-glycero-3-phosphocholine (Sigma) was dissolved in 200 µl of ethanol and oxidized using a mixture of ferric chloride (1 mM), ADP (200 mM), and ascorbate (100 mM) in 5 ml of 50 mM phosphate buffer (pH 7.4)
at 37 °C for 6 h.
Mass Spectrometric Analysis of IsoLGs--
Following oxidation
of arachidonic acid or 1-palmitoyl,
2-arachidonoyl-glycero-3-phosphocholine, compounds were converted to
O-methyloxime derivatives by addition of 3%
methoxyamine·HCl and incubated for 45 min at room temperature.
Compounds formed from the oxidation of phosphatidylcholine were
subsequently subjected to base hydrolysis with 7.5% KOH in 50%
aqueous methanol for 30 min at 37 °C. Samples were then acidified to
pH 3 with 1 N HCl and diluted. 1-4 ng of
bis-[2H3]O-methyloxime-LGE2,
which was formed by treatment of synthetic LGE2 (14) with
[2H3]methoxyamine·HCl (Regis, Morton Grove,
IL), was added as an internal standard. The concentration of the
bis-[2H3]O-methyloxime-LGE2
was standardized against PGF2
by gas chromatography
(GC)/negative ion chemical ionization (NICI)/mass spectrometry (MS).
The sample was then applied to a C18 Sep-Pak column (Waters
Associates, Milford, MA) that had been pre-conditioned with 5 ml of
methanol and 10 ml of pH 3 water. The Sep-Pak was washed sequentially
with 10 ml of pH 3 water and 10 ml of heptane/ethyl acetate (99:1 v/v),
and the IsoLGs were then eluted with 10 ml of heptane/ethyl acetate
(1:1 v/v). The heptane/ethyl acetate was dried under a stream of
N2. The samples were then converted to a pentafluorobenzyl
ester derivative by treatment with 40 µl of 10% pentafluorobenzyl
bromide in acetonitrile and 20 µl of 10%
N,N-diisopropylethylamine in acetonitrile for 20 min at 37 °C. Samples were then subjected to thin layer
chromatography (TLC) using silica gel plates (VWR Scientific, Atlanta,
GA) using the solvent heptane/ethyl acetate (60:40 v/v). Compounds
migrating 2 cm above and 0.5 cm below the bis-O-methyloxime,
pentafluorobenzyl ester derivative of LGE2 were scraped and
extracted from the silica gel with ethyl acetate. IsoLGs were then
converted to trimethylsilyl (TMS) ether derivatives by treatment with
10 µl of dimethylformamide and 10 µl of
N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Supelco,
Bellefonte, PA) and analyzed by GC/NICI/MS. GC was performed with a
12-m DB 1701 fused silica capillary column (J & W Scientific, Folsom,
CA) heated from 190 to 300 °C at 20 °C/min with a 0.2-min hold.
Source temperature was 250 °C.
Determination of functional groups was accomplished by subjecting 2 ml
of an arachidonate oxidation mixture to derivatization with
methoxyamine·HCl or
[2H3]methoxyamine·HCl. No internal standard
was added. Derivatization was continued as above except each sample was
divided equally after TLC and half derivatized with BSTFA as above and
half with [2H9]BSTFA
(Deutero-Regisil-d18, Regis, Morton Grove, IL)
(6 µl + 6 µl dimethylformamide). Samples were then analyzed by
GC/NICI/MS, employing selected ion monitoring for the [M
·CH2C6F5]
ion
of each derivative. Disappearance of signals was monitored as well as
appearance of signals, i.e. the presence of two carbonyl groups was indicated by the disappearance of signals at m/z
481 and the appearance of signals at m/z 487 in samples
treated with [2H3]methoxyamine·HCl and
BSTFA, and the presence of a single hydroxyl group was indicated by the
disappearance of signals at m/z 481 and the appearance of
signals at m/z 490 in samples treated with methoxyamine·HCl and [2H9]BSTFA.
For analysis of IsoLGs by electron impact ionization (EI)/MS,
methoximated IsoLGs were purified by reverse phase HPLC on an Econosil C18 column (Alltech Associates, Deerfield, IL)
utilizing a solvent system of 45% acetonitrile in water with 0.1%
acetic acid. One-ml fractions were collected. Fractions containing
IsoLGs were identified by analysis of aliquots as above by GC/NICI/MS. IsoLGs were detected eluting over approximately 30 fractions. Eight
fractions eluting at approximately 30 ml, which contained a high
concentration of IsoLGs, were pooled. This purification procedure was
repeated several times to obtain sufficient material for analysis by
GC/EI/MS. GC/EI/MS analysis yielded a single prominent chromatographic
peak with a smaller shoulder that contained ions consistent with mass
spectra of IsoLGs at an approximate retention time of 9 min. This
retention time is longer than the retention time of IsoLGs in the
GC/NICI/MS experiments described above (approximately 6 min) because a
longer GC column was used to achieve more effective separation of
IsoLGs from other potentially interfering compounds. Spectra were
displayed by averaging the scans across the chromatographic peaks. The
source temperature was 200 °C, the ionization energy 70 eV, and the
transfer line and injector each held at 250 °C.
Mass Spectrometric Analysis of F2-IsoPs and
D2/E2-IsoPs--
D-ring, E-ring, and F-ring
IsoPs generated from oxidation of arachidonic acid were quantified by
GC/NICI/MS as described (4, 8).
Comparison of the Rate of Adduction of LGE2 and 4-HNE
to Albumin--
0.1 mM LGE2 and 0.1 mM 4-HNE were incubated with bovine serum albumin (BSA) (5 ml at 20 mg/ml in Hanks' balanced salt solution at 37 °C). 100-µl
aliquots were removed at indicated time points, and free unadducted
LGE2 was quantified by GC/NICI/MS as described above. Free
unadducted 4-HNE was measured by colorimetric assay (Oxis
International, Portland, OR).
Formation and Analysis of LGE2-Lysine
Adducts--
LGE2 (1 mM) was incubated with
[3H]lysine (1 mM; 17,000 cpm/mg) in 1 ml of
phosphate-buffered saline at 37 °C for 4 h. Incubations were
also performed in which lysine was replaced with
[U-13C]lysine (Cambridge Isotope Laboratories, Andover,
MA) or N
-acetyl-lysine methyl ester (Sigma).
100-µl aliquots of the incubation mixtures were analyzed by liquid
chromatography (LC)/electrospray ionization (ESI)/MS in the positive
ion mode using a Waters 2.1 × 150 mm C18 column and a
water/acetonitrile gradient (3%/min; hold 5 min) at 0.2 ml/min.
Auxiliary gas pressure was 70 pounds/square inch; sheath gas pressure
was 10 pounds/square inch. The voltage on the capillary was 20.0 V, the
capillary temperature 200 °C, and the tube lens voltage 90 V. Parent
ions were scanned from m/z 400 to m/z 500. Collision-induced dissociation (CID) of molecular ions of the putative
lactam and hydroxylactam adducts formed in these incubations was
performed from
10 to
40 eV, scanning daughter ions from
m/z 50 to m/z 500 (up to m/z 560 for
the N
-acetyl-lysine methyl ester derivative).
Spectra shown were obtained at
28 eV. CID gas was argon with a
pressure set at 2.0 millitorr. Spectra were displayed by averaging
scans across the chromatographic peaks.
Formation and Analysis of IsoLG Lysine Adducts--
100 mg of
arachidonic acid was oxidized as described above in the presence of 100 mg of lysine. The incubation mixture was then loaded onto a
C18 Sep-Pak cartridge in pH 3 water, washed sequentially
with 10 ml of water, 10 ml of heptane, and 10 ml of heptane/ethyl
acetate (1:1 v/v). Adducts were eluted with 10 ml methanol/ethyl
acetate (35:65 v/v). The organic solvents were then dried under a
stream of N2, and an aliquot was analyzed by LC/ESI/MS/MS
using conditions described above except the LC flow rate was 0.1 ml/min. MS/MS analysis was carried out using selected reaction
monitoring (SRM) of daughter ions produced from CID of [MH]+ at
28 eV. 1/20 of this preparation was analyzed
by SRM of daughter ions of the lactam adducts, and approximately 1/3
was analyzed by SRM of daughter ions of the hydroxylactam adducts.
Isolation and Analysis of IsoLG Adducts on Oxidized LDL--
LDL
was isolated from 10 ml of plasma obtained from normal human volunteers
using a low temperature ethanol precipitation procedure (15). Briefly,
the plasma was stirred with a 25% ethanol solution at
5 °C for 15 min and then centrifuged at 3000 × g for 30 min at
5 °C. The precipitated LDL was then suspended in 50 mM
potassium phosphate buffer (pH 7.4) at a concentration of 10 mg of
protein/ml and oxidized with 0.1 mM
2,2'-azobis(2-amidinopropane) HCl (AAPH) (Polysciences, Warrington, PA)
for 4 h. The LDL was subsequently re-precipitated as above and
delipidated (15). Briefly, the LDL was diluted to 10 mg of protein/ml
and then sequentially extracted with ethanol/diethyl ether (2:3 v/v)
for 24 h; ethanol/ether (3:1 v/v) for 24 h; ethanol/ether
(1:1 v/v) for 30 min; ethanol/ether (1:3 v/v) for 30 min; and finally
briefly washed with ether. The temperature was maintained at
15 °C
during the delipidation procedure. The protein was recovered after each
extraction by centrifugation at 850 × g for 20 min.
The protein was re-dissolved by suspending it in ether, and 0.2 N NaOH was then added. The mixture was gently bubbled with
N2 until the ether evaporated and then incubated for 2 h at room temperature to hydrolyze IsoLG protein adducts that may have
formed with IsoLG esterified to LDL lipids. The pH was then adjusted to
7.5, and the protein was subjected to complete enzymatic digestion to
individual amino acids as described (16). Briefly, the protein
concentration was adjusted to 1 mg/ml. 0.1 volume of a 1 mg/ml solution
of Pronase (Calbiochem) was added, and the mixture was incubated at
37 °C for 18 h. The pH was then adjusted to 8.5, and
MgCl2 was added to a final concentration of 4.8 mM. 0.1 volume of leucine aminopeptidase (Sigma) activation solution was subsequently added. Leucine aminopeptidase was activated by incubating 225 µg/ml in 10 mM Tris-HCl buffer (pH 8.5)
with 1 mM MnCl2 for 2-3 h at 40 °C and
subsequently added to the digest, which was then incubated at 37 °C
for 18 h at room temperature. The compounds were then extracted
using a C18 Sep-Pak cartridge as described above. The
eluate was dried under a stream of N2, and IsoLG/lysine
adducts were analyzed by LC/ESI/MS/MS as described for the IsoLG
adducts above. Before analysis, a mixture of
[13C6]lactam and
[13C6]hydroxylactam IsoLG adducts was added
to allow quantification of the amount of IsoLG adducts detected. These
compounds were formed by oxidation of 25 mg of arachidonic acid in the
presence of [3H]lysine (50 × 106 cpm;
10,000 cpm/µg) (NEN Life Science Products) diluted with 1 mg of
[13C6]lysine (Cambridge Isotope Laboratories,
Andover, MA). The [3H]- and
[13C6]lysine-IsoLG lactam, and hydroxylactam
adducts formed were then purified by extraction on C18
Sep-Pak cartridges as above and HPLC (water to acetonitrile in 30 min
with 5-min water pre-wash). HPLC fractions containing the lysyl IsoLG
adducts were detected by LC/ESI/MS analysis, and the concentration was
determined by the specific activity of the
[3H]lysine.
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RESULTS |
Identification of IsoLGs Formed during Oxidation of Arachidonic
Acid in Vitro--
GC/NICI/MS analysis of oxidized arachidonate
mixtures revealed a series of compounds with characteristics of IsoLGs
(Fig. 2). The lower m/z 487 chromatogram displays a series of incompletely resolved peaks
corresponding to the [M
·CH2C6F5]
ion
of the bis-[2H3]O-methyloxime
LGE2 internal standard. These represent the four methoxime
isomers (two syn and two ante) resulting from
methoximation of the two carbonyl groups of the internal standard. At a
similar retention time in the upper m/z 481 chromatogram are
a series of peaks with the [M
·CH2C6F5]
ion
of IsoLGs. The pattern of the putative IsoLG compounds differs from
that seen in the m/z 487 chromatogram, consistent with the formation of multiple IsoLGE2 and IsoLGD2
isomers (see Fig. 1). Virtually identical results were obtained from
the analysis of IsoLGs formed during oxidation of 1-palmitoyl,
2-arachidonoyl-3-glycero-phosphocholine (data not shown).

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Fig. 2.
Selected ion current chromatograms obtained
from GC/NICI/MS analysis for IsoLGs formed during oxidation of
arachidonic acid in vitro. Compounds were analyzed as a
pentafluorobenzyl ester, O-methyloxime, trimethylsilyl ether
derivative monitoring the [M ·CH2C6F5]
ions at m/z 481 for IsoLGs and m/z 487 for the
deuterated internal standard, the
bis-[2H3]O-methyloxime derivative
of synthetic LGE2.
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Additional studies were then undertaken to confirm the identity of
these compounds as IsoLGs. When analyzed as a
[2H3]O-methoxylamine derivative,
all of the m/z 481 peaks shifted upwards 6 Da to
m/z 487, indicating the presence of two carbonyl groups.
When analyzed as a [2H9]TMS ether derivative,
all of the m/z 481 peaks shifted upward 9 Da to
m/z 490, indicating the presence of one hydroxyl group (data
not shown). These compounds were then analyzed by GC/EI/MS after
partial purification by HPLC. Mass spectra were obtained that were
consistent with the formation of both E2- and
D2-IsoLGs. One of the mass spectra obtained is shown in
Fig. 3A. Although it is
unlikely that this is a mass spectrum of a single IsoLG isomer because
the preparation analyzed was only partially purified, this mass
spectrum is consistent with a major component being a 15-series
IsoLGD2 compound. The spectrum is characterized by an
intense molecular ion at m/z 662 and intense ions at
m/z 631 (M
31), loss of ·OCH3
from a methoxime group, m/z 591 (M
71), loss of
·CH2(CH2)3CH3
from fragmentation between C-15 and C-16 on the lower side chain,
m/z 559 (M
71
32), the loss of
·CH2(CH2)3CH3 + HOCH3, m/z 541(M
90
31), loss of
Me3SiOH + ·OCH3, m/z 501 (M
90
71), loss of Me3SiOH + ·CH2(CH2)3CH3,
m/z 489 (M
173), loss of ·CH2
(OSiMe3)(CH2)4CH3 from
fragmentation between C-14 and C-15 on the lower side chain. A mass
spectrum of authentic LGE2 is shown in Fig. 3B.
Notable are the striking similarities in the high mass ions present and
their relative abundances in the spectra in Fig. 3, A and
B. The presence of ions involving the loss of 71 and 173 Da
in both mass spectra resulting from fragmentation adjacent to the
carbon on the lower side chain on which the TMS ether group is attached
supports the identity of a major component of the mass spectrum in Fig.
3A as a 15-series IsoLG. An ion at m/z 418 is
present in both the IsoLG and LGE2 spectra, but its origin
is unclear; it may derive from a minor common decomposition product.
Intense fragmentation ions are present in the mass spectrum of
authentic LGE2 resulting from fragmentation between C-8 and C-12, one representing the upper portion of the molecule
(m/z 392) and one representing the lower portion of the
molecule (m/z 270). These ions are also observed in the
IsoLG spectrum, indicating that it may also contain a 15-series
IsoLGE2 compound as a minor component. The base ion in the
mass spectrum in Fig. 3A is m/z 284. This origin
of this ion would be consistent with the lower portion of a 15-series
IsoLGD2 molecule, as a result of fragmentation between C-8
and C-12. However, unlike the mass spectrum of authentic LGE2, an ion representing the upper portion of the molecule
is not present. Possible reasons for this include different relative abundances of these two ions in different IsoLG isomers or in LGD
versus LGE compounds. The latter possibility cannot be
explored because authentic LGD2 is not available. Salomon
and colleagues have attempted the synthesis of LGD2 but
found it to be highly unstable under the conditions used for its
synthesis (12).

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Fig. 3.
Electron ionization mass spectra obtained
from the analysis of partially purified IsoLGs (A) and
of synthetic LGE2 (B) as pentafluorobenzyl
ester, bis-O-methyloxime, trimethylsilyl ether
derivatives. Interpretations of ions are discussed in detail in
the text.
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Relative Amounts of IsoLGs and IsoPs Produced during Oxidation of
Arachidonic Acid in Vitro--
To evaluate the quantitative relevance
of the formation of IsoLGs, we compared the amounts of IsoLGs with the
amounts of F2-IsoPs and E2/D2-IsoPs
formed during oxidation of arachidonic acid in vitro.
Surprisingly, we found that the amounts of IsoLGs formed were
approximately equivalent with the amounts of F2-IsoPs and were only slightly less than the amounts of
E2/D2-IsoPs (Fig. 4). These data suggest strongly that
IsoLGs can be produced as products of the IsoP pathway at levels that
can potentially have significant biological impact.

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Fig. 4.
Comparison of the relative amounts of IsoLGs,
F2-IsoPs, and E2/D2-IsoPs formed
following oxidation of arachidonic acid in vitro. Five mg of
arachidonic acid was oxidized for 4 h with
iron/ADP/ascorbate.
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LGs Readily Form Covalent Adducts With Proteins--
Having
established that IsoLGs can be formed in significant amounts during
oxidation of arachidonic acid in vitro, we explored whether
we could detect their formation in a number of biological systems
in vitro and in vivo. Despite the fact that
readily detectable levels of F2-IsoPs are present in less
than 1 ml of normal human plasma and urine, we could not detect IsoLGs
in 3 ml of either human plasma or urine. Furthermore, we could not
detect the formation of IsoLGs in vivo in 1 g of liver
from rats treated with CCl4 to induce an intense oxidant
injury to the liver which was associated with a marked increase in the
formation of F2-IsoPs. We also could not detect the
formation of IsoLGs in vitro following (a)
iron/ADP/ascorbate-induced oxidation of liver microsomes (17) prepared
from 5 g of rat liver or (b) following copper-mediated
oxidation of LDL (18) isolated from 2.5 ml of plasma, both of which
again were accompanied by marked increases in F2-IsoP
formation. The overriding difference between these experiments and
those in which we oxidized arachidonic acid in vitro is the
presence of protein. We hypothesized that the failure to detect IsoLGs
in these biological systems may be attributed to extremely rapid
adduction of IsoLGs to proteins. Thus, we attempted to intercept the
formation of IsoLG adducts in the microsome and LDL oxidation mixtures
by adding oxime reagents to a final concentration of 3% to derivatize
the carbonyls, which would prevent IsoLG adduction to amines. Although
this approach effectively converted the carbonyls of
E2/D2-IsoPs that were formed in these
experiments to oxime derivatives, it did not successfully trap free
IsoLGs. This suggested either that IsoLGs were not formed or that under
the experimental conditions used, their reaction with proteins was much
faster than their reaction with the oxime reagents.
In an attempt to gain support for our hypothesis that IsoLGs could not
be detected as free dicarbonyl compounds in biological systems because
they adduct to proteins with extreme rapidity, we compared the rate of
adduction of LGE2 and 4-HNE with BSA, as a model protein.
We thought a comparison of the rate of adduction of LGE2
with that of 4-HNE would be informative because 4-HNE, although
considered to be one of the most highly reactive products of lipid
peroxidation yet identified, can be detected in free form in biological
systems (2). The rate of adduction of these two compounds was assessed
by monitoring the decline in levels of free compounds measured in
aliquots removed at various times during incubations consisting of
LGE2 or 4-HNE with a 3 molar excess of BSA. As shown in
Fig. 5, levels of free LGE2
dropped precipitously during the initial 60 s of the incubation;
notably more than 50% had adducted within 20 s. Of note, free
LGE2 did not decline completely to undetectable levels but
leveled off between 5 and 10% of the amount detected at time 0. This
can likely be attributed to facile migration of the
13
double bond to the
12 position, which may decrease the
reactivity of the molecule (12). A small amount of
12-LGE2 may have been present in the
synthetic preparation, or migration of the double bond may have
occurred during the incubation since the double bond migration has been
shown to be catalyzed by buffer ions (12). In striking contrast to the
remarkably fast rate of adduction of LGE2 to BSA that was
observed, ~50% of 4-HNE had still not adducted after 1 h. The
rate of adduction of 4-HNE to albumin found in this study agrees
closely with that reported previously by Curzio et al. (19).
These data indicated that the rate at which LGE2 adducts to
proteins exceeds that of 4-HNE by several orders of magnitude and
provided a very plausible explanation for our inability to detect
IsoLGs in free form in biological systems containing protein.

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Fig. 5.
Comparison of the rates of covalent adduction
of LGE2 and 4-HNE to BSA. The formation of covalent
adducts was assessed by monitoring the disappearance of free compounds
over time and expressed as a percent of the amount of free compound
present at time 0. The proposed explanation for why the levels of
LGE2 do not continue to fall to undetectable levels but
plateau between 5 and 10% of the amount of LGE2 present at
time 0 is discussed in the text.
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Identification of LGE2-Lysine Adducts--
We then
sought to structurally identify the nature of LG adducts using
LC/ESI/MS/MS. Initial studies were carried out using synthetic
LGE2 as a model for IsoLGs. Based on previous data reported by Salomon and colleagues (20), we predicted that a major protein adduct would be a pyrrole produced from reaction of LGE2
with the
-amino group of lysyl residues. In initial studies, we used free lysine to model this reaction which seemed valid because the
-amino group of free lysine is only about 1/6 as reactive as the
-amino group (16). The predicted [MH]+ ion for the
LGE2-lysine pyrrole adduct is m/z 463. Full
scanning spectrum analysis, however, consistently revealed the presence of intense ions at m/z 479 and 495, rather than an ion at
m/z 463. Selected ion current chromatograms of
m/z 479 and m/z 495 obtained from analysis of an
incubation of LGE2 with lysine is shown in Fig.
6. Notably, these ions are 16 and 32 Da
higher than the [MH]+ ion of the pyrrole adduct. Relevant
to this finding were previous data demonstrating that pyrroles can
undergo autoxidation to form lactams and hydroxylactams, which have
masses 16 and 32 Da higher, respectively, than the corresponding
pyrroles (21). The mechanism by which lactams and hydroxylactams are
predicted to form by autoxidation of pyrroles is shown in Fig.
7. The LGE2 pyrrole would be
expected to be highly susceptible to autoxidation because it is highly alkylated and electron-rich (20). Consistent with such a prediction, Salomon and colleagues (20) found that the pyrrole formed by LGE2 reaction with neopentylamine was highly unstable and
could not be isolated unless air was rigorously excluded. We therefore attempted to prevent oxidation of the LGE2-lysine pyrrole
that was predicted to have been formed by incubating LGE2
with lysine under argon in deaerated buffer. Under these conditions, we
did see a new peak at m/z 463 that eluted at approximately
the same retention volume as the lactam and hydroxylactam adducts,
consistent with an LGE2-lysine pyrrole. However, the CID
mass spectrum of this compound only produced ions at m/z
445, formed by the loss of H2O, and m/z 363, formed by the loss of 100 Da. The loss of 100 Da is speculated to occur
from fission of the bond between C-14 and C-15 with transfer of a
proton as described by Murphy and colleagues (22) during CID of
prostaglandins. Although suggestive, it could not be concluded with
certainty that this compound was in fact the lysyl-LGE2
pyrrole because of the limited structural information provided by the
CID mass spectrum.

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Fig. 6.
Selected ion current chromatograms of
[MH]+ ions m/z 479 and m/z
495 from an LC/ESI/MS analysis of adducts formed following
incubation of LGE2 with lysine.
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Fig. 7.
Proposed mechanism of formation of lactams
and hydroxylactams by autoxidation of pyrroles (adapted from Ref.
21).
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The putative lysyl-LGE2 lactam and hydroxylactam adducts
were then structurally characterized further by CID of the
[MH]+ ions, m/z 479 and m/z 495, respectively. CID of the putative lactam adduct produced informative
daughter ions at m/z 461, m/z 415, m/z
346, m/z 332, and m/z 84 (Fig.
8A). CID of the putative hydroxylactam adduct produced analogous daughter ions at m/z
477, m/z 459, m/z 413, m/z 330, and
m/z 84 (Fig. 8B). Insight into the structures of
these fragment ions was obtained by analysis of the LGE2
adducts formed with [13C6]lysine and
N
-acetyl-lysine methyl ester. The proposed
structures for these ions and the corresponding ions in the CID spectra
of the adducts formed with the lysine analogs are shown in Figs.
9 and 10.
The ion shifts in the CID spectra of the adducts formed with the lysine analogs support the proposed structures of these ions. However, the
precise mechanism of their formation remains speculative, except for
ions at m/z 461 in the lactam CID spectrum and
m/z 477 and m/z 459 in the hydroxylactam CID
spectrum, representing the loss of H2O (m/z 461 and m/z 477) and 2× H2O (m/z
459).

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Fig. 8.
LC/ESI/MS/MS analysis of
LGE2-lysine adducts. The [MH]+ ions of
the putative lactam (A) and hydroxylactam (B)
adducts, m/z 479 and m/z 495, respectively, were
subjected to CID at 28 eV and daughter ions scanned from
m/z 50 to m/z 500. Spectra were obtained by
averaging scans across the peaks observed. The interpretations of the
structures of individual ions are detailed in Figs. 9 and 10.
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Fig. 9.
Interpretations of the structures of
individual CID daughter ions of the lysyl LGE2 lactam
adducts. LGE2 was reacted with free lysine
(column 1), [13C6]lysine
(column 2), or N -acetyl-lysine
methyl ester (column 3), producing the corresponding lactam
adduct [MH]+ ions at m/z 479, m/z
485, and m/z 535, respectively. These ions were then
subjected to LC/ESI/MS/MS, yielding corresponding daughter ions noted
that supported the interpretations of the ion structures shown on the
right.
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Fig. 10.
Interpretations of the structures of
individual CID daughter ions of the hydroxylactam adducts.
LGE2 was reacted with free lysine (column 1),
[13C6]lysine (column 2), or
N -acetyl-lysine methyl ester (column
3), producing the corresponding hydroxylactam [MH]+
ions at m/z 495, m/z 501, and m/z 551, respectively. These ions were then subjected to LC/ESI/MS/MS, yielding
corresponding daughter ions noted that supported the interpretations of
the ion structures shown on the right.
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It is important to mention that we cannot be quantitatively precise
about the yields of the lactam and hydroxylactam adducts for reasons
related to the labile and highly reactive nature of LGE2.
First, the synthetic LGE2 is impure, and thus the weight of
the preparation is an unreliable indicator of the actual amount of
LGE2 present. The LGE2 cannot be purified from
precursors and side products from the final reaction in the synthesis
because it degrades extensively during column chromatography (12). We also cannot accurately quantify the amount of LGE2 present
by selected ion monitoring GC/NICI/MS comparing the amount present to a
known amount of deuterated internal standard. This is because the
double bond at the
13 position, as mentioned above, can
readily migrate to the
12 position, which changes the
reactivity of the molecule (12). GC/NICI/MS analysis cannot distinguish
between the reactive
13-LGE2 and the
relatively unreactive
12-LGE2. In this
regard, we have found that the amount of LGE2 that adducts
decreases significantly over time during storage, even though the
amount of signal seen by GC/NICI/MS analysis remains essentially
constant. This phenomenon is presumably due to conversion of synthetic
13-LGE2 to
12-LGE2 during storage. Thus, it is not
possible in any given experiment to know precisely how much reactive
12-LGE2 is added to the incubation. As
discussed previously, this conversion may also account for the apparent
biphasic rate of adduction of LGE2 to BSA seen in Fig. 5.
The other issue that confounds quantification is related to the
remarkable proclivity of LGE2 to induce cross-links (10,
11). In this regard, HPLC analysis of LGE2 that has been
incubated with a molar excess of tritiated lysine results in a large
amount of radioactivity that chromatographs as multiple unresolved
peaks eluting over many fractions. This broad slur of tritiated
reaction products elutes at a retention volume that is widely separated
from free lysine and elutes over a much broader range than the lactam
and hydroxylactam adducts. Although this material is not amenable to
analysis by standard approaches utilized in these studies, we speculate
that this material represents cross-linked species. Thus lactam and hydroxylactam adducts do not account for all or even most of the adducts formed during LGE2 reaction with proteins.
Identification of IsoLG-Lysine Adducts--
We then utilized the
information obtained from analysis of LGE2-lysine adducts
to characterize the formation of IsoLG-lysine adducts. Arachidonic acid
was oxidized in the presence of lysine, and adducts formed were
analyzed by LC/ESI/MS. The analysis revealed compounds at
m/z 479 and m/z 495 that had LC elution volumes
consistent with the formation of IsoLG-lysine lactam and hydroxylactam
adducts, respectively. LC/ESI/MS/MS analysis revealed that these
compounds formed the same daughter ions that had been observed for
LGE2-lysine lactam and hydroxylactam adducts (Fig.
11). No peaks at m/z 463, indicative of pyrrole adducts, were detected. Experiments excluding air
in an attempt to obtain evidence for the formation of pyrrole adducts
could not be performed because of the requirement of oxygen during
oxidation of arachidonic acid. Notably, the chromatograms obtained from
analysis of IsoLG adducts reveal multiple m/z 479 and
m/z 495 peaks, consistent with the formation of a series of IsoLG isomers.

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Fig. 11.
LC/ESI/MS/MS analysis of IsoLG-lysine
adducts formed during oxidation of arachidonic acid, to generate
IsoLGs, in the presence of lysine. SRM monitoring of the
transitions of m/z 479 (lactam adducts) and m/z
495 (hydroxylactam adducts) to the daughter ions indicated in the
figure was performed. Interpretations of daughter ion structures are
shown in Figs. 9 and 10.
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Detection of IsoLG/Apolipoprotein Adducts in Oxidized LDL--
We
then sought to determine if we could detect IsoLG/apoB-100 adducts
following oxidation of LDL. We considered these experiments to be very
informative since, as mentioned previously, our attempts to detect
unadducted free IsoLGs during oxidation of LDL were not successful. For
these analyses, as outlined under "Experimental Procedures," LDL
was oxidized for 4 h with AAPH and delipidated, and the apoB-100
protein was then digested to individual amino acids by sequential
treatment with Pronase and leucine aminopeptidase (16). Native LDL (not
subjected to oxidation) was also treated in an identical fashion. The
amino acid mixture was then analyzed by LC/ESI/MS/MS, employing SRM. No
adducts were detected in native LDL. However, as shown in Fig.
12, intense signals consistent with the
presence of IsoLG lactam and hydroxylactam adducts were detected in the
hydrolysate prepared from oxidized LDL. These compounds had molecular
ions ([MH]+) of m/z 479 and m/z 495 that transitioned to m/z 84.1 during CID, supporting their
characterization as lactam and hydroxylactam adducts, respectively.
These compounds also had the same LC elution volume as the internal
standards, [13C6]lysine-IsoLG lactam and
hydroxylactam adducts, at m/z 485 and m/z 501 that transitioned to m/z 89, respectively. Multiple peaks representing the [13C6]lysine internal
standard adducts are present because these were generated by oxidation
of arachidonic acid in the presence of [3H]- and
[13C6]lysine and thus consist of multiple
IsoLG-lysine adducts. Approximately 1 nmol of IsoLG adduct was formed
per 500 nmol of apoB-100 (500 ng/250 mg starting lipoprotein) as
calculated by comparing the ratio of intensity of signals for adducts
detected in oxidized LDL to signals for the known amount of
3H- and 13C6-adducts added.

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Fig. 12.
LC/ESI/MS/MS analysis of IsoLG-apoB-100
adducts from oxidized LDL. LDL was subjected to oxidation,
delipidated, and the apo-B-100 protein, then subjected to complete
enzymatic hydrolysis to individual amino acids as described under
"Experimental Procedures." Internal standards of
[13C6]lactam and -hydroxylactam adducts,
formed by oxidation of arachidonate in the presence of
[13C6]lysine, were then added. The
hydrolysate was analyzed by SRM of the following transitions:
m/z 495.4 to m/z 84.1 (IsoLG-lysine hydroxylactam
adducts from oxidized LDL); m/z 501.4 to m/z 89.1 (internal standard IsoLG [13C6]lysine
hydroxylactam adducts); m/z 479.4 to m/z 84.1 (IsoLG-lysine lactam adducts from oxidized LDL); m/z 485.4 to m/z 89.1 (internal standard IsoLG
[13C6]lysine lactam adducts).
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DISCUSSION |
These studies have identified a novel class of extremely reactive
molecules that are generated as products of the IsoP pathway. The
relevance of the formation of these compounds relates to their remarkable proclivity to form covalent adducts with proteins and induce
protein-protein and DNA-protein cross-links. Thus, these compounds
might be expected to be highly injurious as a result of covalent
modification of key biomolecules that are critical to normal cellular
function and replication. Reactive aldehydes generated from lipid
peroxidation, such as 4-HNE and MDA, form covalent adducts and are
thought to be important mediators of some of the adverse sequelae of
oxidant injury, including conversion of LDL to an atherogenic form (2,
3). The finding that IsoLG adducts could be easily detected in apoB
protein following oxidation of LDL in vitro suggests that
IsoLGs may also contribute to this pathogenetic process. Further
studies quantitatively assessing the relative abundance of 4-HNE, MDA,
and IsoLG adducts on apoB following oxidation of LDL should provide
important insight into the relative extent to which these reactive
molecules participate in the conversion of LDL to an atherogenic form.
In addition, 4-HNE is considered to be one of the most reactive
aldehydes generated as a product of lipid peroxidation (2). In this
regard, the data obtained that demonstrated that LGE2
adducts to BSA at a rate that exceeds that of 4-HNE by several orders
of magnitude was a finding that dramatically highlights the highly
reactive nature and potential importance of the generation of
IsoLGs.
These studies open up numerous new avenues for potentially important
scientific inquiry regarding the formation of IsoLGs in settings of
oxidant injury. In addition to their ability to adduct to proteins,
IsoLGs should also readily adduct to DNA. In this regard, Salomon and
colleagues (11) have shown that LGE2 can form DNA-protein
cross-links in cultured cells. It is well recognized that free
radical-induced DNA damage can lead to malignant transformation (23).
However, nothing is known at present about the consequences of the
formation of IsoLG-DNA adducts, e.g. what type(s) of
mutation results from the formation of such adducts, whether these
adducts are repaired, and if they are repaired, the rate at which this occurs.
The chemistry of the IsoLG reaction with amines is similar to other
-dicarbonyl compounds. Thus, information regarding the pharmacological toxicology of
-dicarbonyl compounds can provide a
basis for hypotheses regarding the potential biological ramifications of the formation of IsoLGs. For example, the toxicity of one such compound that has been extensively studied is the hexane metabolite, 2,5-hexanedione. 2,5-Hexanedione has been shown to cause axonal degeneration through formation of pyrrole adducts and subsequent cross-linking of neurofilaments (24). This suggests, therefore, that
similar effects may also result from the formation of IsoLGs during
oxidative neuronal injury. In this regard, we recently described (25)
the formation of IsoP-like compounds, termed neuroprostanes, in
vivo from free radical induced oxidation of docosahexaenoic acid
(C-22:6
T3), which is highly enriched in neurons in the brain. In
light of this finding, it is reasonable to speculate that reactive
IsoLG-like compounds are also generated as products of the
neuroprostane pathway, which in turn may induce neuronal injury.
Potentially highly relevant in this regard is our recent discovery that
levels of both neuroprostanes and IsoPs are significantly increased in
cerebrospinal fluid from patients with Alzheimer's disease compared
with age-matched control subjects (25, 26).
In summary, these studies have elucidated a novel class of extremely
reactive compounds, IsoLGs, that are formed as products of the IsoP
pathway. LGs exhibit a proclivity to adduct to proteins that far
exceeds that of any other known reactive product of lipid peroxidation.
The structural characterization of the nature of the lysyl IsoLG
adducts formed on proteins reported herein provides key information
that will allow us to explore the formation of IsoLGs in
vivo. This should afford ample new avenues for investigation to
elucidate the biological consequences of the formation of IsoLGs in
settings of oxidant injury.