From the Department of Pharmacology, School of
Medicine, and § Department of Chemistry, Vanderbilt
University, Nashville, Tennessee 37232
Received for publication, February 28, 2001
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ABSTRACT |
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The mechanism of formation of
4-hydroxy-2E-nonenal (4-HNE) has been a matter of debate
since it was discovered as a major cytotoxic product of lipid
peroxidation in 1980. Recent evidence points to
4-hydroperoxy-2E-nonenal (4-HPNE) as the immediate
precursor of 4-HNE (Lee, S. H., and Blair, I. A. (2000)
Chem. Res. Toxicol. 13, 698-702; Noordermeer, M. A.,
Feussner, I., Kolbe, A., Veldink, G. A., and Vliegenthart,
J. F. G. (2000) Biochem. Biophys. Res. Commun.
277, 112-116), and a pathway via 9-hydroperoxylinoleic acid and
3Z-nonenal is recognized in plant extracts. Using the 9- and 13-hydroperoxides of linoleic acid as starting material, we find
that two distinct mechanisms lead to the formation of 4-H(P)NE and the
corresponding 4-hydro(pero)xyalkenal that retains the original carboxyl
group (9-hydroperoxy-12-oxo-10E-dodecenoic acid). Chiral
analysis revealed that 4-HPNE formed from
13S-hydroperoxy-9Z,11E-octadecadienoic acid (13S-HPODE) retains >90% S
configuration, whereas it is nearly racemic from
9S-hydroperoxy-10E,12Z-octadecadienoic
acid (9S-HPODE). 9-Hydroperoxy-12-oxo-10E-dodecenoic acid is >90%
S when derived from 9S-HPODE and almost racemic
from 13S-HPODE. Through analysis of intermediates and
products, we provide evidence that (i) allylic hydrogen abstraction at
C-8 of 13S-HPODE leads to a 10,13-dihydroperoxide that
undergoes cleavage between C-9 and C-10 to give 4S-HPNE, whereas direct Hock cleavage of the 13S-HPODE gives
12-oxo-9Z-dodecenoic acid, which oxygenates to racemic
9-hydroperoxy-12-oxo-10E-dodecenoic acid; by contrast, (ii)
9S-HPODE cleaves directly to 3Z-nonenal as a
precursor of racemic 4-HPNE, whereas allylic hydrogen abstraction at
C-14 and oxygenation to a 9,12-dihydroperoxide leads to chiral 9S-hydroperoxy-12-oxo-10E-dodecenoic acid. Our
results distinguish two major pathways to the formation of 4-HNE that
should apply also to other fatty acid hydroperoxides. Slight
(~10%) differences in the observed chiralities from those predicted
in the above mechanisms suggest the existence of additional routes to
the 4-hydroxyalkenals.
The complex processes of lipid peroxidation result in the
formation of multiple products with the potential to interact and influence the outcome of normal cellular processes and control mechanisms. The pioneering work of Esterbauer and co-workers (1-3) on
the production of cytotoxic molecules in peroxidation reactions led to
the discovery of a group of conjugated aldehydes with toxic potential.
Within this group, the most abundant member was identified as
4-hydroxy-2E-nonenal
(4-HNE)1. In the ensuing
years, 4-HNE has achieved status as one of the best recognized and most
studied of the cytotoxic products of lipid peroxidation (4, 5). In
addition to studies on its bioactivity, 4-HNE is commonly used as a
biomarker for the occurrence and/or the extent of lipid peroxidation.
The reviews on the production of 4-HNE include its involvement in cell
cycle control (6), the oxidative alterations in Alzheimer's disease
(7, 8), and its participation in the formation of etheno DNA-base
adducts (9).
Despite the volumes of literature on the occurrence and activities of
4-HNE, there are comparatively few studies on how it is formed. It is
recognized that linoleic acid and arachidonic acid are among the
potential precursors for 4-HNE formation and that the nine carbons of
4-HNE are represented by the last nine carbons of these In the present work, we utilized both 9-HPODE and 13-HPODE as model
fatty acid hydroperoxides to study the mechanisms of nonenzymatic formation of the 4-hydroxyalkenals. Both fatty acid hydroperoxides give
rise to 4-H(P)NE, but each has differing rates and susceptibilities to
inhibition by Preparation of Hydroperoxides--
13S-HPODE was
synthesized from linoleic acid using soybean lipoxygenase (Sigma, Type
V) and purified by preparative SP-HPLC (Alltech Econosil silica,
1.0 × 25 cm, hexane/isopropanol/acetic acid 100:1.5:0.1 by volume
at 4 ml/min). 9S-HPODE was synthesized using a lipoxygenase
preparation from tomato fruit (15) and purified using the same SP-HPLC
conditions as above. The hydroperoxides were stored as a 5 mg/ml stock
solution in acetonitrile or methanol under argon at Autoxidation Reactions--
25-µg aliquots of the linoleic
acid hydroperoxides were transferred into 1.5-ml plastic tubes and
evaporated from the solvent under a stream of nitrogen. In some
experiments, Identification of Autoxidation Products--
4-HPNE was prepared
from an incubation of 1 mg of 9S-HPODE with a crude
bacterial lysate of a hydroperoxide lyase from melon fruit expressed in
Escherichia coli (16). 4-HPNE (retention time 5.7 min) was
isolated using a Waters Symmetry C18 5-µm column (0.46 × 25 cm)
eluted with a solvent of acetonitrile/water/acetic acid (60:40:0.01) at
a flow rate of 1 ml/min. The collected product was evaporated from
acetonitrile, extracted using a 100-mg C18 cartridge (Varian) eluted
with diethyl ether, and dried over Na2SO4. An
aliquot of the 4-HPNE was reduced with triphenylphosphine to 4-HNE,
repurified by RP-HPLC (retention time 4.6 min), and derivatized with
bis(trimethylsilyl)trifluoroacetamide to the trimethylsilyl ether derivative. GC-MS analysis yielded the following
diagnostic fragments: m/z 199 [M+
9-Hydroperoxy-12-oxo-10E-dodecenoic acid was prepared
from a 5-mg reaction of 13S-HPODE with an expressed and
purified recombinant hydroperoxide lyase from melon fruit in 25 ml of
50 mM Tris-HCl, pH 7.5 (16). After 5 min, the reaction was
terminated by adding 1 N HCl up to pH 4.5 and extracted
twice with 30 ml of ethyl acetate containing 250 µg
The 8,13-diHPODEs and 9,14-diHPODEs were isolated from a 5-mg
autoxidation of 13S-HPODE or 9S-HPODE,
respectively, by RP-HPLC (Beckman Ultrasphere ODS 10 µm, 1.0 × 25 cm eluted with a solvent of acetonitrile/water/acetic acid
(50:50:0.01) at a flow rate of 4 ml/min). For 1H NMR
analysis, the collected 8,13-diHPODEs were reduced with NaBH4, methylated, and further purified by SP-HPLC using a
Whatman Partisil 5-µm column (0.46 × 25 cm) and a solvent of
hexane/isopropanol/acetic acid (90:10:0.1 by volume) at a flow rate of
1 ml/min. The 1H NMR spectra were recorded in
C6D6 using residual benzene as internal
reference ( Quantification of 4-HPNE and
9-Hydroperxy-12-oxo-10E-dodecenoic acid--
4-HPNE and
9-hydroperoxy-12-oxo-10E-dodecenoic acid were quantified
using an external calibration curve obtained by injecting aliquots of
4-HNE (5-100 ng) on the RP-HPLC system used for product analysis and
plotting against peak height.
Chiral Resolution of 4-H(P)NE and
9-Hydro(pero)xy-12-oxo-10E-dodecenoic Acid--
100 µg of a racemic
standard of 4-HNE (Cayman Chemical, Ann Arbor, MI) were reacted with a
molar excess of methyl oxime hydrochloride in 20 µl of pyridine at
room temperature overnight. The solvent was evaporated, and the residue
was dissolved in 1 ml of methylene chloride and washed three times with
500 µl of water to remove residual reagent and pyridine. The 4-HNE
methoxime derivatives were separated on a Waters Symmetry C18 5-µm
column (0.46 × 25 cm) eluted with a solvent of
acetonitrile/water/acetic acid (50:50:0.01 by volume) at a flow rate of
1 ml/min and UV detection at 235 nm. The two isomers (syn
and anti) eluted at 10.4 and 11.2 min retention time,
respectively. The later eluting isomer was analyzed by chiral
phase HPLC using a Chiralpak AD (0.46 × 25 cm) column eluted with
hexane/ethanol (90:10 by volume) at a flow rate of 1 ml/min and
monitored using an HP 1040A diode array detector.
20 µg of the chemically synthesized
9-hydroperoxy-12-oxo-10E-dodecenoic acid were reduced with
triphenylphosphine and treated with methyl oxime hydrochloride in
pyridine overnight. The sample was extracted, washed, evaporated, and
dissolved in 20 µl of methanol. To this solution a few drops of
ethereal diazomethane were added, and the sample was evaporated
immediately. The syn- and anti-methoxime (MOX)
isomers (retention times of 10.0 and 10.8 min, respectively) were isolated from RP-HPLC (Waters Symmetry C18 5-µm column 0.46 × 25 cm) using acetonitrile/water/acetic acid (37.5:62.5:0.01 by
volume) as solvent at a flow rate of 1 ml/min. Chiral analysis of the
earlier eluting isomer was performed using the chiral phase HPLC
conditions described above.
From a 5-mg autoxidation of 13S-HPODE and
9S-HPODE (5 h at 37 °C), 4-HPNE and
9-hydroperoxy-12-oxo-10E-dodecenoic acid were isolated by
RP-HPLC using a Beckman Ultrasphere ODS 10-µm column (1.0 × 25 cm) eluted with a solvent of acetonitrile/water/acetic acid
(50:50:0.01) at a flow rate of 4 ml/min. The products were reduced with
an excess of triphenylphosphine and then further derivatized, purified,
and analyzed essentially as described for the racemic standards.
CD Spectroscopy--
The enantiomers of the racemic 4-HNE
methoxime derivative and
methyl-9-hydroxy-12-oxo-10E-dodecenoic acid methoxime
derivative were collected from the chiral phase HPLC separations. The
four products were evaporated from solvent under a stream of nitrogen and dissolved in 50 µl of dry acetonitrile. 1 µl of
1,8-diazabicyclo[5,4,0]undec-7-ene and a few grains of
1-(2-naphthoyl)imidazole (Fluka) were added. The reaction was kept at
room temperature overnight, and the solvent was evaporated. The residue
was dissolved in 1 ml of methylene chloride, washed three times with
water, and evaporated; finally the naphthoate derivatives were purified
by SP-HPLC using a Beckman Ultrasphere Si column (0.46 × 25 cm)
eluted with hexane/isopropanol/acetic acid (100:1:0.1 by volume) at a
flow rate of 1 ml/min. For UV and CD spectroscopy, the collected
products were evaporated from column solvent and dissolved in
acetonitrile to a final A239 nm of 1 absorbance unit (4-HNE derivatives) or 0.2 absorbance units (methyl-9-hydroxy-12-oxo-10E-dodecenoic acid derivatives).
CD spectra were recorded on a JASCO J-700 spectropolarimeter.
Time Course of Degradation of Linoleic Acid
Hydroperoxides--
The 13- and 9-linoleic acid hydroperoxides were
autoxidized in 25-µg aliquots as a dry film in open 1.5-ml plastic
tubes at 37 °C for 1, 2, or 4 h. At each time point column
solvent was added to the tubes, and the complete sample was injected on
RP-HPLC. The time course of the decay of 9- and 13-HPODE in the
presence and absence of RP-HPLC Analysis of Autoxidation Reactions--
The polar products
formed in the autoxidation reactions were analyzed by RP-HPLC (Fig.
2). In these chromatograms, the
autoxidations of 13S-HPODE (Fig. 2A) and
9S-HPODE (Fig. 2B) were analyzed after 1 h,
and the autoxidation of 13S-HPODE in the presence of 5%
(w/w) Identification of Products--
Compound 3 was identified as
4-HPNE based on identical UV spectra (
The UV spectra of compounds 1 and 3 (4-HPNE) were almost
indistinguishable, but compound 1 eluted at a much earlier retention time. The reduction of compound 1 with triphenylphosphine resulted in a
slightly more polar product on RP-HPLC and with a UV spectrum almost
identical to 4-HNE (Fig. 3). Based on the chromatographic and
spectroscopic data, compound 1 was suspected to be
9-hydroperoxy-12-oxo-10E-dodecenoic acid, a C-12 aldehyde
derivative that retains the original carboxyl group of the
starting fatty acid hydroperoxide. An authentic standard of
9-hydroperoxy-12-oxo-10E-dodecenoic acid was synthesized
through the following steps: (i) preparation of 13S-HPODE
from linoleic acid using soybean lipoxygenase, (ii) cleavage of the
hydroperoxide using a recombinant hydroperoxide lyase from melon fruit
(16), (iii) autoxidation of the 12-oxo-9Z-dodecenoic acid
cleavage product in the presence of
Compound 2 was identified by LC-MS and 1H NMR as
11-oxo-9Z-undecenoic acid (data not shown). This product is
not directly involved in the pathways leading to the formation of the
4-hydro(pero)xyalkenals. Further characterization of this product and
its mechanism of formation will be reported elsewhere.
As determined by the RP-HPLC analysis, compounds 4 and 5 (derived from
13S-HPODE; Fig. 2A) and 6 and 7 (derived from
9S-HPODE, Fig. 2B) were formed consistently in
the same relative amount to each other during the 4-h time period of
autoxidation. They showed identical UV spectra indicative of a
trans,trans conjugated diene (
Thus, analysis by LC-MS, GC-MS, UV spectroscopy, and 1H NMR
identified compounds 4 and 5 as
8,13-dihydroperoxyoctadeca-9E,11E-dienoic acids,
presumably a pair of diastereomers with the
8R,13S and 8S,13S
configurations. GC-MS analysis of the equivalent derivatives of
compounds 6 and 7 derived from 9S-HPODE showed major
fragments at m/z 459 [M Time Course of Formation of 4-HPNE and
9-Hydroperoxy-12-oxo-10E-dodecenoic Acid--
In Fig.
6 the time course of the formation of
4-HPNE (compound 3) and 9-hydroperoxy-12-oxo-10E-dodecenoic
acid (compound 1) during the autoxidation of 13S- and
9S-HPODE is shown. Starting with 25 µg of
13S-HPODE, ~100 ng of 4-HPNE are detected after 4 h
of autoxidation, whereas from the same amount of 9S-HPODE, less than half as much 4-HPNE is detected (Fig. 6A). In the
formation of 9-hydroperoxy-12-oxo-10E-dodecenoic acid, more
is generated from 9S-HPODE (~100 ng from 25 µg) than
from 13S-HPODE (~30 ng from 25 µg) (Fig.
6B).
Autoxidation of 13S-HPODE in the Presence of
Chiral Analysis of 4-HPNE--
To provide insights into the
mechanism(s) of formation of 4-HPNE, an HPLC method for the chiral
resolution of the more stable reduction product 4-HNE was developed.
Injection of underivatized 4-HNE on the chiral column used resulted in
the reaction of 4-HNE with the chiral stationary phase (Chiralpak AD).
Therefore, the aldehyde group was derivatized to the MOX derivative.
The resulting syn- and anti-isomers were readily
resolved using RP-HPLC. When the later eluting oxime isomer from the
RP-HPLC separation was injected on chiral phase HPLC, the enantiomers
in a commercial standard of 4-HNE were widely resolved with retention
times of 5.7 and 7.4 min (Fig.
7C). The earlier eluting MOX
isomer was also resolved into two enantiomers, but with less
resolution. The elution order of the enantiomers from the chiral phase
HPLC separation was determined using CD spectroscopy (see below).
From separate 5-mg autoxidations of 13S- and
9S-HPODE, the 4-HPNE product was isolated by RP-HPLC,
reduced with triphenylphosphine, and converted to the MOX
derivative. The syn- and anti-isomers of the MOX
derivative were resolved on RP-HPLC as described above for the racemic
standards. Fig. 7A shows the chiral phase HPLC elution
profile of the later eluting MOX isomer of 4-HNE derived from
13S-HPODE. Integration of the peak areas of the enantiomers gave a 90:10 ratio of 4S-HNE to 4R-HNE. Similar
chiral analysis of the 4-HPNE product from a 9S-HPODE
auto-oxidation showed that it was formed as a virtually racemic mixture
(54% S and 46% R; Fig. 7B).
Chiral Analysis of 9-Hydroperoxy-12-oxo-10E-dodecenoic
Acid--
A method for chiral analysis of
9-hydro(pero)xy-12-oxo-10E-dodecenoic acid was developed for
the methyl ester derivative along the same lines as for 4-HNE.
Injection of the earlier eluting methyl ester MOX isomer from RP-HPLC
resulted in the resolution into two enantiomers as shown in Fig.
8C (retention times of 9.4 and
12.9 min). When the 9-hydroperoxy-12-oxo-10E-dodecenoic acid derived from autoxidation of 5 mg of 13S-HPODE was analyzed
using this method, it was found to be an almost racemic mixture (53:47 S to R) (Fig. 8A). In contrast,
9-hydroperoxy-12-oxo-10E-dodecenoic acid was formed from
9S-HPODE in an S/R enantiomer ratio of
91:9 (Fig. 8B).
CD Spectroscopy--
The absolute configuration of the enantiomers
of the MOX-derivatized 4-HNE and
9-hydroxy-12-oxo-10E-dodecenoic acid was determined by CD
spectroscopy to determine the elution order from the chiral phase HPLC
separations (20). The enantiomers of both products were collected from
chiral phase HPLC, and the hydroxy group was derivatized with
2-naphthoyl-imidazole to introduce a second chromophore at the chiral
center as depicted in Fig. 9. The
exciton-coupled circular dichroism method of CD spectroscopy uses the
interaction of two chromophores at the chiral center to define the
absolute configuration. To delineate the absolute configuration of a
particular chiral molecule from the CD spectrum, the molecule is
represented in the Newman projection. If the chirality of the electric
transition moments of the first to the second chromophore is clockwise,
defined as positive, the CD shows a positive first and a
negative second Cotton effect; if the chirality is counter clockwise,
defined as negative, the CD shows a negative first and a positive
second Cotton effect.
The 2-naphthoate derivative of the first eluting enantiomer of the
MOX-derivatized 4-HNE from the chiral column resolution showed a
positive first Cotton effect at 244 nm (
In the case of 9-hydroxy-12-oxo-10E-dodecenoic acid, the
first eluting enantiomer from the chiral column showed a negative split
CD curve (extremum at 244 nm, Analysis of the stereochemistry of 4-hydroxyalkenals formed from
chiral hydroperoxides together with the detection of some unusual
dihydroperoxides of linoleic acid provide some valuable new insights
into the pathways of 4-H(P)NE formation. We found that the 4-HPNE
formed from 13S-HPODE largely retains the initial S configuration. This retention of configuration can be
explained by the mechanism outlined in Scheme 1. The initial event is
the abstraction of an allylic hydrogen at C-8 of 13S-HPODE.
This yields a radical that can be localized on C-8, C-10, or C-12.
Oxygenation at C-10 forms diastereomeric
10R,S,13S-dihydroperoxides. The
formation of these products entails a bis-allylic oxygenation that is
precedented in autoxidation (21) and in the soybean
lipoxygenase-catalyzed oxygenation of a synthetic substrate,
16,17-dehydro-arachidonic acid (22). The doubly allylic 10-hydroperoxy
group is unstable, and cleavage in a Hock rearrangement between C-9 and
C-10 yields two aldehyde fragments, 9-oxo-nonanic acid and
4S-HPNE. The Hock rearrangement, which is promoted by protic
and Lewis acids, occurs readily for hydroperoxides that have an
unsaturated unit attached to the carbon bearing the hydroperoxide group
(23, 24). Thus, benzylic, allylic, and dienylic hydroperoxides undergo
the rearrangement readily by migration of the unsaturated group from
carbon to oxygen while the weak O-O bond fragments. The initial
13S-hydroperoxy group of 13S-HPODE is not
directly involved in this reaction sequence, and therefore the
4S-HPNE cleavage product retains its absolute configuration.
The 9-hydroperoxy-12-oxo-10E-dodecenoic acid product
(compound 1) from 13S-HPODE is formed through an alternative
mechanism (Scheme 2). The first reaction
is the Hock cleavage of 13-HPODE between carbons 12 and 13 (24). This
yields the two aldehydes hexanal and 12-oxo-9Z-dodecenoic
acid. Neither compound was detected in our analyses, because hexanal is
volatile and virtually nondetectable in the UV, and
12-oxo-9Z-dodecenoic acid rapidly oxygenates to 9-hydroperoxy-12-oxo-10E-dodecenoic acid (13, 25). The
stereochemistry of the 9-hydroperoxy group is predicted to be racemic
according to this mechanism (Scheme 2). Chiral analysis of the product
from 13S-HPODE (Fig. 8) revealed that it is a 53:47 mixture
of the S to the R enantiomer, which is largely in
agreement with this mechanism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-6 essential
fatty acids. It was also reported in the early work (4) that
15-hydroperoxy-eicosatetraenoic acid is a precursor. In 1990, Porter and Pryor (10) presented a hypothesis paper that proposed
several mechanisms of 4-HNE formation involving the 4,5-epoxy
derivative as the intermediate. The first experimental evidence for a
pathway from fatty acid hydroperoxides to 4-HNE stemmed from the work
of Gardner and Hamberg (11) on the biosynthesis of 4-HNE in broad bean
extracts. They established that the aldehydic product of the reaction
of 9-hydroperoxylinoleic acid with hydroperoxide lyase, namely
3Z-nonenal, can be converted to
4-hydroperoxy-2E-nonenal (4-HPNE) by a reaction of molecular oxygen, mainly catalyzed in this case by a 3Z-alkenal
oxygenase. 4-HPNE is a simple reduction step removed from 4-HNE.
Gardner and Hamberg (11) also substantiated an additional route to
4-HNE via peroxygenase reactions utilizing the co-substrates
3Z-nonenal and 4-HPNE; the existence of a nonenzymatic
pathway was also implicated. Subsequent work by Gardner and Grove (12)
showed that 3Z-nonenal is a substrate for soybean
lipoxygenase, which thus could function as a 3Z-alkenal
oxygenase and that the product is 4-HPNE. More recently, Noordermeer
et al. (13) implicated nonenzymatic oxygenation of
3Z-alkenals (via 4-hydroperoxy intermediates) as the major pathway of production of 4-HNE and related 4-hydroxyalkenals in plant
extracts. 4-HPNE also has been detected as a nonenzymatic breakdown
product of 13-HPODE (14).
-tocopherol. The use of chiral starting materials and
analyses of stereochemistry of the products reveal a pathway from
-6
hydroperoxides (13-HPODE) to the 4-hydroxyalkenals and insights into
the stereochemistry of the nonenzymatic reactions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
-tocopherol from a stock solution in ethanol, 5 or 10%
(w/w), was added prior to evaporation. The tubes were placed in a
37 °C oven and removed after a 1-, 2-, or 4-h incubation. 30 µl of
column solvent was added, and the complete content was injected on an
RP-HPLC column (Waters Symmetry C18 5 µm, 0.46 × 25 cm) eluted
with a solvent of acetonitrile/water/acetic acid (60:40:0.01 by volume)
at a flow rate of 1 ml/min. The column effluent was monitored using an
HP 1040A diode array detector. For product identification and chiral
analyses, 5-mg aliquots of 13S- and 9S-HPODE were
autoxidized for 5 h at 37 °C.
CHO]; m/z 157 [CHO-C2H2-CH-OSi(CH3)3+];
and m/z 129 [CHO-C2H2-CH-OSi(CH3)3+
CO]. 1H NMR spectra of 4-HPNE were recorded in
CD3CN on a Bruker WM 400-MHz spectrometer using residual
CH3CN as an internal reference (
= 1.92 ppm): 9.58 ppm, d, J = 7.8 Hz, H1; 6.9 ppm, dd, J = 15.9 Hz, 6.2 Hz, H3; 6.25 ppm, ddd, J = 15.9 Hz, 7.8 Hz, 1.2 Hz, H2; 4.6 ppm, q, J ~ 6.5 Hz, H4.
-tocopherol.
The pooled organic phases were washed with water, dried over
Na2SO4, and evaporated under reduced pressure.
The crude mixture was kept under an atmosphere of oxygen at 37 °C
for 7 days. 9-Hydroxy-12-oxo-10E-dodecenoic acid and 9-hydroperoxy-12-oxo-10E-dodecenoic acid were isolated by
RP-HPLC using a Beckman Ultrasphere ODS column (1.0 × 25 cm)
eluted with acetonitrile/water/acetic acid (37.5:62.5:0.01 by volume)
at 4 ml/min (retention times of 5.4 and 7.3 min, respectively). The collected fractions were evaporated from acetonitrile, and the products were extracted using a 100-mg C18 cartridge (Varian) eluted
with ethyl acetate and dried over Na2SO4. For
the GC-MS analysis, 9-hydroperoxy-12-oxo-10E-dodecenoic acid
was reduced with triphenylphosphine, treated with methoxime
hydrochloride and ethereal diazomethane, and purified by RP-HPLC. The
syn- and anti-isomers gave essentially the same
fragment ions at m/z 240 [M+
OCH3], 114 [COC2H2CH=NOCH3]+,
and 86 [C2H2CH=NOCH3]+.
1H NMR spectra were recorded in CDCl3 on a
Bruker WM 400 MHz spectrometer using residual CHCl3 as
internal reference (
= 7.26 ppm).
9-Hydroperoxy-12-oxo-10E-dodecenoic acid: 9.61 ppm, d,
J = 7.7 Hz, H12; 6.79 ppm, dd, J = 15.9 Hz, 6.3 Hz, H10; 6.30 ppm, ddd, J = 15.9 Hz, 7.7/7.8
Hz, 1.0 Hz, H11; 4.65 ppm, dt, J = 6.2 Hz, 0.9 Hz, H9;
2.36 ppm, t, J = 7.4 Hz, H2.
9-Hydroxy-12-oxo-10E-dodecenoic acid: 9.59 ppm, d,
J = 7.8 Hz, H12; 6.82 ppm, dd, J = 15.7 Hz, 4.7 Hz, H10; 6.31 ppm, ddd, J = 15.7 Hz, 7.8 Hz,
1.4 Hz, H11; 4.43 ppm, m, 1H, H9.
= 7.24 ppm). Aliquots of the samples collected from
the initial RP-HPLC separation were used for analysis by LC-ESI-MS.
LC-coordination ion spray-MS of the Ag+ adduct ions of the
linoleic acid dihydroperoxides was performed on a triple-stage
quadrupole TSQ7000 instrument (Finnigan, San Jose, CA) using conditions
essentially as described (17). The HPLC parameters were: Beckman
Ultrasphere Si column (0.2 × 25 cm) eluted with
hexane/isopropanol/acetic acid (90:10:0.1 by volume) at a flow rate of
0.15 ml/min. A solution of AgBF4 in isopropanol (0.3 mM) was mixed to the column effluent before the ESI
interface using a syringe pump at a pump rate of 75 µl/min. For the
GC-MS analysis the pairs of diastereomers were collected from RP-HPLC, reduced with triphenylphosphine, methylated using ethereal
diazomethane, and further purified by SP-HPLC using a Beckman
Ultrasphere Si column (0.46 × 25 cm) eluted with
hexane/isopropanol/acetic acid (90:10:0.1 by volume) at 1 ml/min. The
collected products were hydrogenated using 5% palladium on alumina and
treated with bis(trimethylsilyl)trifluoroacetamide/pyridine. GC-MS was
performed on a Finnigan Incos 50 mass spectrometer connected to an
HP5890A gas chromatograph. For GC, an 8-m OV1701 column was used with a
temperature program starting at 150 °C (1 min isotherm) and a
rate of 15 °C/min to 300 °C (4 min isotherm).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-tocopherol is shown in Fig.
1. Over the course of 4 h at
37 °C the plain hydroperoxides are about 90% degraded, whereas in
the presence of
-tocopherol the degradation is slowed down to about
70-80% remaining hydroperoxides after 4 h.
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Fig. 1.
Time course of the degradation of linoleic
acid hydroperoxides in the presence and absence of
-tocopherol. 25-µg aliquots of the
hydroperoxides were autoxidized with or without 5% (w/w)
-tocopherol (
-toc.) at 37 °C and analyzed as
described under "Experimental Procedures." The remaining HPODEs are
expressed as a percentage of the starting amount (t = 0, set at
100%).
-tocopherol was analyzed after 4 h (Fig. 2C).
The polar products with distinctive UV chromophores were designated as
1-7. Compounds 1 and 3 were formed from both hydroperoxides. Compounds
2, 4, and 5 were products of the 13-hydroperoxide, and 6 and 7 were products from 9-HPODE. The arrows in Fig. 2 indicate the
retention time of 4-HNE, detected only as a minor product from
13S- and 9S-HPODE in these experiments (<5 ng/25
µg HPODE). Over the course of the 4-h period of autoxidation, no
additional abundant products were formed, and there were only minor
changes in the product pattern.
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Fig. 2.
RP-HPLC analysis of the autoxidation
reactions of linoleic acid hydroperoxides. A,
autoxidation of 13S-HPODE. B, autoxidation of
9S-HPODE. C, autoxidation of 13S-HPODE
in the presence of 5% (w/w) -tocopherol (
-toc.).
25-µg aliquots of the hydroperoxides were auto-oxidized at 37 °C
for 1 h (A and B) or 4 h
(C), and the complete reaction mixture was analyzed on
RP-HPLC (Waters Symmetry C18 5-µm, 0.46 × 25 cm,
acetonitrile/water/acetic acid 60:40:0.01 by volume, at a flow rate of
1 ml/min). The column effluent was monitored using an HP 1040A diode
array detector. The chromatograms shown were recorded at 220 nm (full
scale absorbance, 25 milliabsorbance units).
max 223 nm in
RP-HPLC column solvent; Fig. 3) and
co-chromatography with a synthesized standard. Furthermore, treatment
of compound 3 with triphenylphosphine yielded a product that
co-chromatographed on RP-HPLC with authentic 4-HNE.
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Fig. 3.
UV spectra of 4-HNE, compound 3 (4-HPNE), compound 4 (8,13-diHPODE), and
13S-HPODE. The UV spectra were recorded in column
solvent (acetonitrile/water/acetic acid 60:40:0.01 by volume) using an
HP 1040A diode array detector. The spectra are normalized to
max.
-tocopherol, and (iv) isolation
of the 9-hydroperoxy-12-oxo-10E-dodecenoic acid by RP-HPLC.
The identification of compound 3 as
9-hydroperoxy-12-oxo-10E-dodecenoic acid was confirmed
by 1H NMR and by GC-MS analysis of the
triphenylphosphine-reduced methyl ester methoxime derivative.
max
231 nm; Fig. 3) (18), giving the strong implication that these products
were pairs of diastereomers. LC-ESI-MS analysis of the
Ag+-adduct ion revealed two [M + Ag+] adduct
ions at m/z 451 and 453 for compounds 4, 5, 6, and 7 (Fig. 4). This corresponds to a
molecular weight of 344, which is compatible with linoleic acid
dihydroperoxides. To reveal the position of the hydroperoxide groups on
the fatty acid carbon chain, GC-MS analysis (electron impact
mode) was performed on the reduced, methylated, and hydrogenated
bis-trimethylsilyl-ether derivatives. The mass spectra of
derivatized compounds 4 and 5 (derived from 13S-HPODE)
showed characteristic ions at m/z 459 [M
CH3]+, 245 [CH3CO2 C8H13OSi(CH3)3]+
and 331 [HCOSi(CH3)3
C5H9OSi(CH3)3 C5H11]+
(indicating the C-8 hydroxyl), and 403 [CH3CO2C8 H13OSi(CH3)3C5H9OSi(CH3)3]+
and 173 [HCOSi(CH3)3 C5H11]+
(indicating the C-13 hydroxyl) (Fig.
5A). Finally, 1H
NMR of the reduced compounds 4 and 5 (8,13-dihydroxyoctadecadienoates) fully supported the structures. 1H NMR (400 MHz, in
deuterated benzene using 7.24 ppm for the residual protons in the
solvent) gave for the methyl ester of the
8,13-dihydroxyoctadecadienoate derivative of compound 4:
(ppm)
0.94, t, 3 protons, H18; 0.98 (d, two protons, -OH,
J8,-OH = J13,-OH = 3.5 Hz); 1.2-1.8 (m, 18 protons, H3-H7, and H14-H17); 2.14 (t, 2 protons, H2); 4.00 (m, 2 protons, H8, H13); 5.67 (m, 2 protons, H9, H12); and 6.24 (m, 2 protons, H10, H11). These data confirm the symmetry of the 1,6-dihydroxy-2,4-diene system; the olefinic region shows two complex
multiplets, each comprised of two superimposed protons (H10/H11 at 6.24 ppm and H9/H10 at 5.67 ppm), and similarly a superimposed signal for
the two geminal hydroxy protons (H8, H13 at 4.00 ppm). The fact that
these signals consist of overlapping pairs of protons of identical
chemical shift results in nonlinear effects that precluded a ready
assignment of the coupling constant across the double bonds. For
example, decoupling of the signal for H9/H12 at 5.67 ppm caused the
downfield signal for the internal pair of olefinic protons to simplify
to a singlet (rather than a doublet), a predictable result based on the
lack of coupling between the two superimposed proton signals for
H10/H11 (19). Despite their complexity, the 1H NMR spectra
were entirely supportive of the structures deduced from the UV and MS
data.
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Fig. 4.
LC-coordination ion spray
(Ag+) mass spectrum of
8,13-dihydroperoxy-9E,11E-octadecadienoic
acid (compound 5). Compound 5 was isolated from the autoxidation
of 5 mg of 13S-HPODE by RP-HPLC and analyzed by LC-CSI-MS
with AgBF4 using the conditions described under
"Experimental Procedures."
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Fig. 5.
Mass spectra (electron impact mode) of
8,13-dihydroperoxy-9E,11E-octadecadienoic
acid (A) and
9,14-dihydroperoxy-10E,12E-octadecadienoic
acid (B) of the reduced, methylated, and hydrogenated
trimethylsilyl ether derivative. A, mass spectrum of
compound 5 isolated from the autoxidation of 13S-HPODE after
reduction, hydrogenation, and derivatization with diazomethane and
bis(trimethylsilyl)trifluoroacetamide. B, mass spectrum of
compound 7 isolated from the autoxidation of 9S-HPODE after
similar derivatization.
CH3]+, 259 [CH3CO2C9H15OSi(CH3)3]+
and 317 [HCOSi(CH3)3C5H9OSi(CH3)3C4H9]+
(indicating the C-9 hydroxyl), and 417 [CH3CO2C9H15OSi(CH3)3C5H9OSi(CH3)3]+
and 159 [HCOSi(CH3)3C4H9]+
(indicating the C-14 hydroxyl) (Fig. 5B). Compounds 6 and 7 were thus identified as
9,14-dihydroperoxyoctadeca-10E,12E-dienoic acids,
also presumably a pair of diastereomers with the
9S,14S and 9S,14R configuration.
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Fig. 6.
Time course of the formation of 4-HPNE and
9-hydroperoxy-12-oxo-10E-dodecenoic acid from
13S-HPODE (A) and
9S-HPODE (B). 25-µg aliquots
of the hydroperoxides were autoxidized at 37 °C for the time
indicated and analyzed as described under "Experimental
Procedures." 4-HPNE and
9-hydroperoxy-12-oxo-10E-dodecenoic acid were quantified
based on external calibration using 4-HNE as a standard.
-Tocopherol--
Autoxidations of 25-µg aliquots of
13S-HPODE as a dry film were carried out in the presence of
5%
-tocopherol for 1, 2, and 4 h. As shown in Fig. 1, the rate
of decay of the hydroperoxide is decreased in the presence of
-tocopherol. A representative chromatogram obtained after 4-h
autoxidation at 37 °C shows the formation of compound 1 (9-hydroperoxy-12-oxo-10E-dodecenoic acid) as the major
product with absorbance at 220 nm (Fig. 2C). 4-HPNE (compound 3) and the UV-absorbing conjugated diHPODEs are only minor
products in these experiments.
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Fig. 7.
Chiral phase HPLC analysis of 4-H(P)NE
(methoxime derivative) derived from 13S-HPODE
(A) and 9S-HPODE
(B). 4-HPNE isolated from autoxidations was
reduced with triphenylphosphine and derivatized and purified by RP-HPLC
as described under "Experimental Procedures." A,
analysis of 4-HPNE isolated from the autoxidation of 5 mg of
13S-HPODE. B, analysis of 4-HPNE isolated from
the autoxidation of 5 mg of 9S-HPODE. C, racemic
reference of 4-HNE. Chiral resolution was performed on a Chiralpak AD
column (0.46 × 25 cm) eluted with hexane/ethanol (90:10 by
volume) at a flow rate of 1 ml/min and UV detection at 235 nm. The
elution order was determined by CD spectroscopy of the collected
enantiomers.
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Fig. 8.
Chiral phase HPLC analysis of
9-hydroxy-12-oxo-10E-dodecenoic acid methyl ester
(methoxime derivative) derived from 13S-HPODE
(A) and 9S-HPODE
(B).
9-Hydroperoxy-12-oxo-10E-dodecenoic acid isolated from
autoxidations was reduced with triphenylphosphine and derivatized and
purified by RP-HPLC as described under "Experimental Procedures."
A, analysis of
9-hydroperoxy-12-oxo-10E-dodecenoic acid isolated from the
autoxidation of 5 mg of 13S-HPODE. B, analysis of
9-hydroperoxy-12-oxo-10E-dodecenoic acid isolated from the
autoxidation of 5 mg of 9S-HPODE. C, racemic
reference of 9-hydroxy-12-oxo-10E-dodecenoic acid. Chiral
resolution was performed on a Chiralpak AD column (0.46 × 25 cm)
eluted with hexane/ethanol (90:10 by volume) at a flow rate of 1 ml/min
and UV detection at 235 nm. The elution order was determined by CD
spectroscopy of the collected enantiomers.
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Fig. 9.
Derivatization of the 4-HNE methoxime
derivative to the 2-naphthoate. The chromophoric derivatives for
CD spectroscopy were synthesized by derivatization of the enantiomers
of methoxime 4-HNE, collected from the chiral phase separation, with
1-(2-naphthoyl)imidazole. The sign of the Cotton effects of the two
enantiomers can be predicted from the left- ( ) and right-handed (+)
sense between the transition moments (thick black line) of
the chromophores as indicated by the curved arrows. In this
case, the S enantiomer of the derivatized 4-HNE has positive
chirality.
, +32.4) and a negative
second Cotton effect at 226 nm (
,
27.0) (Fig.
10). Thus, the CD spectrum of this
enantiomer has a clockwise (positive) sense between the transition
moments of the two chromophores as depicted in the projection in Fig.
9; therefore its absolute configuration is S. The later eluting enantiomer exhibited a mirror-image
CD spectrum with extrema at 244 nm (
,
33.1) and 226 nm (
,
+27.0), resulting in a negative chirality; the absolute configuration is R (Fig. 10).
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Fig. 10.
CD and UV spectra of R- and
S-4-HNE (2-naphthoate, methoxime derivative) in
acetonitrile. The enantiomers of the methoxime derivative of 4-HNE
were resolved using chiral phase HPLC and further derivatized to the
2-naphthoates (Fig. 9 and "Experimental Procedures"). The CD
spectrum of the first eluting enantiomer from the chiral phase
separation of methoxime 4-HNE has a positive first and a negative
second Cotton effect (solid line); therefore it has positive
chirality (S configuration; Fig. 9) (20).
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Scheme 1.
,
3.4) which defines R configuration for this enantiomer. Accordingly, the second enantiomer had a positive split CD curve (extrema at 244 nm,
, +3.2, and at
225 nm,
,
1.7), which reveals S configuration.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Scheme 2.
The reactions and products observed upon autoxidation of
9S-HPODE were mechanistically equivalent. In an initial Hock
rearrangement, 9S-HPODE is cleaved into 9-oxo-nonanic acid
and 3Z-nonenal. 3Z-Nonenal is very rapidly
oxygenated to 4-HPNE, which in this case is formed as a racemic mixture
(Fig. 7B and Scheme
3A). In plants, the cleavage of the 9S-hydroperoxide is catalyzed by a hydroperoxide
lyase (26), and the subsequent nonenzymatic oxygenation of
3Z-nonenal has been described as the source for the
formation of 4-HNE in plant tissue (13). There is also, however, an
initial allylic hydrogen abstraction at C-14, which is analogous to the
H abstraction at C-8 of 13-HPODE. The resulting radical rearranges and
can be oxygenated at different positions (i.e. on carbons
10, 12, and 14). Hock cleavage of the 9S,12-dihydroperoxide
yields 9S-hydroperoxy-12-oxo-10E-dodecenoic acid
and hexanal (Scheme 3B). The predicted S
configuration of the 9-hydroperoxy group was confirmed by chiral phase
analysis (Fig. 8B).
|
An initial hydrogen abstraction at C-8 of 13S-HPODE, as
postulated in Scheme 1, predicts the formation of positional isomers of
diastereomeric linoleic acid dihydroperoxides. We identified a pair of
diastereomeric 8,13-dihydroperoxides, providing evidence of the C-8
hydrogen abstraction. The corresponding 8,13-dihydroxy derivatives of
linoleic acid have been characterized previously as minor end products
of the heme-catalyzed degradation of 13S-HPODE (27). These
dihydroxy derivatives were formed via synthesis of a leukotriene type
of allylic epoxide (12,13-epoxy-8,10-octadecadienoic acid) that
hydrolyzed to the 8,13-dihydroxides. This mechanism is quite distinct
from the route to the 8,13-dihydroperoxides that were the major
products under the conditions used in our study. The fact that these
8,13-dihydroperoxides have a trans,trans conjugated diene also provides strong circumstantial evidence for
oxygenation at the C-10 position. Such a reaction at C-10 is required
to account for the change in configuration of the original 9,10 cis double bond to the trans configuration found in the 8,13-dihydroperoxides (Scheme 4)
(28, 29). The change in stereochemistry is allowed by the occurrence of
a peroxyl radical at C-10. This permits rotation around the 9,10 bond.
The subsequent loss of O2 and formation again of the carbon
radical aligns the carbon backbone in the more stable
trans,trans configuration. Oxygenation at C-8 and
formation of the hydroperoxide gives the stable 8,13-dihydroperoxide
diastereomers that we isolated and identified (Scheme 4). The
equivalent reactions occurred starting with 9S-HPODE,
resulting in characterization of the two 9,14-dihydroperoxides by LC-MS
and GC-MS.
|
The presence of -tocopherol during the autoxidation of
13S-HPODE caused a slowing in the loss of this substrate and
a noticeable change in the pattern of products. As an antioxidant,
-tocopherol intercepts peroxyl radicals and in the process forms an
-tocopheroxyl radical and the hydroperoxide. This accounts for the
slowing of the disappearance of the 13S-HPODE starting
material. The tocopheroxyl radical becomes the dominant free radical
chain carrier, but it is not a sufficiently strong oxidant to abstract
an allylic hydrogen at C-8 of 13-HPODE (30). This results in a
selective absence of the dihydroperoxides discussed above and of the
4-HPNE cleavage product. On the other hand, direct Hock cleavage of the
13S-HPODE can still occur, providing
12-oxo-9Z-dodecenoic acid. The tocopheroxyl radical can
abstract a doubly allylic hydrogen from C-11 of this intermediate, thus
forming racemic 9-hydroperoxy-12-oxo-10E-dodecenoic acid as
the major polar product of 13S-HPODE detected in the
presence of
-tocopherol.
Interesting small deviations from the stereochemistries predicted in
the mechanisms in Schemes 1 and 3 point to the existence of additional
routes to the 4-hydroxyalkenals. We found that the 4-HPNE formed from
13S-HPODE was significantly less than 100% S in
stereochemistry. The hydroperoxide starting materials were 98%
S configuration, so ~10% of the R enantiomer
was formed during the reaction by a pathway that is yet to be
elucidated. Initial 9/13 hydroperoxide isomerizations occurring prior
to chain cleavage may contribute to the product profiles. It was
apparent also that the 4-HPNE formed from the 9S-HPODE was
not racemic, but rather it showed a slight preference of the
S enantiomer. In this case there must be some transfer of
chirality from the 9S starting material to the 4-hydroperoxy
product. The same consideration holds true for the formation of the
9-hydroperoxy-12-oxo-10E-dodecenoic acids; they showed
similar small deviations from the predicted chiralities.
In conclusion, we provide evidence for at least two independent
mechanisms leading from isomeric -6 fatty acid hydroperoxides to
4-H(P)NE. Here, we used the two isomeric linoleic acid hydroperoxides as model compounds. Analogous reactions are to be expected with hydroperoxides from other
-6 fatty acids, especially arachidonic acid. With arachidonic acid, 11- and 15-hydroperoxy-eicosatetraenoic acid are the precursors to form 4-HPNE via the analogous mechanisms. The chiral analysis method of 4-HNE we developed here should be useful
in further studies to investigate the mechanism of 4-hydroxyalkenal synthesis. For example, the possibility of an involvement of enzymes such as lipoxygenases or cytochrome P450s could result in biosynthesis of chiral 4-HNE and its analogues. A potential enzyme-initiated pathway
to 4-HNE becomes increasingly important as physiological activities of
4-HNE in the sub-micromolar range are uncovered.
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ACKNOWLEDGEMENTS |
---|
We thank Markus Voehler for expert help with the NMR analyses and William Boeglin for help with the HPLC analyses.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants GM-53638 and GM-15431.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt University School of Medicine, 23rd Ave. at Pierce, Nashville, TN 37232-6602. Tel.: 615-343-4495; Fax: 615-322-4707; E-mail: alan.brash@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.M101821200
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ABBREVIATIONS |
---|
The abbreviations used are: 4-HNE, 4-hydroxy-2E-nonenal; 4-HPNE, 4-hydroperoxy-2E-nonenal; HPODE, hydroperoxy-octadecadienoic acid; 13S-HPODE, 13S-hydroperoxy-9Z,11E-octadecadienoic acid; 9S-HPODE, 9S-hydroperoxy-10E,12Z-octadecadienoic acid; SP, straight phase; HPLC, high pressure liquid chromatography; RP, reverse phase; GC, gas chromatography; MS, mass spectrometry; ESI, electrospray ionization; LC, liquid chromatography; MOX, methoxime; CD, circular dichroism.
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REFERENCES |
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