Manganese Lipoxygenase
DISCOVERY OF A BIS-ALLYLIC HYDROPEROXIDE AS PRODUCT AND
INTERMEDIATE IN A LIPOXYGENASE REACTION*
Mats
Hamberg
§,
Chao
Su¶, and
Ernst
Oliw¶
From the
Division of Physiological Chemistry II,
Department of Medical Biochemistry and Biophysics, Karolinska
Institutet, S-171 77 Stockholm and the ¶ Division of Biochemical
Pharmacology, Department of of Pharmaceutical Biosciences, Uppsala
Biomedical Center, Uppsala University, S-751 24 Uppsala, Sweden
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ABSTRACT |
Linoleic acid was incubated with manganese
lipoxygenase (Mn-LO) from the fungus Gäumannomyces
graminis. The product consisted of
(13R)-hydroperoxy-(9Z,11E)-octadecadienoic
acid ((13R)-HPOD) and a new hydroperoxide,
(11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD). Incubation of
(11R)-[2H]- and
(11S)-[2H]linoleic acids with Mn-LO led to
the formation of hydroperoxides that largely retained and lost,
respectively, the deuterium label. Conversion of the
(11S)-deuteriolinoleic acid was accompanied by a primary
isotope effect, which manifested itself in a strongly reduced rate of
formation of hydroperoxides and in a time-dependent accumulation of deuterium in the unconverted substrate. These experiments indicated that the initial step catalyzed by Mn-LO consisted of abstraction of the pro-S hydrogen of linoleic
acid to produce a linoleoyl radical. (11S)-HPOD was
converted into (13R)-HPOD upon incubation with Mn-LO.
The mechanism of this enzyme-catalyzed hydroperoxide rearrangement
was studied in experiments carried out with
18O2 gas or
18O2-labeled hydroperoxides. Incubation of
[11-18O2](11S)-HPOD with Mn-LO
led to the formation of (13R)-HPOD, which retained 39-44%
of the 18O label, whereas (11S)-HPOD incubated
with Mn-LO under 18O2 produced
(13R)-HPOD, which had incorporated 57% of 18O.
Furthermore, analysis of the isotope content of (11S)-HPOD remaining unconverted in such incubations demonstrated that
[11-18O2](11S)-HPOD suffered a
time-dependent loss of 18O when exposed to
Mn-LO, whereas (11S)-HPOD incorporated 18O when
incubated with Mn-LO under 18O2. On the basis
of these experiments, it was proposed that the conversion of
(11S)-HPOD into (13R)-HPOD occurred in a
non-concerted way by deoxygenation into a linoleoyl radical. Subsequent
reoxygenation of this intermediate by dioxygen attack at C-13 produced
(13R)-HPOD, whereas attack at C-11 regenerated
(11S)-HPOD. The hydroperoxide rearrangement occurred by
oxygen rebound, although, as demonstrated by the 18O
experiments, the oxygen molecule released from (11S)-HPOD
exchanged with surrounding molecular oxygen prior to its
reincorporation.
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INTRODUCTION |
Lipoxygenase-catalyzed dioxygenation of polyunsaturated
fatty acids leads to the formation of reactive fatty acid
hydroperoxides. Mammalian lipoxygenases can catalyze oxygenation at
carbons 5, 8, 12, and 15 of their predominant substrate,
i.e. arachidonic acid (1). Many plant lipoxygenases can also
utilize arachidonic acid, although their most important substrates are
the C-18 fatty acids linoleic acid and
-linolenic acid (2). Interest
in lipoxygenases stems partly from the fact that fatty acid
hydroperoxides can be further metabolized into biologically active
oxylipins such as leukotrienes and jasmonates.
Lipoxygenases contain ferrous iron, which is oxidized into the ferric
state by, e.g., hydroperoxides. The ferric form of
lipoxygenases is catalytically active (3) and catalyzes the
stereospecific abstraction of one hydrogen from the bis-allylic
methylene group of the (1Z,4Z)-pentadiene
structure of the substrate as the initial step (4). Attack by dioxygen
at one of the terminal positions of the resulting pentadienyl radical
results in the formation of a hydroperoxide having one pair of
E/Z-conjugated double bonds. Studies of the
regio- and stereochemistry of the steps occurring in the oxygenation of
8,11,14-eicosatrienoic acid by soybean lipoxygenase-1 revealed that the
pro-S hydrogen was stereospecifically removed from C-13 and
that dioxygen was regio- and stereospecifically inserted at C-15 to
produce a (15S)-hydroperoxide (4). This finding, and results
of similar studies carried out with linoleic acid
(9S)-lipoxygenase from corn (5), arachidonic acid
(12S)-lipoxygenases from human platelets (6) and a red alga
(7), arachidonic acid (5S)-lipoxygenases from rat basophil
leukemia cells and potato (8), and arachidonic acid
(8S)-lipoxygenase from mouse epidermis (9) indicated the
existence of an antarafacial relationship between hydrogen abstraction
and oxygen insertion as a common feature of dioxygenations catalyzed by
lipoxygenases. Interestingly, such a steric relationship has also been
found for the dioxygenation catalyzed by an "R"
lipoxygenase, i.e. (12R)-lipoxygenase from sea
urchin (10), as well as for dioxygenations catalyzed by prostaglandin
endoperoxide synthases I (11) and II (12), and by ferrylmyoglobin (13).
Mammalian and plant lipoxygenases so far studied catalyze production of
hydroperoxides that have the "S" absolute configuration.
In contrast, a number of marine invertebrates, such as starfish, sea
urchin, and the coral Plexaura homomalla, express
lipoxygenases, which catalyze formation of "R"
hydroperoxides, as demonstrated by the configuration of hydroperoxides
formed by oxygenation of arachidonic acid at the C-5, C-8, C-11, and C-12 positions. One of these enzymes, arachidonic acid
(8R)-lipoxygenase, was recently cloned and sequenced (14).
Manganese lipoxygenase
(Mn-LO)1 was purified from
the fungus Gäumannomyces graminis as described in the
accompanying paper (15). The protein was found to be heavily
glycosylated and to contain 0.5-1 atom of manganese per molecule. The
molecular mass of the native protein (~135 kDa) was comparable to
that of arachidonic acid (15S)-lipoxygenase purified from
the fungus Saprolegnia parasitica (145-150 kDa) (16).
Interestingly, the molecular mass of the deglycosylated Mn-LO (~73
kDa) (15) was similar to those of mammalian lipoxygenases. The present
paper is concerned with the stereochemistry and mechanism of the
Mn-LO-catalyzed dioxygenation. In the course of the work, it was
unexpectedly found that the enzyme catalyzes bis-allylic oxygenation of
polyunsaturated fatty acids. Thus, oxygenation of linoleic acid
resulted in the formation of 11-hydroperoxylinoleic acid, a new member
of the oxylipin family of compounds.
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EXPERIMENTAL PROCEDURES |
Materials--
[1-14C]Linoleic acid was purchased
from NEN Life Science Products. Dilution with unlabeled material
(Nu-Chek-Prep, Elysian, MN) followed by purification by
SiO2 chromatography afforded a specimen having a specific
radioactivity of 5.0 kBq/µmol.
(11R)-[2H]Linoleic acid (37.2% monodeuterated
and 62.8% undeuterated molecules) and
(11S)-[2H]linoleic acid (32.6% monodeuterated
and 67.4% undeuterated molecules) were prepared by lithium aluminum
deuteride reduction of the p-tolylsulfonyl derivatives of
methyl (11S)- and (11R)-hydroxystearates,
respectively, followed by biological desaturation of the resulting
stereospecifically deuterated stearic acids as described in detail
elsewhere (17, 18). The optical purities of the chiral
11-hydroxystearates were: (11R)-hydroxystearate (precursor
of (11S)-[2H]linoleic acid), 98.3%
R; (11S)-hydroxystearate (precursor of (11R)-[2H]linoleic acid), 95.4% S.
(9S)-HPOD and (13S)-HPOD were prepared by
incubation of linoleic acid with tomato lipoxygenase (19) and soybean
lipoxygenase (20), respectively. The corresponding alcohols,
(9S)-HOD and (13S)-HOD, were obtained from the
hydroperoxides by reduction with sodium borohydride.
(11R)-HOD and 11-ketolinoleic acid were prepared by
incubation of linoleic acid with an enzyme preparation of the red alga
Lithothamnion corallioides (21). 18O2, 99.4 atom %, was obtained from Isotec,
Miamisburg, OH. Glutathione peroxidase, reduced glutathione, and
soybean lipoxygenase were purchased from Sigma. Mn-LO was prepared as
described in the accompanying paper (15). Solutions of the enzyme (22 µg/ml) could be stored at 4 °C for several weeks without
appreciable loss of activity.
Geometrical Isomers of Methyl
11-Hydroxy-9,12-octadecadienoate--
The methyl ester of
(11R,11S)-HOD (i.e. the natural
(9Z,12Z)-isomer) was prepared by treatment of
linoleic acid (1 mM) with myoglobin (100 µM)
and cumene hydroperoxide (3 mM) at 0 °C as recently
described (22). The methyl ester of
(9E,12E)-(11R,11S)-HOD was
prepared in an analogous way starting with
(9E,12E)-octadecadienoic acid, although the poor
solubility of this acid in aqueous buffer made it necessary to carry
out the hydroxylation at 23 °C. A mixture of
(9E,12Z)- and
(9Z,12E)-octadecadienoic acids was obtained from a commercially available mixture of the four geometrical isomers of
9,12-octadecadienoic acid (Sigma) using RP-HPLC. Treatment of the acids
with myoglobin/cumene hydroperoxide at 0 °C followed by
esterification and isolation by RP-HPLC afforded a mixture of
comparable amounts of the methyl esters of
(9E,12Z)-(11R,11S)-HOD and
(9Z,12E)-(11R,11S)-HOD; UV
spectrum (methanol), featureless in the region 210-340 nm; FT-IR
spectrum (film), absorption bands inter alia at 3200-3620
cm
1 (hydroxyl), 1742 cm
1 (ester carbonyl),
and 968 cm
1 (E double bond). The four isomeric
methyl 11-hydroxy-9,12-octadecadienoates were separable as their
Me3Si derivatives by GLC, i.e. derivative of
(11R,11S)-HOD, C-19.41; derivative of
(9E,12E)-(11R,11S)-HOD, C-19.62; and derivatives of
(9E,12Z)-(11R,11S)-HOD and
(9Z,12E)-(11R,11S)-HOD, C-19.48 and C-19.56 (these two isomers have not yet been individually correlated with the two C-values). The mass spectra of the four isomers
were similar and showed strong ions at m/z 382 (M), 311 (M
71; loss of
(CH2)4-CH3), and 225 ([CH=CH-CH(OSiMe3)-CH=CH-(CH2)4-CH3]+).
Incubations--
Most incubations were carried out by stirring
[1-14C]linoleic acid (50-700 µM) or
[1-14C](11S)-HPOD (40-60 µM)
with 0.5-2.2 µg of Mn-LO at 23 °C in 0.5-2.1 ml of one of the
following buffers: 0.1 M potassium phosphate buffer, pH 7.4 (buffer A); 0.1 M sodium borate buffer, pH 10.0 (buffer B);
or 10 mM triethanolamine acetate, 1 mM EDTA, 2 mM sodium azide, 0.04% Tween 20, pH 7.0 (buffer C). An
aliquot of the reaction mixture was transferred to a cuvette (path
length, 1 or 10 mm; temperature, 23 °C) and the reaction progress
was followed by monitoring the absorbance at 235 nm versus
time. Incubations under 18O2 were conducted at
23 °C in a reaction vessel connected to a high vacuum line and a
supply of argon. Buffer A (2 ml) and Mn-LO were added to the reaction
vessel, and the solution was de-aerated at 0 °C by five cycles of
vacuum and argon purging prior to introduction of
18O2 gas and substrate. Isolation of reaction
products was performed by extraction with diethyl ether at pH 5, followed by RP-HPLC.
Chemical Methods--
Preparation and purification of
(
)-menthoxycarbonyl (MC) derivatives (23), and steric analysis of
9-HOD and 13-HOD (23) and of 11-HOD (21) were performed as indicated.
Catalytic hydrogenation was performed with platinum catalyst (3 mg)
using methanol (1 ml) as the solvent. Partial hydrogenation of the
methyl ester of 11-HOD (25 µg) was carried out by stirring with 5%
palladium-on-calcium carbonate (5 mg) in ethyl acetate (3 ml) under
hydrogen gas for 4 min (cf. Ref. 21). Oxidative ozonolysis
was carried out as described (23) using an ozone generator (model T-12)
purchased from TriO3 Industries, Fort Pierce, FL.
Chromatographic and Instrumental Methods--
RP-HPLC was
performed with a column of Nucleosil 100-5 C18 (250 × 4.6 mm) purchased from Macherey-Nagel (Düren, Germany). The
solvent system used consisted of acetonitrile/water/2 M
hydrochloric acid (60:40:0.02, v/v/v). Straight phase high performance
liquid chromatography was carried out with a column of Nucleosil 50-5 (200 × 4.6 mm) and a solvent system of 2-propanol/hexane (1:99, v/v). The absorbance (217 nm) and radioactivity of high performance liquid chromatography effluents were determined on-line using a
Spectromonitor III ultraviolet detector (Laboratory Data Control, Riviera Beach, FL) and a liquid scintillation counter (IN/US Systems, Tampa, FL), respectively. GLC was performed with a Hewlett-Packard (Avondale, PA) model 5890 gas chromatograph equipped with a methyl silicone capillary column (length, 25 m; film thickness, 0.33 µm). Helium at a flow rate of 25 cm/s was used as the carrier gas.
Retention times were converted into C-values using standards of
saturated fatty acid methyl esters (24). GC-MS was carried out with a
Hewlett-Packard model 5970B mass selective detector connected to a
Hewlett-Packard model 5890 gas chromatograph. LC-MS was performed as
described in the accompanying paper (15). Ultraviolet absorption as a
function of wavelength or time was recorded with a Hitachi (Tokyo,
Japan) model U-2000 UV-visible spectrophotometer. Infrared spectrometry
was carried out using a Perkin-Elmer model 1650 FT-IR
spectrophotometer. Radioactivity was determined with a Packard Tri-Carb
model 4450 liquid scintillation counter (Packard Instruments, Downer's
Grove, IL).
 |
RESULTS |
Oxidation of Linoleic Acid by Manganese Lipoxygenase
Isolation of Reaction Products of Linoleic
Acid--
[1-14C]Linoleic acid (350 µM)
was stirred for 30 min with Mn-LO (1.5 µg) in buffer A (2.1 ml) at
23 °C. Material isolated by extraction with diethyl ether was
subjected to RP-HPLC radiochromatography. As seen in Fig.
1, three peaks of radioactivity appeared.
The least polar material (29%; 81.1 ml of effluent) was identical to
[1-14C]linoleic acid remaining unconverted. Compound A
(12%; 14.1 ml of effluent) was intermediate in polarity to references
of authentic (11R,11S)-HOD (12.1 ml of effluent)
and (13S)-HOD (15.4 ml of effluent). Compound B (59%; 17.0 ml of effluent) cochromatographed with authentic
(13S)-HPOD.

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Fig. 1.
RP-HPLC radiochromatogram of reaction product
formed from linoleic acid incubated with Mn-LO.
[1-14C]Linoleic acid (350 µM) was treated
with Mn-LO (1.5 µg) in 2.1 ml of buffer C at 23 °C for 30 min, and
the reaction product was isolated by extraction with diethyl ether.
Solvent system, acetonitrile/water/2 M hydrochloric acid
(60:40:0.02, v/v/v); flow rate 1 ml/min. A, compound A;
B, compound B; C, linoleic acid; BHT,
2,6-di-tert-butyl-4-methylphenol, antioxidant added at a
level of 10 ppm to the diethyl ether used for extraction.
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Identification of Compound B--
Analysis of compound B by LC-MS
demonstrated a peak (10.4 min effluent) giving prominent ions at
m/z 311 (M
1; loss of H), 295 (M
17; loss of OH), and 293 (M
(1 + 18); loss of H plus H2O). When compound B was dissolved in
D2O/CD3OD (1:1, v/v), the M
1 ion
shifted to m/z 312. An aliquot of compound B was
reduced by treatment with sodium borohydride. Analysis of the
esterified product by straight phase high performance liquid
chromatography produced the methyl esters of 13-HOD (97.1%; 11.6 ml of
effluent) and 9-HOD (2.9%; 14.7 ml of effluent) as judged by their
chromatographic behaviors, UV spectrometry, and mass spectrometry. The
two hydroxyoctadecadienoates were converted into their MC derivatives
and subjected to oxidative ozonolysis. Analysis of the esterified
product by GLC demonstrated the formation of dimethyl azelate as well
as the MC derivative of methyl 2-hydroxyheptanoate
(R/S, 97.5:2.5) from the derivative of 13-HOD.
The derivative of 9-HOD afforded the MC derivative of dimethyl
2-hydroxysebacate (R/S, 37.0:63.0). These
experiments confirmed the results of the accompanying paper (15), which identified the major hydroperoxide produced from linoleic acid as
(13R)-HPOD. In addition, the analyses showed that the
Mn-LO-catalyzed oxygenation of linoleic acid resulted in small amounts
of the following hydroperoxyoctadecadienoate isomers:
(13S)-HPOD (2.4%), (9R)-HPOD (1.1%), and
(9S)-HPOD (1.8%).
Structure of Compound A--
The retention time of compound A upon
LC-MS analysis was 8.3 min, and the mass spectrum showed prominent ions
at 311 (M
1; loss of H), 295 (M
17; loss of OH), and
293 (M
(1 + 18); loss of H plus H2O). This result
indicated that compound A was a hydroperoxyoctadecadienoic acid
(molecular weight, 312). The UV spectrum of compound A was featureless
(Fig. 2) demonstrating the absence of,
e.g., conjugated double bonds. Treatment of compound A with
a small amount of perchloric acid resulted in a rapid appearance of UV
absorption bands at 259, 268, and 279 nm (Fig. 2). Analysis of the
esterified reaction product by GLC and GC-MS demonstrated the presence
of geometrical isomers of methyl 8,10,12- and
9,11,13-octadecatrienoates as judged by their molecular weight (292)
and retention times (C-19.00 (21%), C-19.13 (21%), C-19.42 (28%),
and C-19.46 (30%)). The fact that a virtually identical mixture of
geometrical isomers of methyl 8,10,12- and 9,11,13-octadecatrienoates
was formed upon acid treatment of the methyl ester of
(11R)-HOD (21) suggested that compound A was a derivative of
linoleic acid substituted at the bis-allylic position (C-11). Treatment
of compound A with sodium borohydride followed by esterification
afforded the methyl ester of 11-HOD as judged by GLC and GC-MS. The
retention time corresponded to C-19.41, a value identical to that
observed for the Me3Si derivative of the methyl ester of
authentic 11-HOD but different from the values recorded for the
Me3Si derivatives of the methyl esters of
(9E,12Z)-11-HOD and
(9Z,12E)-11-HOD (C-19.48/C-19.56) and
(9E,12E)-11-HOD (C-19.62). The mass spectrum of
the reduced product obtained from compound A was identical to that of
the Me3Si derivative of the methyl ester of 11-HOD and
showed prominent ions at m/z 382 (M, 38%), 311 (M
71; loss of
(CH2)4-CH3, 32%), 253 (9%),
and 225 ([CH=CH-CH(OSiMe3)-CH=CH-(CH2)4-CH3]+,
44%). Reduction of compound A with sodium borodeuteride afforded 11-HOD without significant incorporation of deuterium (less than 0.5%), thus excluding the possibility of a 11-keto group in compound A. Catalytic hydrogenation of compound A using platinum as catalyst resulted in the formation of a 1:1 mixture of 11-hydroxyoctadecanoate and 11-ketooctadecanoate as shown by GC-MS analysis using the authentic
compounds as references. On the basis of these data, compound A was
identified as a 11-hydroperoxy derivative of linoleic acid.

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Fig. 2.
Acid-catalyzed elimination of hydrogen
peroxide from (11S)-HPOD. (11S)-HPOD (15 µg) was added to acetonitrile (1 ml; 23 °C) in a cuvette having 10 mm path length. UV spectra were recorded before (solid line)
and 10 min after (dashed line) addition of 2 µl of 70%
(w/w) perchloric acid.
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Partial hydrogenation of compound A followed by oxidative
ozonolysis performed on the MC derivative was used to determine the
double bond positions and the configuration of C-11. Analysis of the
esterified ozonolysis product by GLC and GC-MS demonstrated the
presence of the MC derivatives of methyl
(2S)-hydroxynonanoate (less than 2% of the
(2R)-hydroxynonanoate; fragment originating in the MC
derivative of methyl 11-hydroxy-9-octadecenoate) and of dimethyl
(2R)-hydroxydodecane-1,12-dioate (less than 2% of the
(2S)-hydroxydodecanedioate; fragment originating in the MC derivative of methyl 11-hydroxy-12-octadecenoate). This experiment thus
established that the two double bonds of compound A were localized in
the
9 and
12 positions, and that the
absolute configuration of the alcohol group at C-11 was
"S." The data presented demonstrated that compound A was
identical to
(11S)-hydroperoxy-(9Z,12Z)-octadecadienoic acid ((11S)-HPOD).
Conversion of (11S)-HPOD into (13R)-HPOD
Formation of (11S)-HPOD and (13R)-HPOD as a Function of Time and
Substrate Concentration--
Linoleic acid was incubated with Mn-LO
and the product compositions at different times of incubation and
varying substrate concentrations were determined by RP-HPLC
(cf. Fig. 1). Because of the different extinction
coefficients of (13R)-HPOD, (11S)-HPOD, and
linoleic acid at the wavelength used for detection (217 nm), the peak
areas had to be divided with relative response factors in order to
obtain the molar composition of reactions products. These relative
response factors, i.e. 0.0732 (linoleic acid), 0.578 ((11S)-HPOD), and 1 ((13R)-HPOD, were calculated
by performing repeated injections of mixtures containing the three
14C-labeled compounds onto the high performance liquid
chromatography column and separately determining the peak areas of
aborbance at 217 nm and the radioactivity present in the corresponding
effluents.
As seen in Fig. 3, when a relatively low
concentration of linoleic acid (175 µM) was incubated
with Mn-LO, (11S)-HPOD was transient in appearance. The
maximum level was observed at 10-20 min of incubation, and at 30 min
only a trace amount of the hydroperoxide was detectable. In contrast,
when the linoleic acid concentration was increased to 526 µM, the amounts of (11S)-HPOD continued to increase during the entire incubation period of 30 min. Interestingly, analyses by RP-HPLC of products from experiments, where
(11S)-HPOD had been generated from linoleic acid and then
allowed to disappear, did not show any new peak(s) of UV absorption
ascribable to the further conversion of (11S)-HPOD. It was
conceivable that (11S)-HPOD had been converted into
non-UV-absorbing compound(s); however, analysis by RP-HPLC
radiochromatography of reaction products generated from
[1-14C]linoleic acid showed the presence of
(13R)-HPOD as the single labeled product. These results
suggested that (11S)-HPOD was convertible to
(13R)-HPOD in the presence of Mn-LO, and that this
conversion was slowed down in the presence of high concentrations of
linoleic acid.

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Fig. 3.
Time courses of formation of
(11S)-HPOD and (13R)-HPOD. Panel A,
linoleic acid (175 µM) in buffer C (2.1 ml) was treated
with Mn-LO (1.1 µg) at 23 °C. The absorbance at 235 nm was
measured using a cuvette having 1 mm path length. Aliquots (0.65 ml)
were removed at 6.5, 15, and 30 min of incubation and subjected to
RP-HPLC for determination of the molar ratio of
(11S)-HPOD/(13R)-HPOD (Q). The amounts of
(13R)-HPOD ( ) were calculated from the absorbance values
using = 26,000, and the amounts of (11S)-HPOD ( ) were
obtained from the amounts of (13R)-HPOD multiplied with Q. A
second addition of enzyme (0.5 µg) was made, as indicated by
arrow. Panel B, same as A, but
conducted with 526 µM linoleic acid. Aliquots for RP-HPLC
analysis were removed at 7, 14, and 30 min. A second addition of enzyme
(0.8 µg) was made, as indicated by arrow.
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Metabolism of (11S)-HPOD by
Mn-LO--
[1-14C](11S)-HPOD (60 µg) was
obtained by incubations of a high concentration of
[1-14C]linoleic acid (701 µM) with Mn-LO
(2.2 µg) followed by isolation by RP-HPLC. That no degradation of the
collected hydroperoxide had taken place was verified by analysis of an
aliquot of the specimen by RP-HPLC radiochromatography. Treatment of
(11S)-HPOD (52 µM) with Mn-LO resulted in the
formation of (13R)-HPOD as judged by RP-HPLC
radiochromatography (cf. Fig. 1). The identity of the
hydroperoxide produced from (11S)-HPOD as
(13R)-HPOD was based on its UV spectrum (solvent methanol;
max = 234 nm), its conversion into 13-HOD upon reduction
with sodium borohydride, its conversion into 13-hydroxystearate upon
reduction followed by catalytic hydrogenation, and on results of its
chemical degradation by oxidative ozonolysis. As was the case with
(13R)-HPOD isolated following incubation of linoleic acid
with Mn-LO, the (13R)-HPOD produced from
(11S)-HPOD was accompanied by small amounts of
9-hydroperoxyoctadecadienoates, i.e. (9R)-HPOD
(1.2%) and (9S)-HPOD (1.7%).
Conversion of (11S)-HPOD into (13R)-HPOD was
conveniently monitored by recording the UV absorbance at 235 nm
versus time (Fig. 4). At the
concentrations used, the maximum rate of formation of
(13R)-HPOD from (11S)-HPOD, i.e. 18.3 nmol min
1 µg
1, was ~50% greater than
that of formation of (13R)-HPOD from linoleic acid,
i.e. 12.1 nmol min
1 µg
1.
Interestingly, a slight lag phase was consistently observed when
(11S)-HPOD was used as the substrate (Fig. 4).

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Fig. 4.
Time courses of formation of
(13R)-HPOD from (11S)-HPOD (A) and
linoleic acid (B). A, (11S)-HPOD (52 µM) in buffer C (1.05 ml) was treated with Mn-LO (1.1 µg) at 23 °C. The absorbance at 235 nm was measured using a
cuvette having 10 mm path length. The maximum rate of increase of the
absorbance at 235 nm was 0.50 absorbance unit/min corresponding to a
rate of formation of (13R)-HPOD equal to 18.3 nmol
min 1 µg 1. B, linoleic acid (53 µM) in buffer C (1.05 ml) was treated with Mn-LO (1.1 µg) at 23 °C and the absorbance was measured as described in
A. The maximum rate of increase of the absorbance at 235 nm
was 0.33 absorbance unit/min corresponding to a rate of formation of
(13R)-HPOD equal to 12.1 nmol min 1
µg 1.
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Incubations in the Presence of Glutathione
Peroxidase--
Glutathione peroxidase and reduced glutathione
catalyze reduction of fatty acid hydroperoxides into hydroxy acids.
Trapping of hydroperoxides by this enzyme has been used in previous
studies to confirm the existence of fatty acid hydroperoxides as
intermediates in the biosynthesis of epoxy-hydroxy acids (25) and
dihydroxy acids (26, 7). If the Mn-LO-catalyzed formation of
(13R)-HPOD from linoleic acid took place by the sequence
linoleic acid
(11S)-HPOD
(13R)-HPOD,
inclusion of glutathione peroxidase and glutathione would be expected
to result in high yields of 11-HOD, the reduction product of
(11S)-HPOD, and in low yields of 13-HOD, the reduction
product of (13R)-HPOD. In a typical experiment, a mixture of
linoleic acid (224 µM), glutathione peroxidase (3 units)
and reduced glutathione (3 mM) in 0.8 ml of buffer C was treated with Mn-LO (0.86 µg) at 23 °C. Spectrophotometric assay of
the reaction mixture showed that the rate of formation of 13-H(P)OD was
2.1 nmol min
1 µg
1, a rate considerably
lower than that observed in a control incubation carried out in the
absence of glutathione peroxidase and reduced glutathione,
i.e. 9.8 nmol min
1 µg
1. This
reduced rate of formation of (13R)-HPOD in the presence of
glutathione peroxidase was not due to a selective interference with
formation of this hydroperoxide but to partial inhibition of linoleic
acid oxygenation. Thus, analysis of the reaction product (20 min of
incubation) by RP-HPLC showed that unconverted linoleic acid was the
main component (75%). The remaining part of the product was due to
13-HOD (21%; formed by reduction of (13R)-HPOD) and a small
percentage of 11-HOD (4%; formed by reduction of
(11S)-HPOD).
Incubations of (11S)-HPOD with Soybean Lipoxygenase--
In order
to test the possibility that (11S)-HPOD served as a
substrate for soybean lipoxygenase, the hydroperoxide (48 µM) in 1 ml of buffer A was treated with soybean
lipoxygenase (750 units) at 23 °C. No increase in the absorbance at
235 nm could be detected (Fig. 5). In
another experiment, soybean lipoxygenase (600 units) in 1 ml of buffer
B was treated with (11S)-HPOD (3 or 5 µM) for
2 min. Subsequently, linoleic acid (73 µM) was added. Measurement of the absorbance at 235 nm showed that the rate of conversion of linoleic acid by soybean lipoxygenase treated with (11S)-HPOD was virtually identical to that of untreated
lipoxygenase (Fig. 5).

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Fig. 5.
Tests of (11S)-HPOD as substrate
or inhibitor of soybean lipoxygenase. (11S)-HPOD (48 µM) was dissolved in buffer A (1 ml) at 23 °C, and
soybean lipoxygenase (750 units) was added at time indicated by
arrow. The absorbance at 235 nm was followed
versus time ( ). (11S)-HPOD (3 µM) in buffer B (1 ml) was preincubated with soybean
lipoxygenase (600 units) at 23 °C for 2 min. Linoleic acid (73 µM) was added, and the absorbance at 235 nm was monitored
( ). As a control, linoleic acid (73 µM) in buffer B (1 ml) was treated with soybean lipoxygenase (600 units) ( ).
|
|
Isotope Experiments
18O2 Experiments--
The mechanism of
formation of hydroperoxides catalyzed by Mn-LO was studied in
experiments where linoleic acid, (11S)-HPOD, or
(13R)-HPOD were incubated with Mn-LO in the presence of
18O2. The products were isolated by RP-HPLC
(cf. Fig. 1), and aliquots were reduced with sodium
borohydride, esterified, and analyzed by GC-MS as the Me3Si
derivatives. In other experiments,
[11-18O2](11S)-HPOD and
[13-18O2](13R)-HPOD were
biosynthesized from linoleic acid and reincubated with Mn-LO under
ambient air.
Incubation of linoleic acid under 18O2 led to
the formation of (11S)-HPOD and (13R)-HPOD that
had incorporated 18O2 (Table
I). Incubations of
[11-18O2](11S)-HPOD with Mn-LO led
to the formation of (13R)-HPOD that retained 39-44% of the
18O2 label. In addition, 9-HPOD, a minor
hydroperoxide of Mn-LO catalysis, was labeled at the same level
(incubations 2 and 4, Table I). In another experiment,
(11S)-HPOD was incubated with Mn-LO under
18O2. As seen (incubation 5, Table I), this led
to the formation of (13R)-HPOD that had incorporated a
significant amount of 18O2. Furthermore,
analysis of the 18O content of (11S)-HPOD
remaining not converted in incubations of
[11-18O2](11S)-HPOD conducted
under air, and in an incubation of (11S)-HPOD under an
atmosphere of 18O2, demonstrated a
time-dependent exchange of the hydroperoxide oxygens with
O2 (incubations 4 and 5, Table I). Such oxygen exchange was
not observed when (13R)-HPOD was treated with Mn-LO
(incubation 6, Table I).
Incubations of Stereospecifically Deuterated Linoleic
Acids--
(11S)-HPOD and (13R)-HPOD
biosynthesized from (11R)-[2H]- and
(11S)-[2H]linoleic acids were isolated by
RP-HPLC and their isotope content determined by GC-MS after reduction,
esterification, and conversion into the Me3Si derivatives.
As seen in Table II, hydroperoxides produced from (11R)-[2H]linoleic acid retained
most of the deuterium label, whereas hydroperoxides generated from
(11S)-[2H]linoleic acid lost most of the
label. These results demonstrated that the hydrogen abstracted from the
C-11 methylene group by Mn-LO had the pro-S configuration.
The isotope contents of linoleic acid remaining unconverted in
incubations of (11R)-[2H]- and
(11S)-[2H]linoleic acids were also determined.
As seen (Table II), incubation of
(11S)-[2H]linoleic acid was accompanied by a
time-dependent enrichment of deuterium in unconverted
linoleic acid. The presence of a kinetic isotope effect in the
enzyme-catalyzed abstraction of the (11S) deuterium
indicated by this experiment also manifested itself in the time course
of formation of (13R)-HPOD from
(11S)-[2H]linoleic acid measured
spectrophotometrically. As seen in Fig. 6, production of (13R)-HPOD
from (11S)-[2H]linoleic acid (32.6%
deuterated and 67.4% undeuterated molecules) occurred by a biphasic
time course. This was explainable if it is assumed that undeuterated
linoleic acid present in the mixture incubated was mainly oxygenated in
the early phase of the incubation (segment A in Fig. 6),
whereas the gradually accumulating deuterated substrate was oxygenated
at a much slower rate during the later phase (segments B and
C in Fig. 6). Estimates of the rates of conversion of
undeuterated and deuterated molecules could be made from the slopes of
segments A-C, i.e. 7.9 nmol min
1
µg
1 for segment A (roughly corresponding to oxygenation
of undeuterated linoleic acid), and 0.4-0.5 nmol min
1
µg
1 for segments B and C (roughly corresponding to
oxygenation of deuterated linoleic acid). The magnitude of the kinetic
isotope effect estimated from these rates was
kH/kD = 15-22. The reported
value of the isotope effect in the soybean lipoxygenase-catalyzed
oxygenation of linoleic acid dideuterated at C-11 (94-95%
dideuterated molecules) is kH/kD = 8-9 (27, 28); however, much larger values have been reported recently (29-31).

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Fig. 6.
Rates of formation of (13R)-HPOD
from deuterated linoleic acids.
(11R)-[2H]Linoleic acid (411 µM)
in buffer A (1.05 ml) was treated with Mn-LO (1.5 µg) at 23 °C. An
aliquot was transferred to a cuvette having 1 mm path length, and the
absorbance at 235 nm was monitored versus time ( ).
Aliquots (0.3 ml) were removed at 3.5, 11.5, and 16 min of incubation
and subjected to RP-HPLC. Hydroperoxides (3.5-min sample) and linoleic
acid were collected, derivatized, and subjected to GC-MS analysis for
determination of isotope content.
(11S)-[2H]Linoleic acid (321 µM)
was treated with Mn-LO (1.5 µg) in buffer A (1.05 ml) at 23 °C. An
aliquot was transferred to a cuvette having 1 mm path length, and the
absorbance at 235 nm was monitored versus time ( ).
Aliquots (0.3 ml) were removed at 4.5, 15, and 49 min of incubation.
Products were isolated by RP-HPLC, derivatized, and subjected to GC-MS
analysis. Arrow indicates addition of additional Mn-LO (1.5 µg). The estimated rates of formation of (13R)-HPOD in
time segments A, B, and C were 7.9, 0.5, and 0.4 nmol min 1 µg 1,
respectively.
|
|
An interesting finding in the experiments with the stereospecifically
deuterated linoleic acids was the slightly different deuterium contents
of (11S)-HPOD and (13R)-HPOD biosynthesized from
(11R)-[2H]linoleic acid (Table II). The
optical purity of the (11S)-hydroxystearate used to prepare
the (11R)-[2H]linoleic acid was 95.4% (18);
therefore, the percentage retentions of deuterium observed for
(11S)-HPOD (98%) and (13R)-HPOD (86%) were
somewhat higher and lower, respectively, compared with the theoretical
value (~95%). A secondary isotope effect has been described for the
oxygenation of (10R)-[3H]arachidonic acid
catalyzed by human platelet 12-lipoxygenase (32), and it is tempting to
speculate that the slightly different extent of labeling of the two
hydroperoxides formed from (11R)-[2H]linoleic
acid in the presence of Mn-LO is due to a secondary isotope effect in
the conversion of [11-2H](11S)-HPOD into
[11-2H](13R)-HPOD (cf. Fig.
7).

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Fig. 7.
Mechanism proposed for oxygenation of
linoleic acid catalyzed by Mn-LO. In the initial step, Mn-LO
abstracts the pro-S hydrogen from C-11 of linoleic acid. The
resulting linoleoyl radical is reversibly oxygenated at C-11 to produce
an (11S)-peroxy radical that can be further converted into
(11S)-HPOD. Alternatively, oxygen attack at the C-13
position of the linoleoyl radical results in irreversible formation of
(13R)-HPOD. Free radical conversions are shown, although it
is conceivable that heterolytic reactions may partly be involved
(cf. Ref. 37). R1,
(CH2)7-COOH; R2,
(CH2)4-CH3.
|
|
 |
DISCUSSION |
Mn-LO purified from the fungus G. graminis as described
in the accompanying paper (15) catalyzes conversion of linoleic acid
into (13R)-HPOD as the major product. This transformation consisted of dioxygenation of a fatty acid possessing a
(1Z,4Z)-pentadiene moiety into a fatty acid
hydroperoxide having a
1-hydroperoxy-(2E,4Z)-pentadiene structure, thus
satisfying the requirements for classifying Mn-LO as a lipoxygenase
enzyme. The aim of the present study was originally to determine the
stereochemistry of the biosynthesis of (13R)-HPOD using
stereospecifically deuterated linoleic acids. In the course of
this work, it became apparent that Mn-LO not only catalyzes transformation of linoleic acid into (13R)-HPOD but
also other reactions, which are not characteristic of traditional
lipoxygenases.
Experiments with linoleic acids labeled with 2H in the
(11R) and (11S) positions demonstrated that
Mn-LO, like soybean lipoxygenase-1, catalyzed abstraction of the
pro-S hydrogen from C-11 of linoleic acid (Table II). As was
found previously for soybean lipoxygenase (4, 27-31), a pronounced
primary isotope effect resulting in accumulation of 2H in
the unconverted substrate was noted in these experiments (Table II,
Fig. 6). This result indicated that the first step of the
Mn-LO-catalyzed oxygenation, like that of the soybean
lipoxygenase-catalyzed oxygenation, consisted of hydrogen abstraction
from the bis-allylic methylene group to produce a pentadienyl moiety.
The overall steric course of formation of (13R)-HPOD from
linoleic acid in the presence of Mn-LO consisted of hydrogen
abstraction and oxygen insertion occurring in a suprafacial
way (Fig. 7). This was in contrast to the antarafacial stereochemistry
repeatedly observed for oxygenations catalyzed by soybean lipoxygenase
and other lipoxygenases (4-10).
Analysis of the hydroperoxide product isolated following incubation of
linoleic acid with Mn-LO demonstrated the presence of a second, less
abundant component in addition to (13R)-HPOD (compound
A, Fig. 1). The structure of this compound was determined by
ultraviolet spectroscopy and mass spectrometry and by chemical methods.
An important clue to the structure was provided by the finding that the
hydroperoxy group of compound A was rapidly eliminated upon acid
treatment to provide a mixture of 8,10,12- and
9,11,13-octadecatrienoates (Fig. 2). This type of conversion had
earlier been observed with 11-HOD (21, 22), thus indicating that
compound A was a bis-allylic hydroperoxide. Reduction of compound A by
treatment with sodium borohydride afforded 11-HOD. The geometry of the
two double bonds of this product was rigorously established as
Z,Z by comparison with authentic 11-HOD and with
chemically prepared (9E,12Z)-, (9Z,12E)-, and
(9E,12E)-11-HOD. Partial hydrogenation of the
methyl ester of 11-HOD derived from compound A followed by treatment with (
)-menthoxycarbonyl chloride and oxidative ozonolysis resulted in the formation of chiral fragments whose structures localized the
double bonds of compound A to the
9 and
12 positions and demonstrated that the absolute
configuration of C-11 was "S." The experiments thus
allowed compound A to be formulated as
(11S)-hydroperoxy-(9Z,12Z)-octadecadienoic
acid ((11S)-HPOD). This bis-allylic hydroperoxide was
a new oxylipin, although a geometrical isomer of the compound,
i.e. methyl
(11R,11S)-hydroperoxy-(9Z,12E)-octadecadienoate, had been prepared by a singlet oxygen reaction (results referred to in
Ref. 33). Furthermore, a more unsaturated analog, i.e. 11-hydroperoxyoctadec-12-en-9-ynoic acid, was recently isolated following incubation of the lipoxygenase inhibitor
octadec-(12Z)-en-9-ynoic acid with soybean lipoxygenase
(34). Biosynthesis of (11S)-HPOD catalyzed by Mn-LO was
accompanied by selective loss of the pro-S hydrogen from
the carbon dioxygenated (Table II) and thus proceeded with
retention of absolute configuration (Fig. 7).
The proportion between (11S)-HPOD and (13R)-HPOD
depended on the time of incubation and the substrate concentration
(Fig. 3). Short times of incubation of high substrate concentrations gave the highest yields of (11S)-HPOD and a ratio
(11S)-HPOD/(13R)-HPOD equal to 0.31. As shown by
RP-HPLC radiochromatographic analysis, the only product present in
incubations where (11S)-HPOD had been allowed to disappear
was (13R)-HPOD, thus suggesting that (11S)-HPOD was converted into (13R)-HPOD in the presence of Mn-LO. This
could be directly demonstrated in experiments where
(11S)-HPOD was incubated with Mn-LO and the ultraviolet
absorbance due to (13R)-HPOD was monitored versus
time. The time course of formation of (13R)-HPOD showed a
characteristic sigmoidal shape because of the presence of a lag phase
(Fig. 4). The maximum rate of formation of (13R)-HPOD from
(11S)-HPOD was ~50% greater than that of formation of
(13R)-HPOD from linoleic acid. Conversion of
(11S)-HPOD into (13R)-HPOD was suppressed in the
presence of high concentrations of linoleic acid (Fig. 3), indicating
that (11S)-HPOD and linoleic acid competed for the same
catalytic site of Mn-LO. The fact that (11S)-HPOD was
converted into (13R)-HPOD in the presence of Mn-LO raised the question whether (11S)-HPOD served as an obligatory
intermediate in the formation of (13R)-HPOD. The enzyme
glutathione peroxidase, which traps fatty acid hydroperoxides as the
corresponding hydroxy compounds, has been successfully utilized to
prove the existence of hydroperoxides as intermediates in the
biosynthesis of various oxylipins (7, 25, 26). Inclusion of glutathione
peroxidase and reduced glutathione in incubations of linoleic acid with
Mn-LO resulted in a decreased rate of formation of
(13R)-H(P)OD; however, this was not caused by trapping of
(11S)-HPOD but by partial inhibition of oxygenation of
linoleic acid. Importantly, the ratio of the reduction products,
(11S)-HOD/(13R)-HOD, did not increase in the presence of glutathione peroxidase, thus disfavoring the hypothetical sequence linoleic acid
(11S)-HPOD
(13R)-HPOD.
The mechanism of the conversion of (11S)-HPOD into
(13R)-HPOD was studied by 18O experiments (Table
I). In one set of incubations, linoleic acid was treated with Mn-LO
under 18O2 and the hydroperoxides formed were
isolated by RP-HPLC. Aliquots were reduced and analyzed by GC-MS as the
methyl ester/Me3Si derivatives. As expected,
(11S)-HPOD and (13R)-HPOD had both incorporated
18O. Reincubation of
[11-18O2](11S)-HPOD with Mn-LO
resulted in the formation of (13R)-HPOD that retained
39-44% of the 18O label. In a complementary experiment,
incubation of (11S)-HPOD under 18O2
led to the formation of (13R)-HPOD that had incorporated
57% 18O. The findings that (13R)-HPOD only
partially retained the 18O label when formed from
[11-18O2](11S)-HPOD, and that the
hydroperoxide had partially incorporated 18O when
biosynthesized from (11S)-HPOD under
18O2, showed that the oxygen molecule migrating
from C-11 to C-13 during the hydroperoxide rearrangement was subject to
exchange with surrounding molecular oxygen. This fact, in turn,
necessitated that the conversion of (11S)-HPOD into
(13R)-HPOD took place in a stepwise way involving
a deoxygenated intermediate. Interestingly, the oxygen exchange also
manifested itself in the isotope content of (11S)-HPOD
remaining not converted. As seen in Table I, there was a
time-dependent loss of 18O from
[11-18O2](11S)-HPOD incubated with
Mn-LO under ambient air, and an incorporation of 18O from
18O2 into (11S)-HPOD incubated under
18O2. These results gave further support for
the existence of a deoxygenated intermediate and additionally indicated
that this intermediate could be reversibly oxygenated into
(11S)-HPOD. Fig. 7 shows the mechanism proposed for the
Mn-LO-catalyzed oxygenation of linoleic acid. The initial step
consisted of abstraction of the pro-S hydrogen from C-11 to
produce a linoleoyl radical. This intermediate was reversibly converted
into (11S)-HPOD via the corresponding
(11S)-peroxy radical, or irreversibly converted into
(13R)-HPOD via the corresponding (13R)-peroxy
radical. Chemical studies on fatty acid autoxidation have demonstrated
that conversion of carbon-centered fatty acid radicals into peroxy
radicals occurs by reversible binding of dioxygen (35). Furthermore,
non-enzymatic free radical rearrangements of fatty acid hydroperoxides
have been described (33, 36). In a study of rearrangement of
hydroperoxides derived from linoleic acid (36), the methyl ester of
(9S)-HPOD (incorrectly referred to as (9R)-HPOD
in Ref. 36) was found to yield a mixture of the
E,Z- and E,E-isomers of 9- and 13-HPOD methyl esters when kept in hexane solution under
O2. When the reaction was performed under
18O2, partial incorporation of 18O
into the hydroperoxides was observed. Although the 18O
experiments of the present study demonstrated the existence of a
non-concerted pathway involving a deoxygenated intermediate for the
transformation of (11S)-HPOD into (13R)-HPOD,
they did not exclude the possibility of an additional mechanism
contributing to the formation of (13R)-HPOD, i.e.
a direct conversion of the (11S)-peroxy radical into the
(13R)-peroxy radical by concerted transfer of the peroxy
radical oxygen from C-11 to C-13. Studies of the viability of such a
contributing pathway are under way.
It is uncertain whether the mechanism proposed for Mn-LO involving a
bis-allylic peroxy radical and a bis-allylic hydroperoxide has any
relevance for other lipoxygenases such as soybean lipoxygenase. 11-Hydroperoxyoctadec-12-en-9-ynoic acid has been isolated as one of
several products formed from an acetylenic inhibitor,
octadec-(12Z)-en-9-ynoic acid, upon incubation with soybean
lipoxygenase (34). However, despite extensive studies of soybean
lipoxygenase-catalyzed oxygenations, formation of bis-allylic
hydroperoxides from polyunsaturated fatty acids has never been
reported. In the present study, (11S)-HPOD was tested as a
substrate for soybean lipoxygenase with negative result. The possible
roles of the corresponding (11S)-peroxy radical, and of
(11R)-HPOD or its corresponding peroxy radical, as
intermediates in soybean lipoxygenase catalysis remains to be
examined.
 |
ACKNOWLEDGEMENT |
We gratefully acknowledge the expert technical
assistance of G. Hamberg.
 |
FOOTNOTES |
*
This work was supported by Swedish Medical Research Council
Projects 03X-5170 and 6523 and by a grant from Vesical AB.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. Tel.: 46-8-728-7640;
Fax: 46-8-736-0439; E-mail: mats.hamberg{at}mbb.ki.se.
1
The abbreviations used are: Mn-LO, manganese
lipoxygenase; 9-H(P)OD,
9-hydro(pero)xy-(10E,12Z)-octadecadienoic acid;
11-H(P)OD, 11-hydro(pero)xy-(9Z,12Z)-octadecadienoic acid;
13-H(P)OD;
13-hydro(pero)xy-(9Z,11E)-octadecadienoic acid;
GLC, gas-liquid chromatography; GC-MS, gas-liquid chromatography-mass spectrometry; FT-IR, Fourier transform-infrared; LC-MS, liquid chromatography-mass spectrometry; MC, (
)-menthoxycarbonyl;
Me3Si, trimethylsilyl; RP-HPLC, reversed phase high
performance liquid chromatography.
 |
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