10(S)-hydroxy-8(E)-octadecenoic acid, an intermediate in the conversion of oleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid

Article abstract of DOI:10.1007/s11746-000-0015-7

The new microbial isolate Pseudomonas aeruginosa (PR3) has been reported to produce from oleic acid a new compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD), with 10-hydroxy-8-octadecenoic acid (HOD) being a probable intermediate. The production of DOD involves the introduction of two hydroxyl groups at carbon numbers 7 and 10, and a rearrangement of the double bond from carbons 9-10 to 8-9. It has been shown that the 8-9 unsaturation of HOD was possibly in the cis configuration. Now we report that the rearranged double bond of HOD is trans rather than cis, as determined by spectral data. Also, it was found that the 10-hydroxyl was in the S-configuration as determined by gas chromatographic separation of R- and S-isomers after preparation of the (-)-menthoxycarbonyl derivative of the hydroxyl group followed by oxidative cleavage of the double bond and methyl esterification. This latter result coincides with our recent finding that the main final product, DOD, is in the 7(S),10(S)-dihydroxy configuration. In addition, a minor isomer of HOD (about 3%) with the 10(R)-hydroxyl configuration was also detected. From the data obtained herein, we concluded that 10(S)-hydroxy-8(E)-octadecenoic acid is the probable intermediate in the bioconversion of oleic acid to 7(S),10(S)-dihydroxy-8(E)-octadecenoic acid by PR3.

Full text of DOI:10.1007/s11746-000-0015-7

10(S)-Hydroxy-8(E)-octadecenoic Acid, an Intermediate
in the Conversion of Oleic Acid to 7,10-Dihydroxy-8(E)-
octadecenoic Acid
Hakryul Kima, Harold W. Gardnerb, and Ching T. Houa,*
aOil Chemical Research and bBioactive Agents Research, ARS, USDA, NCAUR, Peoria, Illinois 61604
ABSTRACT: The new microbial isolate Pseudomonas aerugi- crobial conversion of unsaturated fatty acids has been widely
nosa (PR3) has been reported to produce from oleic acid a new
compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD), with
10-hydroxy-8-octadecenoic acid (HOD) being a probable inter-
mediate. The production of DOD involves the introduction of
two hydroxyl groups at carbon numbers 7 and 10, and a re-
arrangement of the double bond from carbons 9-10 to 8-9. It has
been shown that the 8-9 unsaturation of HOD was possibly in
the cis configuration. Now we report that the rearranged double
bond of HOD is trans rather than cis, as determined by spectral
data. Also, it was found that the 10-hydroxyl was in the S-con-
figuration as determined by gas chromatographic separation of
exploited to produce new, value-added hydroxy fatty acids.
Microbial oxidation of unsaturated fatty acids was recently
reviewed (3). Much research has been done in this and other
laboratories to investigate the microbial conversion of veg-
etable oils and their component fatty acids to value-added
products. For example, Hou et al. (4) reported the discovery
of a new compound, 7,10-dihydroxy-8(E)-octadecenoic acid
(DOD), which was produced from oleic acid in >60% yield
by a bacterial isolate Pseudomonas sp. PR3 (5). Strain PR3
was identified subsequently as Pseudomonas aeruginosa (6).
R- and S-isomers after preparation of the (−)-menthoxycarbonyl Recently the yield of DOD by strain PR3 was improved to
derivative of the hydroxyl group followed by oxidative cleavage
of the double bond and methyl esterification. This latter result
coincides with our recent finding that the main final product,
DOD, is in the 7(S),10(S)-dihydroxy configuration. In addition, a
minor isomer of HOD (about 3 %) with the 10(R)-hydroxyl con-
figuration was also detected. From the data obtained herein, we
concluded that 10(S)-hydroxy-8(E)-octadecenoic acid is the
probable intermediate in the bioconversion of oleic acid to
7(S),10(S)-dihydroxy-8(E)-octadecenoic acid by PR3.
>80% yield by modifying the culture medium and reaction
parameters (7). The formation of a similar compound by
Pseudomonas 42A2 also was reported and studied (8,9).
The bioconversion of oleic acid into DOD is unique in that
it involves the introduction of hydroxyl groups at carbon
atoms 7 and 10 and a rearrangement of the double bond from
carbons 9-10 to 8-9. Based on the complexity of these reac-
tions, DOD formation seems to involve more than one trans-
formation. To elucidate the metabolic pathway leading to
DOD from oleic acid, Hou and Bagby (10) reported isolation
Paper no. J9210 in JAOCS 77, 95–99 (January 2000).
KEY WORDS: Bioconversion, dihydroxy octadecenoic acid, of two additional products from the reaction mixture, of
monohydroxy octadecenoic acid, oleic acid, Pseudomonas
aeruginosa, stereo configuration.
which one was identified as phenazine-1-carboxylic acid and
the other as 10-hydroxy-8(Z)-octadecenoic acid (HOD). HOD
was thought to be an intermediate in the bioconversion of
oleic acid to DOD. Recently, another group reported the con-
version of oleic acid by Pseudomonas sp. 42A2 into HOD and
DOD, both with an 8,9-trans double bond (11).
Here we report that the double bond of HOD is trans rather
than cis by nuclear magnetic resonance (NMR) and Fourier
transform infrared (FTIR) data and that the 10-hydroxyl
group is mainly in the S-configuration with a minor percent-
age in the R-configuration. Some aspects of the pathway in-
volved in this bioconversion are discussed as well.
Hydroxy fatty acids are known to be produced in nature
mainly by plant systems. The hydroxyl group is known to
give a fatty acid special properties, such as higher viscosity
and reactivity compared with other nonhydroxylated fatty
acids (1). As a result, hydroxy fatty acids are used in a wide
range of industrial products, such as resins, waxes, nylons,
plastics, lubricants, cosmetics, and additives in coatings and
paintings. At present, imported castor oil and its derivatives
are the major commercial source of hydroxy fatty acids. How-
ever, the fluctuating supplies and prices for castor oil cause
users to seek alternative raw materials.
EXPERIMENTAL PROCEDURES
Since Wallen et al. (2) reported the first bioconversion of
oleic acid to 10-hydroxystearic acid by a pseudomonad, mi-
Microorganisms. Pseudomonas aeruginosa NRRL strain B-
18602 (PR3) was originally isolated from a water sample in
Morton, IL. The culture was aerobically grown at 28°C in a
125-mL Erlenmeyer flask containing 50 mL of medium at
200 rpm. The medium used contained 4 g dextrose, 2 g
*To whom correspondence should be addressed at ARS, USDA, NCAUR,
1815 N. University St., Peoria, IL 61604.
E-mail: houct@mail.ncaur.usda.gov
Copyright © 2000 by AOCS Press
95
JAOCS, Vol. 77, no. 1 (2000)

96
H. KIM ET AL.
K2HPO4, 2 g (NH4)2HPO4, 0.5 g yeast extract, 1 g NH4NO3, vapor and then spraying the plate with 50% sulfuric acid and
0.01 g FeSO4·7H2O, 0.008 g MnSO4·7H2O, 0.014 g ZnSO4, heating in a 100°C oven for 10 min.
and 0.01 g nicotinic acid per liter. The medium was adjusted
The HPLC-purified HOD was characterized by GC/mass
to pH 7.0 with dilute phosphoric acid. Cultures were main- spectrometry (GC/MS), nuclear magnetic resonance (NMR),
tained on an agar slant with the above medium except for the and Fourier transform infrared (FTIR) spectrometry. Elec-
addition of 1.5% agar.
tron-impact mass spectra were obtained with a Hewlett-
Chemicals. Dimethyl octanedioate and oleic acid with Packard (Avondale, PA) 5890 GC coupled to a Hewlett-
99+% purity by gas chromatography (GC) were purchased Packard 5972 series mass selective detector. The column out-
from Sigma Chemical Co. (St. Louis, MO). Racemic methyl let was connected directly to the ion source. Compounds were
2-hydroxydecanoate was obtained from Matreya Inc. (Pleas- separated on a methylsilicone column (30 m × 0.25 mm i.d.,
ant Gap, PA) and (−)-menthoxycarbonylchloride [(−)-men- 0.25-µm film thickness) using a temperature gradient of
thyl chloroformate] was from Aldrich (Milwaukee, WI). 20°C/min from 70 to 170°C, holding 1 min at 170°C, and then
Kieselgel 60 and thin-layer precoated Kieselgel 60F254 plates 5°C/min to 250°C, followed by holding for 15 min at 250°C
were obtained from EM Science (Cherry Hill, NJ). Other (helium at 0.67 mL/min). For GC/MS trimethylsilyloxy
reagent-grade chemicals were purchased from Sigma and (TMSiO) derivatives were prepared by treatment of methyl
used without further purification.
esters with pyridine/hexamethyldisilazane/trimethylchlorosi-
Bioconversion. Oleic acid (0.3 g) was added to a 24-h-old lane (2:2:3, vol/vol/vol) for 15 min at room temperature. 1H
culture in the above medium and the flasks were incubated and 13C NMR spectra were determined in deuterated chloro-
under the same conditions for an additional 48 h. At the end form with a Bruker ARS-400 spectrometer (Billerica, MA),
of the cultivation, the culture broth was acidified to pH 2 with operated at a frequency of 400 and 100 MHz, respectively.
6 N hydrochloric acid. The culture broth was extracted once FTIR analysis of the free acid products were run as films on
with an equal volume of ethyl acetate followed by extraction KBr with a PerkinElmer FTIR model 1750 spectrometer
with an equal volume of ether. The solvent was evaporated (PerkinElmer Inc., Oakbrook, IL).
from the combined extracts with a rotary evaporator.
Determination of stereochemistry. For chiral analysis, 1
Analysis of products. The crude extract (200 mg) was sub- mg of HOD methylated with diazomethane was reacted with
jected to high-performance liquid chromatography (HPLC) for (−)-menthoxycarbonylchloride, and then the menthoxycar-
analysis and isolation of HOD. HPLC was conducted with a bonyl (MCO) derivative was isolated by TLC using
Hewlett-Packard (Palo Alto, CA) series 1100 equipped with a hexane/ethyl acetate (185:15 vol/vol) developed as previously
Hewlett-Packard 1100 series diode array detector (DAD) and a described (12). This method was modified to oxidatively
Microsorb silica column (41.4 mm i.d. × 25 cm) from Rainin cleave the MCO derivative with 12 mg KMnO4 in 0.3 mL
Instrument Co. (Woburn, MA). Purification was carried out by acetic acid for 1 h at 37°C (13). After oxidative cleavage, the
injecting crude extracts (200 mg) dissolved in methylene chlo- acetic acid was evaporated with a stream of nitrogen, and the
ride/methanol (2:1 vol/vol) onto the column equilibrated with products were methyl esterified with diazomethane. The
hexane/ethyl acetate (95:5 vol/vol). Separation was achieved methyl 2-MCO-decanoate and dimethyl octanedioate ob-
by eluting the column with a linear gradient from hexane/ethyl tained by this procedure were dissolved in hexane and ana-
acetate (95:5 vol/vol), after an initial hold of 20 min, to lyzed by GC/MS. Methyl 2(R,S)-MCO-decanoate was pre-
hexane/ethyl acetate (50:50 vol/vol) for a total running time of pared from racemic methyl 2-hydroxydecanoate by the
200 min at a flow rate of 7 mL/min. Fractions were collected method described above (12). GC/MS for this experiment
with the peaks being monitored at 240 nm wavelength. Al- was completed with a Hewlett-Packard model 5890 (Palo
though the 240 nm wavelength was not satisfactory for detect- Alto, CA) gas chromatograph equipped with a Hewlett-
ing HOD directly, a characteristic pattern of other eluting com- Packard model 5971 mass selective detector operating at 70
pounds detected at this wavelength was used to locate the HOD ev. The capillary column used was a Hewlett-Packard HP-
peak reliably. The collected fractions were analyzed by GC and 5MS cross-linked 5% phenyl methyl silicone, 30 m × 0.25
thin-layer chromatography (TLC). For GC analysis, the sam- mm, film thickness 0.25 m. The oxidative cleavage products
ples were first methylated with ethereal diazomethane for 2 min methyl 2-MCO-decanoate and dimethyl octanedioate were
at room temperature. The methyl esters, from which the sol- separated by temperature programming from 65 to 260°C at a
vent was evaporated under a nitrogen stream, were dissolved rate of 10oC/min (helium at 0.67 mL/min). In order to sepa-
in methylene chloride/methanol (95:5 vol/vol) and injected into rate diastereomeric methyl 2(S)- and 2(R)-MCO-decanoates
a Hewlett-Packard model 5890 GC (Hewlett-Packard, Avon- for determination of stereo configuration, the temperature
dale, CA). The GC was equipped with a flame-ionization de- program had to be changed from 100 to 260°C at a rate of
tector (FID), a Supelco SPB-1™ capillary column (15 m × 0.32 2%C/min (helium at 0.67 mL/min).
mm i.d., 0.25-µm thickness; Supelco Inc., Bellefonte, PA), and
a Hewlett-Packard 3392A integrator. Compounds were eluted
isothermally at 200°C. The retention time of HOD was 9.3 min.
RESULTS AND DISCUSSION
TLC plates were developed with toluene/dioxane/acetic acid Purification of HOD. After incubating P. aeruginosa PR3 in
(79:14:7 vol/vol/vol). The spots were visualized first by iodine the presence of oleic acid, a crude organic extract was obtained
JAOCS, Vol. 77, no. 1 (2000)

BIOCONVERSION OF OLEIC ACID
97
NMR analysis. Purified HOD was also subjected to 1H and
13C NMR analyses to confirm the structure of HOD. The pro-
ton and carbon resonance signals (ppm) and corresponding
molecular assignments are given in Table 1. 1H NMR absorp-
tion for the C8 and C9 olefinic protons (–CH=CH–) were ob-
served at 5.61 and 5.42 ppm, respectively, with a coupling con-
stant of 15.4 Hz strongly suggesting a trans double bond. One
proton (–CH–O–) was observed at 4.02 ppm. Because of the
downfield shift, compared to alcohols adjacent to saturated car-
bons, and the doublet of triplet multiplicity, the hydroxyl-bear-
from the culture. A sample of the extract was injected onto the
HPLC column and separated by eluting the column with the
gradient system described in the Experimental Procedures sec-
tion. The collected fractions were analyzed by TLC and GC
revealing that most of the HOD eluted between 165–185 min
after injection. Fractions containing HOD were combined,
concentrated, and reanalyzed by HPLC. After the second
HPLC separation, 5 mg of purified HOD (>95% by GC) was
obtained. TLC of the purified sample showed one spot.
Structure determination. The location of the hydroxyl
group and double bond was deduced from the fragment ions
observed in the electron-impact GC/MS data of the silylated
methyl ester of HOD (Fig. 1). The intense fragment (m/z 271)
arising from alpha cleavage to the silylated hydroxyl group
gave a fragment ion containing both the silyl group and the
double bond. This fragment ion, together with the m/z 241
ion, located the hydroxyl group at either C10 or C8, but the
greater intensity of the m/z 271 ion suggested that the hy-
droxyl group was localized at C10 as shown for a similar silyl-
ated methyl ester, methyl 13-hydroxy-11(E)-octadecenoate
(14). This result was also supported by the fact that the MCO
derivative of HOD subjected to oxidative cleavage of the dou-
ble bond by KMnO4 followed by methylation generated
methyl 2-MCO-decanoate and dimethyl octanedioate (see the
experiments described below). These data are consistent with
the TMSiO derivative of a methylated 10-hydroxy-8-octade-
cenoic acid with a molecular mass of 384.
ing carbon was suggested to be vicinal to the double bond. 13
C
NMR showed the presence of the following carbon atoms: car-
bonyl carbon at 174 ppm (C1), a double bond at 131.97 ppm
(C8) and 133.14 ppm (C9), and a –CHOH– carbon at 73.27
ppm (C10). The chemical shifts of the remaining proton and
carbon are listed in Table 1. The data obtained from GC/MS,
FTIR, NMR, and oxidative cleavage confirmed that the puri-
fied compound was 10-hydroxy-8(E)-octadecenoic acid.
Determination of stereochemistry. Purified HOD was sub-
jected to chiral analysis by the MCO method (see Experimen-
tal Procedures section). Oxidative cleavage of the MCO deriv-
ative of methyl HOD followed by methyl esterification gave
two major peaks by GC at retention times of 10.65 and 20.2
min using the appropriate temperature program (see Experi-
mental Procedures section). The first eluting peak was deter-
mined to be dimethyl octanedioate by comparison with the
standard, and the second peak was determined to be methyl 2-
MCO-decanoate by comparison with standard racemic methyl
2(R,S)-MCO-decanoate.
FTIR analysis. Purified HOD (as the free fatty acid) was
analyzed by FTIR. The presence of hydroxyl group(s) was in-
dicated by the IR absorption at 3605 cm−1. An absorption at
1706 cm−1 showed the presence of a carbonyl group, and the
absorption at 965 cm−1 indicated trans unsaturation (15).
Although the above temperature programming was useful
in detecting the two cleavage fragments, dimethyl octane-
dioate and methyl 2-MCO-decanoate, the program had to be
changed in order to separate diastereomeric methyl 2(S)- and
TABLE 1
Proton and 13C Nuclear Magnetic Resonance Signals and Molecular
Assignments for HODa
Resonance signal (ppm)
Carbon number
1H
13C
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
174.2
2.3
1.62
1.25
1.25
34.01
24.62
28.57
28.78
29.26
32.01
131.97
133.14
73.27
37.28
25.48
28.81
29.55
29.55
31.86
22.66
14.1
1.25
2.01
5.61 (J8,9 = 15.4 Hz)
5.42
4.02
1.46
1.52
1.25
1.25
1.25
1.25
1.25
0.86
FIG. 1. Electron-impact mass spectrum of trimethylsilyl derivatives of pu-
rified and methylated 10-hydroxy-8-octadecenoic acid (HOD) obtained
with a Hewlett-Packard 5890 (Avondale, PA) gas chromatograph coupled
to an HP 5972 series mass selective detector. Compounds were separated
using a temperature gradient of 20°C/min from 70 to 170°C, holding 1
min at 170°C, and then raised 5°C/min to 250°C, followed by holding at
250°C for 15 min (helium at 0.67 mL/min). OTMS, trimethylsilyloxy.
aHOD, 10-hydroxy-8-octadecenoic acid.
JAOCS, Vol. 77, no. 1 (2000)

98
H. KIM ET AL.
2(R)-MCO-decanoates for determination of stereo configura- (17). The injection of standard racemic methyl 2(R,S)-MCO-
tion (see Experimental Procedures section). With this pro- decanoate separated into two peaks, one [methyl 2(S)-MCO-
gram, methyl 2-MCO-decanoate separated into a major peak decanoate] at 60.17 min and the other [methyl 2(R)-MCO-dec-
at 60.15 min with a minor peak at 60.37 min (about 3%) (Fig. anoate] at 60.38 min (Fig. 2B). Coinjection of the racemate
2A). It is known from previous work that methyl 2(S)-MCO- with the MCO derivative from HOD demonstrated that the
alkanoates elute earlier than methyl 2(R)-MCO-alkanoates main stereoisomer from HOD augmented the methyl 2(S)-
(12,13,17), specifically including methyl 2-MCO-decanoate MCO-decanoate peak from the racemic standard (Fig. 2C).
As expected, the GC-separated diastereomeric methyl 2(S)-
and 2(R)-MCO-decanoates afforded virtually identical mass
spectra essentially as reported previously (18), regardless of
their origin from HOD or the racemic methyl 2-hydroxydec-
anoate. In analogy with the elution order determined previ-
ously of these diastereomeric methyl 2(S)- and 2(R)-MCO-
decanoates (17,18), it was clear that the first major peak of
the MCO derivative from HOD was the methyl 2(S)-MCO-
decanoate and the second minor peak, the methyl 2(R)-MCO-
decanoate, suggesting that the hydroxyl group at C10 of HOD
was mainly in the S-configuration with a minor isomer of R-
configuration (about 3%). These results coincided with our
recent findings (18) that the main final product, DOD, was in
the 7(S),10(S)-dihydroxy stereoconfiguration. A minor DOD
isomer (about 3%) with a similar mass spectrum was sepa-
rated from the main DOD isomer by HPLC (Kim, H., and
C.T. Hou, unpublished data). It follows from the findings of
3% 10(R) HOD that the minor DOD isomer may have the
7(S),10(R)-dihydroxy configuration.
Based on the results of this study and previous work (10),
the overall bioconversion pathway of oleic acid to DOD by
strain PR3 is postulated as shown in Figure 3. Oleic acid is
first converted to HOD by PR3, during which one hydroxyl
group is introduced at C10(S) and a double bond is shifted
from C9,10 cis to C8,9 trans. Although Hou (19,20) reported
a hydratase-catalyzed C10-hydroxylation without retention
of the C9,10-double bond by Flavobacterium sp. DS5, it is
unlikely that a hydratase is involved in the PR3 bioconver-
sion inasmuch as the double bond at C9,10 is not lost, but it
is shifted to C8,9 as the trans isomer. The hydration of oleic,
linoleic, and linolenic acid to their corresponding 10-hydroxy
FIG. 2. (A) Total ion chromatogram of methyl 2-menthoxycarbonyl
(MCO)-decanoate derived from the purified HOD by oxidative cleav-
age after MCO derivatization followed by methylation. First peak is cen-
tered at 60.15 min and the second peak is centered at 60.37 min. (B)
Total ion chromatogram of methyl 2(S,R)-MCO-decanoate standard pre-
pared by MCO derivatization of the racemic methyl 2(S,R)-hydroxydec-
anoate followed by methylation. First peak is the 2(S)-isomer (centered
at 60.17 min) and the second peak is the 2(R)-isomer (centered at 60.37
min). (C) Total ion chromatogram of the coinjected MCO derivatives
derived from the purified HOD (see A) and the standard racemic methyl FIG. 3. Postulated bioconversion pathways of oleic acid leading to
2(S,R)-hydroxydecanoate. First peak is centered at 60.16 min and the
second peak is centered at 60.37 min. See Figure 1 for abbreviation.
7,10-dihydroxy-8(E)-octadecenoic acid (DOD) by Pseudomonas aeru-
ginosa (strain PR3). For other abbreviation see Figure 1.
JAOCS, Vol. 77, no. 1 (2000)

BIOCONVERSION OF OLEIC ACID
99
8. Mercade, E., M. Robert, M.J. Espuny, M.P. Bosch, M.A. Man-
reesa, J.L. Parra, and J. Guinea, New Surfactant Isolated from
Pseudomonas sp. 42A2, Ibid. 65:1915–1916 (1988).
9. De Andres, C., E. Mercade, J. Guinea, and A. Manresa, 7,10-
Dihydroxy-8E-octadecenoic Acid Produced by Pseudomonas
sp. 42A2: Evaluation of Different Cultural Parameters of the
Fermentation, World J. Microbiol. Biotechnol. 10:106–109
(1994).
10. Hou, C.T., and M.O. Bagby, 10-Hydroxy-8(Z)-octadecenoic
Acid, an Intermediate in the Bioconversion of Oleic Acid to
7,10-Dihydroxy-8(E)-octadecenoic Acid, J. Ind. Microbiol.
9:103–107 (1992).
11. Guerrero, A., I. Casals, M. Busquets, Y. Leon, and A. Manresa,
Oxidation of Oleic Acid to (E)-10-Hydroperoxy-8-octadecenoic
and (E)-10-Hydroxy-8-octadecenoic Acids by Pseudomonas sp.
42A2, Biochim. Biophys. Acta 1347:75–81 (1997).
12. Hamberg, M., Steric Analysis of Hydroperoxides Formed by
Lipoxygenase Oxygenation of Linoleic Acid, Anal. Biochem.
43:515–526 (1971).
products have been reported from various microbes as re-
viewed by Hou (20). Another group reported that a lipoxyge-
nase-type enzyme located in the periplasm of Pseudomonas
sp. 42A4 was involved in the formation of (E)-10-hydroper-
oxy-8-octadecenoic and (E)-10-hydroxy-8-octadecenoic
acids from oleic acid (11). Although there is little evidence
for oleic acid-specific lipoxygenases in the literature, a free
radical mechanism of lipoxygenase would explain oxidation
at C10 and the double bond shift. Allylic hydroxylations with
double bond migration also are known to occur as a result of
cytochrome P450 oxidation of fatty acids (21). The resulting
plausible intermediate, HOD, is then subject to another hy-
droxylation at C7(S) by possibly the same enzyme(s) in-
volved in the first hydroxylation reaction resulting in DOD
formation. Further work should be done to investigate the
bacterial enzyme(s) involved in hydroxylation.
13. Hamberg, M., R.P. Herman, and U. Jacobsson, Stereochemistry
of Two Epoxy Alcohols from Saprolegnia parasitica, Biochim.
Biophys. Acta 879:410–418 (1986).
14. Kleiman, R., and G.F. Spencer, Gas Chromatography–Mass
Spectrometry of Methyl Esters of Unsaturated Oxygenated Fatty
Acids, J. Am. Oil. Chem. Soc. 50:31–38 (1973).
ACKNOWLEDGMENT
We gratefully acknowledge the excellent technical assistance of
A.L. Ashbury. We thank Dr. David Weisleder for NMR, and Alan
Lanser for FTIR analysis.
15. Official and Tentative Methods of the AOCS, 3rd edn., edited by
R.O. Walker, American Oil Chemists’ Society, Champaign,
1981, Method Cd 14-61.
16. Tulloch, A.P., and M. Mazurek, 13C Nuclear Magnetic Reso-
nance Spectroscopy of Saturated, Unsaturated, and Oxygenated
Fatty Acid Methyl Esters, Lipids 11:228–234 (1976).
17. Fahlstadius, P., and M. Hamberg, A Gas–Liquid Chromato-
graphic Method for Steric Analysis of Epoxy Acids, Chem.
Phys. Lipids 51:15–22 (1989).
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[Received April 21, 1999; accepted September 24, 1999]
JAOCS, Vol. 77, no. 1 (2000)