Preparation of methyl cis-9, trans-11- and trans-9, trans-11-octadecadienoate-17,17,18,18-d4, two of the isomers of conjugated linoleic acid

Article abstract of DOI:10.1016/S0009-3084(97)00053-4

A multi-step synthesis was used to prepare the cis-9, trans-11- and trans-9, trans-11-isomers (in a ratio of 46/54) of conjugated linoleic acid (cis-9, trans-11-octadecadienoic acid) labeled with deuterium atoms on the 17- and 18-carbon atoms (17,17,18,18-d₄). The methyl cis/trans-9, trans-11-octadecadienoate-17,17,18,18-d₄ isomer pair were obtained from the Wittig coupling of trans-2-nonenyltriphenyl-phosphonium bromide (8,8,9,9-d₄) and methyl 9-oxononanoate. To prepare the phosphonium bromide. 5-hexyn-1-o1 was reduced with deuterium gas/Wilkinson's catalyst to yield 1-hexanol-5,5,6,6-d₄. The alcohol was converted to the iodide with phosphorous pentoxide/phosphoric acid/potassium iodide. Coupling of the iodide with 2-propyn-1-o1 via lithium amide in liquid ammonia gave 2-nonyn-1-o1-d₄. The acetylenic alcohol was reduced with lithium metal in liquid ammonia to yield the trans-2-nonen-1-o1-d₄. The alcohol was converted to the bromide (using triphenylphosphine dibromide) and then converted to the phosphonium salt. The aldehyde ester was prepared by the reductive ozonization of methyl 9-cis-octadecenoate. The two conjugated linoleic acid isomers, formed during the final Wittig coupling reaction, were readily separated by a combination of reversed-phase and silver resin chromatography. Isotopic and chemical purities were > 95% for each geometric isomer. Overall yield (both isomers) from the 8-step synthesis was 12%.

Full text of DOI:10.1016/S0009-3084(97)00053-4

Chemistry and Physics of Lipids
88 (1997) 107–112
Preparation of methyl cis-9, trans-11- and trans-9,
trans-11-octadecadienoate-17,17,18,18-d4, two of the isomers of
conjugated linoleic acid1
R. Adlof *
USDA, Agricultural Research Ser6ice, National Center for Agricultural Utilization Research, Food Quality and Safety Research,
1815 N. Uni6ersity Street, Peoria, IL 61604, USA
Received 15 May 1997; accepted 11 July 1997
Abstract
A multi-step synthesis was used to prepare the cis-9, trans-11- and trans-9, trans-11-isomers (in a ratio of 46/54)
of conjugated linoleic acid (cis-9, trans-11-octadecadienoic acid) labeled with deuterium atoms on the 17- and
18-carbon atoms (17,17,18,18-d4). The methyl cis/trans-9, trans-11-octadecadienoate-17,17,18,18-d4 isomer pair were
obtained from the Wittig coupling of trans-2-nonenyltriphenyl-phosphonium bromide (8,8,9,9-d4) and methyl
9-oxononanoate. To prepare the phosphonium bromide, 5-hexyn-1-ol was reduced with deuterium gas/Wilkinson’s
catalyst to yield 1-hexanol-5,5,6,6-d4. The alcohol was converted to the iodide with phosphorous pentoxide/phospho-
ric acid/potassium iodide. Coupling of the iodide with 2-propyn-1-ol via lithium amide in liquid ammonia gave
2-nonyn-1-ol-d4. The acetylenic alcohol was reduced with lithium metal in liquid ammonia to yield the trans-2-nonen-
1-ol-d4. The alcohol was converted to the bromide (using triphenylphosphine dibromide) and then converted to the
phosphonium salt. The aldehyde ester was prepared by the reductive ozonization of methyl 9-cis-octadecenoate. The
two conjugated linoleic acid isomers, formed during the final Wittig coupling reaction, were readily separated by a
combination of reversed-phase and silver resin chromatography. Isotopic and chemical purities were \95% for each
geometric isomer. Overall yield (both isomers) from the 8-step synthesis was 12%. © 1997 Elsevier Science Ireland
Ltd.
Keywords: Fats; Deuterium; Conjugated; Cis; Trans; Isomers
* Tel.: +1 309 6816278; Fax: +1 309 6816679; E-mail: adlofro@mail.ncaur.usda.gov
1 Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of
the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be
suitable.
0009-3084/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-3084(97)00053-4

108
R. Adlof / Chemistry and Physics of Lipids 88 (1997) 107–112
1. Introduction
m×0.32 mm SP2380 (Supelco, Bellefonte, PA)
capillary column and flame ionization detector
(FID). Helium was utilized as carrier gas. Methyl
ester peaks were identified by comparison with
standard FAME mixtures of known composition.
Gas chromatography/mass spectrometry (GC/
MS) was used to determine the quantity of
deuterated and non-deuterated FAME. Analyses
were made on a Hewlett-Packard Model 5889
GC/MS (quadrapole; positive chemical ionization
mode; isobutane as ionizing gas) equipped with a
30 m×0.25 mm Supelcowax 10 fused silica capil-
lary column (Supelco, Bellefonte, PA). Data col-
lection and manipulation have been described
previously (Rohwedder et al., 1985).
To study the metabolism and interaction of
fatty acid (FA) cis and trans isomers in humans
(Emken, 1979; Emken et al., 1980), we have syn-
thesized various deuterium-labelled monoenoic
(Adlof and Emken, 1978), dienoic (Adlof and
Emken, 1981, 1985, 1987), and trienoic (Adlof
and Emken, 1985, 1987) FAs (Rakoff, 1982).
Conjugated linoleic acid (CLA; cis-9, trans-11-oc-
tadecadienoic acid) has been associated with the
reduction of chemically-induced cancers in mice
and rats and the suppression of atherosclerosis in
rats (Steinhart, 1996; Chin et al., 1992; Belury et
al., 1996; Ip et al., 1996). The cis-9, trans-11-iso-
mer is considered to be the active constituent
(Chin et al., 1992), but contributions by other
isomers (trans-9, trans-11-) have not been ruled
out. Deuterium-labeled CLA isomers can be
safely used in humans to trace their metabolism
and interaction(s) with other fats. A synthesis was
therefore developed to generate both CLA iso-
mers, deuterium atom-labeled, in sufficient yield
and of high enough isotopic and chemical purity
for use in humans.
2.3. Liquid chromatography:
Silver resin chromatography for preparative
separation of CLA isomers was done on a 2.5×
45 cm glass column packed with 200/270 mesh,
silver ion-saturated Rohm and Hass XN1010
macroreticular sulfonic acid resin (Emken et al.,
1978). For silver ion incorporation and packing of
the glass columns see Adlof (1994). Samples were
applied by needle-tipped syringe through a sep-
tum to the top of the column. The C18 RP system
consisted of a 50×250 mm stainless steel column
(5 vm particle size; Serva Feinbiochemica, Heidel-
berg, Germany) and a Rheodyne 7125 injector
(Rheodyne, Cotati, CA) with a 2 ml injection
loop. Both systems utilized Waters 510 HPLC
pumps and R403 refractive index detectors (both
Waters Associates, Milford, MA).
2. Experimental
2.1. Reagents
Lindlar catalyst, n-butyl lithium (2.6 M in hex-
ane), triphenylphosphine, lithium metal, magne-
sium turnings, ethyl bromide, and 2-propynol
were obtained from Aldrich (Milwaukee, WI).
5-Hexynol was purchased from Farchan Labora-
tories (Gainesville, FL) and tris-(triphenylphos-
phine) chlororhodium from Strem Chemicals
(Newburyport, MA). Silica gel (60-200 mesh) was
obtained from J.T. Baker (Jackson, TN) and deu-
terium gas, 98%, from Matheson Gas Products
(Secaucus, NJ).
2.4. Ag-HPLC:
A Spectra-Physics P2000 solvent delivery sys-
tem (Spectra-Physics Analytical, Fremont, CA), a
Rheodyne 7125 injector (Rheodyne, Cotati, CA)
with a 20 vl injection loop, and an ISCO V4
Absorbance Detector (Isco, Lincoln, NE) were
used. The ChromSpher Lipids columns (Cat. No.
28313; 4.6 mm i.d.×250 mm stainless steel; 5
micron particle size; silver ion impregnated) were
purchased from Chrompack International, Mid-
delburg, The Netherlands, and used as received.
Two Lipids columns connected in series resulted
2.2. Analyses of HPLC fractions
Fatty acid methyl esters (FAMEs) were ana-
lyzed with a Varian 3400 GC (Varian Instru-
ments, Palo Alto, CA) equipped with a 30

R. Adlof / Chemistry and Physics of Lipids 88 (1997) 107–112
109
in improved peak-to-peak resolutions. Solvent
flow was standardized at 1.0 ml/min; isocratic
conditions [0.35% acetonitrile (ACN) in hexane;
23°C] were used to minimize variations in FAME
retention(s) and resolution(s). The void volume of
this pair of columns was 4.2 ml.
Preparation: 2-(5-hexynyloxy)tetrahydropyran,
1. The tetrahydropyranyl (THP) ether of 5-
Hexyn-1-ol was prepared [dihydropyran, p-tolue-
nesulfonic acid in diethyl ether (EE)] in 85% yield
as described previously (Adlof and Emken, 1978)
(b.p. 73–76°C/0.15 mmHg).
Preparation: 2-(5,5,6,6-tetradeutero-hexyloxy)
tetrahydropyran, 2. Compound 1 (47.6 g; 550
mmol) in benzene (1000 ml) was treated with
deuterium (D2) gas at atmospheric pressure in the
presence of tris-(triphenylphosphine) chloro-
rhodium (8 g) in the manner described previously
for a 5-carbon analogue (Adlof and Emken,
1981). The residue was eluted with petroleum
ether (PE) through a 5×50 cm glass column
packed with silica gel. Removal of the solvent by
rotary evaporator yielded a colorless oil (92 g;
92% yield). Deuterium distribution by GC/MS:
2.0% d0, 1.3% d3, 96.7% d4.
3. Synthetic procedures
Both compounds were synthesized from a single
synthetic sequence (Fig. 1) involving a combina-
tion of acetylenic and Wittig coupling reactions.
Experimental details for most of these steps (or
for the preparation of analogues) have been pub-
lished elsewhere (Adlof and Emken, 1978, 1981,
1985, 1987).
Preparation: 1-Iodohexane-5,5,6,6-d4, 3. The
halide was prepared from compound 2 using
phosphoric acid and potassium iodide (KI) as
described previously for a 5-carbon analogue.
(Adlof and Emken, 1981). Purification by distilla-
tion through a short-path column (b.p. 50–51°C
at 3.0 mmHg) gave compound 3 (30.9 g; 143
mmoles) in a yield of 65%.
Preparation: 2-Nonyn-1-ol-8,8,9,9-d4, 4. Liquid
ammonia (approx. 180 ml) was added to a 500 ml,
three-neck flask equipped with a mechanical stir-
rer, a dry ice/acetone cooled condenser, and im-
mersed in a dry ice/acetone bath (temperature
maintained at approx. −40°C). As catalyst for
the formation of lithium amide, Fe(NO3)3 ·9H2O
(0.06 g) was added. Lithium metal (1.0 g; 140
mmol) was added in small pieces over 15 min.
After the grey slurry appeared, 2-propynol (3.9 g;
69 mmol) in tetrahydrofuran (THF; 40 ml) was
added dropwise over 15 min. Compound 3 (10 g;
46 mmol) in THF (35 ml) was added dropwise
over 15 min. The slurry was stirred for 1 h at
−35°C, then allowed to warm slowly to room
temperature (22°C) over 3.5 h. The slurry was
then warmed to 30°C, 100 ml of THF was added,
and stirring was continued (under argon atmo-
sphere) at room temperature for 16 h. The flask
was cooled in an ice bath and water (50 ml) was
added dropwise. The reaction mixture was trans-
Fig. 1. Synthetic scheme for preparation of methyl cis-9,
trans-11- and trans-9, trans-11-octadecadienoate-17,17,18,18-
d4. Compound numbers are underlined; yields for each step
are given in brackets.

110
R. Adlof / Chemistry and Physics of Lipids 88 (1997) 107–112
ferred to a round-bottomed flask and the THF
removed by rotary evaporation. The residue was
transferred to a separatory funnel with EE (150
ml) and the layers were separated. The water layer
was acidified with 5N sulfuric acid (H2SO4) and
extracted with 4×50 ml EE. The EE fractions
were combined and washed with 50 ml saturated
ammonium chloride (NH4Cl), 50 ml of 5N
H2SO4, 2×50 ml water and dried over sodium
sulfate (Na2SO4). After removal of the drying
agent and ether, the residue was eluted through
100 g silica gel with PE, then 75/25 PE/EE to give
4 (5.8 g; 86% yield).
Preparation: 2-Trans-nonen-1-ol-8,8,9,9-d4, 5.
Compound 5 was prepared by modification of a
procedure described in Otsuki et al. (1986). Com-
pound 4 (2.0 g; 14 mmol) in 40 ml THF was
added to a 200 ml 3-neck round-bottomed flask
equipped with a thermometer, argon inlet, me-
chanical stirrer, dry ice/acetone reflux condenser,
and a drying tube (calcium chloride; CaCl2). The
flask was cooled in a dry ice/acetone bath at
−35°C. Liquid ammonia (approx. 100 ml) was
introduced slowly, with stirring, and then lithium
metal (1.0 g; 140 mmol) was added in small pieces
over 15 min. The blue-black solution was stirred
at reflux conditions (−33°C) for 3 h, then slowly
warmed to room temperature over 1.5 h. The
slurry was heated to 40°C, stirred for another
hour, then cooled to 10°C and water (20 ml) was
slowly added via syringe. After the vigorous evo-
lution of gas had ceased, the slurry was acidified
with 50 ml 6 M hydrochloric acid (HCl) and
transferred to a separatory funnel with 100 ml
EE. The layers were separated and the water layer
was extracted with 4×25 ml EE. The EE frac-
tions were combined and dried over Na2SO4. Af-
ter removal of the drying agent and EE, the
residue was eluted through 15 g silica gel with
95/5 PE/EE to give 5 (1.8 g; 88% yield).
at a temperature of 10°C. Compound 5 (4.2 g; 29
mmol) dissolved in methylene chloride (5 ml) was
added over 15 min at a temperature of 10°C. The
cooling bath was removed and the solution was
stirred at room temperature for 0.5 h. PE (35 ml)
was added and the slurry was chromatographed
on 25 g silica gel (PE as solvent). Removal of the
solvent resulted in 5.1 g of 6 (99% pure; 88%
yield).
Preparation: 8,8,9,9-Tetradeutero-trans-2-non-
enyltriphenylphosphonium bromide, 7. Com-
pound 6 (2.5 g; 12 mmol), triphenylphosphine (3.9
g; 15 mmol) and ACN (50 ml) were combined in
a 100 ml, 1-neck round bottomed flask equipped
with a reflux condenser and an argon inlet. The
mixture was heated to reflux (oil bath) and stirred
magnetically for 23 h. The homogeneous solution
was cooled to room temperature, the acetonitrile
was removed on a rotary evaporator and the
viscous residue was triturated several times with
fresh portions of EE. After crystallization, the
residue was isolated by vacuum filtration and
dried in a vacuum oven to give compound 7 (5.1
g; 91% yield). Compound 7 had a m.p. (uncor-
rected) of 166–169°C.
Preparation: Methyl 9-oxo-nonenoate, 8. This
compound [prepared by ozonolysis of methyl cis-
9-octadecenoate and subsequent zinc/acetic acid
reduction (Pryde et al., 1960)] was freshly distilled
before use.
Preparation: Methyl cis/trans-9, trans-11-oc-
tadecadienoate-17,17,18,18-d4, 9. Compound 7
(5.1 g; 11 mmol) was dissolved in 80 ml hexam-
ethylphosphoramide/THF (1:9 v/v) in a 200 ml
3-neck flask equipped with an argon inlet, a me-
chanical stirrer, a low temperature thermometer,
and a CaCl2 drying tube. The apparatus was
cooled to −74°C with a dry ice/acetone bath and
n-butyl lithium (2.0 M in hexanes; 6.5 ml; 13
mmol) was added by syringe over 25 min. The
orange-brown slurry was stirred at −74°C for 1.5
h, then compound 8 (90% pure; 2.5 g; 13 mmol)
was added dropwise over 0.5 h. The slurry was
stirred at −74°C for another 5 h, the dry ice/ace-
tone bath was removed, and the slurry was al-
lowed to warm to room temperature overnight.
Saturated sodium chloride solution (40 ml) was
added and the slurry was transferred to a separa-
Preparation: 1-Bromo-trans-2-nonen-8,8,9,9-d4,
6. Triphenylphosphine (7.6 g; 29 mmol) was dis-
solved in methylene chloride (20 ml) in a 100 ml
3-neck flask equipped with a mechanical stirrer, a
burette, a thermometer, an inert gas inlet and a
calcium chloride drying tube. The apparatus was
flushed with a stream of argon and immersed in
an ice bath. Bromine (4.6 g; 29 mmol) was added

R. Adlof / Chemistry and Physics of Lipids 88 (1997) 107–112
111
tory funnel with water. The layers were separated
and the water layer was extracted with 50 ml PE.
The organic layers were combined, washed with
2×50 ml water, then dried over Na2SO4. Re-
moval of the drying agent and solvents provided
the residue 9 (3.6 g). Residue 9 was purified by a
combination of reversed-phase and silver resin
chromatography (see Section 2 for details).
Initial purification was accomplished on a C18
reversed-phase chromatography column (6.5 ml
ACN/min; 1.9 g sample size per injection; see
Section 2 for details) to yield a 1.75 g mixture of
the isomer pair (44% cis/trans; 54% trans/trans).
The isomer pair were separated by silver resin
chromatography (7 ml methanol/min; 500 mg
sample size per injection; see Section 2 for details.)
The isolated yields of the isomers were: 687 mg
cis-9, trans-11- (97% pure; 20% yield; 2.5% trans/
trans and 0.5% trans/cis) and 808 mg 9-trans,
11-trans- (25% yield; 99% pure).
Fig. 2. GC analysis of synthesized CLA-d4 using an SP2340
capillary column (see Section 2 for details). [Methyl cis-9,
trans-11- (Peak c 1); trans-9, cis-11- (c2); cis-9, cis-11-
(c3); trans-9, trans-11-(c4) octadecadienoate-17,17,18,
18-d4 ].
readily isolated (m.p. 134–136°C, uncorrected)
and used in the subsequent Wittig coupling reac-
tion. Conditions for the Wittig reaction were iden-
tical to those described for the preparation of 9.
The reaction proceeded more rapidly than when
the bromide was used and total CLA isomer
yields were comparable for the bromide and io-
dide salts. When product 9 (prepared using the
iodide salt precursor) was analyzed by GC, two
by-products were noted (Fig. 2). Eluting just
after the methyl cis-9, trans-11-octadecadienoate-
17,17,18,18-d4 (peak 1) and before the methyl
trans-9, trans-11-octadecadienoate-17,17,18,18-d4
(peak 4), they are tentatively identified (by coin-
jection of GC standards) as methyl trans-9, cis-
11-octadecadienoate-17,17,18,18-d4 (peak 2) and
methyl cis-9, -3-cis-11-octadecadienoate-17,17,
18,18-d4 (peak 4). The two components respec-
tively comprised 9.2% and 8.5% of the total
CLA mixture. While the cis-9, cis-11- isomer
could be separated by silver resin chromatogra-
phy, the trans-9, cis-11- isomer could not be
separated from the cis-9, trans-11- isomer by re-
versed-phase or silver resin chromatography.
Dual-column Ag-HPLC (10 vg sample size;
0.35% ACN in hexane; see Section 2 for de-
tails) was also unsuccessful in separating these
isomers. Methyl trans-9, cis-11-octadecadienoate-
4. Results and Discussion
Several points of interest were noted during this
synthesis. 5-Hexynol was converted to the THP
ether (1) to eliminate the possibility of hydrogen
exchange during deuteration. The THP ether
could also be readily converted to the iodide. The
lower-than-expected yield of 3 was due to some
loss of material during solvent removal. The deu-
terium atoms were located on the 17- and 18-
positions to minimize potential isotope effects.
The deuterium atoms may be located from car-
bon-14 to carbon-18, the number of deuterium
atoms varied from d2 (olefinic alcohol precursor)
to d6 (‘ene-yne-ol’ precursor). The overall CLA-d4
yield of 12% was accomplished without pre-purifi-
cation of solvents, etc.
We had previously found that use of the iodide
rather than the bromide (6) for preparation of the
phosphonium salt resulted in more easily purified
phosphonium salts and higher yields of product in
the subsequent Wittig coupling reaction (Adlof
and Emken, 1985). Our initial synthesis of CLA
thus included conversion of the olefinic bromide 6
to the iodide (NaI in acetone, reflux conditions).
The triphenylphosphonium salt of the iodide was

112
R. Adlof / Chemistry and Physics of Lipids 88 (1997) 107–112
Adlof, R.O., Emken, E.A., 1981. Preparation of methyl cis-9,
cis-12- octadecadienoate-16,16,17,17-d4. Chem. Phys.
Lipids 29, 3–9.
17,17,18,18-d4 and methyl cis-9, cis-11-octadeca-
dienoate-17,17,18,18-d4 were present at only 0.3
and 0.8%, respectively, when the alkenyl phospho-
nium bromide (6), rather than the iodide, was
used in the final Wittig coupling step.
Adlof, R.O., Emken, E.A., 1985. Preparation of methyl
8c,11c-octadecadienoate-17,17,18,18-d4
8c,11c,14c-octadecatrienoate-17,17,18,18-d4.
Comp. Radiopharm. 23, 149–160.
and
methyl
Label.
J.
Wittig coupling reactions between a moderate
(semi-stabilized), h-unsaturated phosphonium salt
with a saturated aldehyde (n-BuLi catalyst; THF
as solvent; −30°C) tend to produce high (50% or
more) percentages of the trans isomer at the newly
formed double bond and to cause some isomeriza-
tion at the existing double bond (approx. 20%
conversion of trans to cis) (Ideses and Shani,
1989). Applying the composition data provided in
Ideses and Shani (1989) to our Wittig coupling
reactions, the calculated theoretical ratio of the
CLA isomers should be 40% cis/trans, 9% trans/
cis, 9% cis/cis and 40% trans/trans. This data is in
excellent agreement with our observed ratio of
40.0, 9.2, 8.5 and 42.3% when the h-unsaturated
phosphonium iodide salt was used. While both
our Wittig reactions were run at −74°C, use of
the less reactive bromide salt yielded a CLA mix-
ture with the more favorable ratio of 46.6, 0.3, 0.8
and 52.3% respectively. A single synthesis may
thus be used to prepare roughly equal amounts of
two CLA isomers (labelled with four deuterium
atoms) in high isotopic and chemical purity.
Adlof, R.O., Emken, E.A., 1987. Preparation of methyl
8c,11c-eicosadienoate-17,17,18,18-d4, methyl 8c,11c,14c-ei-
cosatrienoate-17,17,18,18-d4 and methyl 5c,8c,11c-eicosa-
trienoate-17,17,18,18-d4. J. Label. Comp. Radiopharm. 24,
699–711.
Belury, M.A., Nickel, K.P., Bird, C.E., Wu, Y., 1996. Dietary
conjugated linoleic acid modulation of phorbol ester skin
tumor promotion. Nutr. Cancer 26, 149–157.
Chin, S.F., Liu, W., Storkson, J.M., Ha, Y.L., Pariza, M.W.,
1992. Dietary sources of conjugated dienoic isomers of
linoleic acid, a newly recognized class of anticarcinagens. J.
Food Comp. Anal. 5, 185–197.
Emken, E.A., 1979. In: Emken, E.A., Dutton, H.J. (Eds.),
Geometrical and Positional Fatty Acid Isomers, American
Oil Chemists’ Society, Champaign, IL. pp. 99–129.
Emken, E.A., Dutton, H.J., Rohwedder, W.K., Adlof, R.O.,
1980. Distribution of Deuterium-labelled cis- and trans-12-
octadecenoic acids in human plasma and lipoprotein lipids.
Lipids 15, 864–871.
Emken, E.A., Hartman, J.C., Turner, C.R., 1978. Separation
of saturated, unsaturated, and acetylenic fatty acid isomers
by silver ion chromatography. J. Am. Oil Chem. Soc. 55,
561–563.
Ideses, R., Shani, A., 1989. The Wittig reaction: comments on
the mechanism and application as a tool in the synthesis of
conjugated dienes. Tetrahedron 45, 3523–3534.
Ip, C., Briggs, S.P., Haegele, A.D., Thompson, H.J., Storkson,
J., Scimeca, J.A., 1996. The efficacy of conjugated linoleic
acid in mammary cancer prevention is independent of the
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Acknowledgements
Otsuki, T., Brooker, R.F., Funk, M.O., 1986. Two geometric
isomers of linoleic acid: Improved total synthesis. Lipids
21, 178–181.
E. Walter is thanked for technical assistance.
Pryde, E.H., Anders, D.E., Teeter, H.M., Cowan, J.C., 1960.
The ozonization of methyl oleate. J. Am. Oil Chem. Soc.
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