Conjugated Linoleic Acid Oils: Bioriginal Food and Science Corp. Saskatoon, Saskatchewan, Canada

1
Conjugated Linoleic
Acid Oils
Rakesh Kapoor, Martin Reaney, and Neil D. Westcott
Bioriginal Food and Science Corp.
Saskatoon, Saskatchewan, Canada
1. INTRODUCTION
The discovery of conjugated linoleic acid (CLA) dates back to 1933, when it was
found that treatment of polyunsaturated fatty acids with alkali increased the UV
absorbency (13). It was later found that the treatment produced a one to one
mixture of cis-9-, trans-11- (9,11-ct) and trans-10-, cis-12 (10,12-tc) CLA. In
1935, it was noted that UV absorbency at 230 nm of milkfats was higher in milk
from cows fed polyunsaturated fatty acids than cows fed saturated fats (4). This
phenomenon was shown to be a result of conjugation of the double bonds of the
polyunsaturated fatty acids (5). The predominant isomer in dairy products (milk,
cheese, butter) or meat is 9,11-ct CLA (6).
Since the time of their discovery, conjugated fatty acids have been the subject of
intense investigation. Spectroscopic analysis using ultraviolet light was the major
analytical instrument available to researchers in the 1930s. As a result of high absor-
bance of UV light at 230 nm or higher, conjugated fat became a useful research tool
for the study of fat metabolism. The rst animal study used naturally conjugated
fats of tung oil, but it was poorly tolerated (7). During this period, essential fatty
Baileys Industrial Oil and Fat Products, Sixth Edition, Six Volume Set.
Edited by Fereidoon Shahidi. Copyright # 2005 John Wiley & Sons, Inc.
1
acids were discovered. Aaes-Jorgensen (8) studied the possibility of using CLA to
treat/prevent the symptoms of essential fatty acid-deciency. They observed that in
essential fatty acid-decient animals, CLA could not prevent deciency but showed
toxicity. Synthetic conjugated fatty acids produced from linoleic acid (LA), usually
mixtures of 9,11-ct and 10,12-tc CLA, replaced the natural substances as a pre-
ferred biological marker.
Over the last two decades, the conjugated fats and, particularly, conjugated linoleic
acid (CLA) have been intensively studied for their biological activity. As a result
of the ease of synthesis, blends of two CLA isomers, namely 9,11-ct and 10,12-tc-
CLA, have been the focus of most research into biological activity. Recent research,
however, has been expanding to include pure or enriched isomer preparations.
2. METABOLISM
Early studies using a CLA mixture revealed that animals absorb and incorporate
some of the CLA in their tissues in phospholipid, glycolipid, and acylglycerol
fractions. In 1950, Reiser (9) observed faster and better absorption and incorpora-
tion of CLA when administered as triacylglycerol (TAG) versus free fatty acid.
Following administration of CLA as TAG, the maximal levels appear in blood,
liver, and organs at 16 hours compared with 24 hours for the free fatty acid
form. Incorporation was higher in mesentery fat followed by perirenal and subcu-
taneous fat (9). Barnes et al. (10), in 1941, reported the kinetics of CLA absorption
from the mixture. The absorption rate was highest in the rst hour following admin-
istration and then gradually declined. Following a single oral dose, over 50% of
conjugated CLA was absorbed in neutral mucosal lipids in the rst hour followed
by a decline, whereas in mucosal phospholipids, the incorporation was much
slower, reaching maximum at about 8 hours followed by decline. In 1951, it was
found that poultry incorporate CLA into egg lipids (5). Recent studies conrm
the earlier nding of preferential incorporation in neutral lipids followed by phos-
pholipids in liver (11) and mammary tissues (11, 12). Incorporation of CLA in tis-
sues is associated with a reduction in the amounts of arachidonic acid and linoleic
acid in neutral lipids (11) and in the liver TAG levels with an increase in the levels
of 18:0 (13). In pig heart lipids, 11,13-ct isomer was the major CLA isomer
followed by 9,11-ct isomer following an oral administration of CLA mixture
(14). CLA isomers compete with desaturases and elongases to produce desaturated
and elongated products maintaining the geometry of double bonds (1517). CLA
has been shown to inhibit delta-9-desaturase enzyme in vitro (18) and in vivo
(19). CLA feeding also resulted in a decrease in arachidonic acid, and also
reduced the desaturation of linoleic acid without affecting the desaturation of
alpha-linolenic acid to any signicant level. In rat, 9,11-ct isomer was preferen-
tially metabolized to conjugated C20:3, whereas 10,12-tc isomer was metabolized
to conjugated C16:2 and 18:3 compounds (19). The CLA mixture and its indivi-
dual isomers (9,11-ct and 10,12-tc) inhibited basal and calcium ionophore stimu-
lated production of prostaglandin from human sapheneous vein endothelial cells in
2 CONJUGATED LINOLEIC ACID OILS
a dose-dependent manner. The mixture of CLA isomers was reported to inhibit the pro-
duction of eicosanoids at all doses, whereas 10,12-tc isomer was shown to inhibit pro-
duction at lower dose but stimulate at higher dose (20).
3. PHYSIOLOGICAL ACTIONS OF CLA
3.1. Effect on Body Composition
While working with essential fatty acid decient rats in 1951, Holman observed
that CLA-fed rats had signicantly less total fat than control rats, and that they
lost weight (21). Subsequent studies demonstrated that CLA inhibited fat accumu-
lation and promoted lean muscle mass in growing animals, including pigs and mice
(2224). The results on the effect of CLA on body fat composition in animals are
unequivocal, whereas studies in humans are providing mixed results. In a double
blind, randomized clinical trial on obese and overweight humans, Blankson et al.
(25) observed a clinically signicant reduction in body fat mass in groups adminis-
tered various doses of CLA ranging from 1.7 g per day to 6.8 g per day. Reduction
in body fat mass was signicant for groups administered 3.4 g CLA per day and 6.8 g
CLA per day. Interestingly, this study found that 3.4 g of CLA per day provided
maximum reduction in body fat mass; increasing the dose above this level provided
no additional effect (25). Lean body mass and body mass index were similar in all
the groups, although there was a slight increase in lean body mass in the CLA
group. The increase in lean body mass did not achieve signicance compared
with a placebo group administered olive oil. Additionally, the CLA group presented
a signicant reduction in total-, LDL-, and HDL-cholesterol (25). In another study,
Riserus et al. (26) observed a reduction in saggital abdominal diameter in abdom-
inally obese humans without affecting total body weight. A reduction in total fat
mass in healthy, nonobese, exercising males was observed when CLA was given
at a total daily dose of 1.8 g (divided in 3 doses) for 12 weeks. Body weight was
not affected in this double blind clinical trial (27). A recent study in type II diabetic
patients who were not on any medication found an inverse relationship between
plasma CLA levels and weight loss and serum leptin levels (28). The inverse rela-
tionship was signicant for 10,12-tc isomer of CLA and not for 9,11-ct isomer. A
study in nonobese individuals using a mixture of CLA isomers containing about
20% each of 9,11-ct and 10,12-tc-CLA isomers, along with 20% to 25% other iso-
mers, did not observe a reduction in body fat mass (29). Another study investigated
the effect of CLA on weight regain after weight loss in overweight subjects (30).
This study observed no effect of CLA on weight gain after weight loss; however,
the weight gain in the CLA group was a result of an increase in fat free mass and
was independent of dose (30). Comparison between studies is difcult as these stu-
dies differed in the degree of obesity of the subjects, duration of treatment, dose,
and the isomer composition of the CLA preparation. Earlier commercial products
of CLA contained equal amounts of 9,11-ct and 10,12 tc isomers with other iso-
mers in small amounts. The other isomers include all trans-isomers as well as other
PHYSIOLOGICAL ACTIONS OF CLA 3
cis-, trans-isomers, including 11,13-ct, 11,13-tc, 8,10 ct,8,10-tc, etc. This illustrates
the need for research on specic isomers and standardized protocols.
These reported actions of CLA could be mediated through a number of physio-
logical mechanisms including increased fat oxidation (31) or inhibition of lipid
accumulation in fat cells. Recent studies have started to investigate the physiological
actions of individual isomers. It appears that the 10,12-tc isomer of CLA is mainly
responsible for the effect of CLA on adiposity (3234).
It was demonstrated that 10,12-tc-CLA reduces leptin, a hormone involved in
regulation of fat deposition, in cultured fat cells (35), and in mice (36). In the lat-
ter study, feeding 10,12-tc-CLA to mice caused a comparatively small gain in
weight with no gain in adipose fat. The 10,12-tc isomer of CLA was also shown
to inhibit differentiation of preadipocytes in murine (3T3-L1) (37) and human
preadipocytes (38). This was associated with decreased accumulation of TAGs
in differentiating preadipocytes; inhibition of peroxisome proliferator-activated
receptor g (PPAR-g) gene (38) and its downstream gene products including lipo-
protein lipase (LPL), GLUT-4 (glucose transporter gene 4), and inhibited expres-
sion of fatty acid synthase (FAS) gene. CLA isomer 9,11-ct, on the other hand,
increased accumulation of TAGs in adiposities and also stimulated GLUT-4 and
LPL. This suggests that isomer 10,12-tc may be responsible for inhibition of glu-
cose uptake and oxidation in the adipocytes, leading to decreased TAG accumula-
tion. These actions also underlie the effect of 10,12-tc isomer in inducing insulin
resistance leading to lipoatrophic diabetes observed in animal (39, 40) and human
studies (41).
3.2. Anticancer Properties
In 1985, Pariza and Hargrave discovered an antimutagenic fraction in cooked and
raw beef during their studies on identication of carcinogenic compounds present
in cooked beef (42). This fraction was identied to be a mixture of 4 isomers of
CLA (9,11-ct, 9,11-tt, 10,12-tc, and 10,12-tt) (43). Studies in animal models and
cell lines demonstrated the antimutagenic activity of a CLA mixture against known
chemical carcinogens (7,12-dimethylbenz[a]anthracene, DMBA, and benzo (a) pyr-
ene) (4347). CLA has been shown to have anticancer effects against breast, colon,
and prostate cancer cell lines (4850). In rat models of breast cancer, CLA was
reported to affect the breast structure when given during development stages. In
this study, dietary CLA reduced the proliferation of terminal end bud and lobuloal-
veolar bud structures, whereby breast tissue became resistant to neoplastic transfor-
mations associated with cancer at later stages in life (4850).
The exact mechanism of anticancer effects of CLA is not clear, and several pos-
sible mechanisms could underlie the anticancer properties of CLA. These actions
may include its ability to interfere with the proliferation of cancer cells, increased
apoptotic cell death, inhibition of angiogenesis, or increased oxidative stress. In a
study comparing the effects of CLA on estrogen receptor positive human breast
cancer cells (MCF-7) and estrogen receptor negative (MDA-MB 231) cells, CLA
was shown to selectively inhibit proliferation of estrogen receptor positive cells.
CLA-treated MCF-7 cells selectively remained in G0/G1 phase and the expression
of c-myc was inhibited. CLA had no effect on the growth of MDA-MB 231 cells.
This study suggests that CLA acts by interfering with the estrogen-mediated second
messenger system (51). CLA is also known to interfere with eicosanoids pathway
and inhibits production of prostaglandin E
2
(PGE
2
) (50, 52). Reduced production of
PGE
2
may play a role in anticancer actions of CLA. CLA is also shown to stimulate
apoptotic death of cancer cells (50, 53, 54). CLA isomers increased apoptosis
by stimulating the expression of caspase 3 and 9 activities and by reducing the
expression of Bcl-2, an apoptosis repressor gene. The 10,12-tc isomer of CLA
was found to be more potent in mediating these actions than either the 9,11-ct iso-
mer or a mixture of the two (54). The other possible mechanism for anticancer
properties of CLA is its ability to inhibit angiogenesis. In a mouse model of breast
cancer, both isomers inhibited angiogenesis in mammary fat pad and reduced
the concentration of vascular endothelium-derived growth factor (55). The CLA
isomer 10, 12-tc also inhibited secretion of leptin and induced apoptosis in white
and brown adipocytes, whereas 9,11-ct isomer was without effect on these para-
meters. CLAwas also shown to reduce cell proliferation by reducing the expression
of proteins involved in cell cycle regulation (p16 and p27) and DNA synthesis (56).
The above discussion focused on the role of CLA as a chemoprotective agent.
Information regarding its effect on cancer treatment is limited. Feeding CLA for
four or eight weeks after carcinogen administration was reported to be ineffective in
preventing tumor formation, whereas continuous administration protected against
tumor development (12, 57). A recent case control study in Finnish women suggested
that dietary CLA may be protective against breast cancer (58). The role of CLA in
prevention or treatment of cancer in humans is not clear and requires more research.
3.3. Insulin Resistance and Diabetes
CLA has been shown to normalize impaired glucose tolerance and improve hyper-
insulinemia in prediabetic ZDF rats (59). These actions appeared to be mediated
through PPAR-g pathway as CLA treatment induced expression of mRNA for
aP2. Recently, it has been observed in Zucker diabetic rats that either a 50:50 mix-
ture of 9,11-ct-CLA and 10,12-tc-CLA isomers or a 90% 9,11-ct-CLA isomer sti-
mulated insulin action in fat and muscle cells (60). These observations suggest that
CLA can prevent or delay the onset of diabetes and that the 9,11-ct isomer may
contribute much of this activity. Recently, 10,12-tc isomer of CLA was shown to
induce insulin resistance and hyperinsulinemia in mice, whereas 9,11-ct isomer
had no effect (61). Both these isomers were shown to be equally efcient in stimu-
lating PPAR-a and g receptors, indicating that the hyperinsulinemia may not be
mediated through nuclear receptor pathway. In another study using either high
metabolic rate mice or low metabolic rate mice, Hargrave et al. (62) demonstrated
that CLA increased the insulin resistance in high metabolic rate mice only, whereas
in Zucker diabetic rat, CLA was shown to prevent a rise in insulin and glucose
levels that might have been mediated through an increased production of adiponec-
tin, a hormone released by adipose tissues (63). In pigs, dietary CLA had no effect
on plasma glucose or insulin levels or on the ability of insulin to mobilize plasma
glucose (64). These studies indicate that the effect of CLA in diabetes is not clear
and the reported differences may be species specic. In human subjects, the effects
of CLA on insulin resistance and glucose homeostasis are not well studied. In one
clinical trial, the 10,12-tc isomer, but not a mixture of 9,11-ct and 10,12-tc isomers,
was shown to increase the insulin resistance and blood glucose levels in abdomin-
ally obese people (41), whereas another study on normolipidemic subjects failed to
observe any effect of either a 50:50 or 80:20 mixture of 9,11-ct and 10,12-tc iso-
mers on blood glucose or insulin levels (65). Belury et al. (28) observed a reduction
in fasting plasma glucose levels in type II diabetics when they were treated with
6.0 g of a mixture of CLA for eight weeks. This necessitates the need for controlled
studies in human to delineate the effect of CLA and its isomers on blood glucose
homeostasis and insulin resistance.
3.4. Cardiovascular Actions
An isomeric mixture of CLA was reported to inhibit the development of athero-
sclerosis in rabbits (66, 67) or hamsters (68) fed a high cholesterol diet. The CLA mix-
ture caused a regression of atherosclerosis in rabbits (67) and Apo E
-/-
mouse (69).
In hamsters, CLA was shown to lower the levels of total- and LDL-cholesterol and
TAG levels (33, 70). The reduction in plasma levels of cholesterol could be
mediated via an increase in LDL receptor expression in the liver leading to
increased clearance from the circulation (71). CLA has also been reported to reduce
secretion of apolipoprotein B in animals (72). The effect of CLA could be mediated
through induction of the PPAR-g pathway. CLA has been shown to inhibit cyclo-
oxygenase enzyme in vitro (69). Its anti-inammatory actions might be playing a
role in prevention of atherosclerosis but does not appear to play a role in regression
of atherosclerosis in animals. The 9,11-ct, and 10,12-tc isomers of CLA and the
metabolite of 9,11-ct isomer (13-hydroxy-9c,11t- octadecadienoic acid) have
been reported to inhibit arachidonic acid and collagen-induced platelet aggregation
(73), which could have been mediated through inhibition of thromboxane A2 for-
mation from arachidonic acid. CLA was also shown to prevent the development of
hypertension in Zucker diabetic rats (63) that was associated with increased expres-
sion of mRNA for adiponectin, a hormone released by adipose tissues. Similar
results on prevention of hypertension were shown in Otsuka Long-Evans Tokush-
ima fatty (OLETF) rats (74). In this rat model, the 10,12-tc isomer of CLA was
shown to inhibit angiotensinogen production from adipose tissues. The effect of
CLA on plasma cholesterol levels and atherogenic potential in human is not clear.
In healthy, normocholesterolemic human subjects, Benito et al. (75) reported no
effect of CLA on cholesterol levels, platelet aggregation, or bleeding time, whereas
Noone et al. (65) reported a TAG lowering effect of 50:50 mixture of CLA isomers.
These differences could be due to differences in the composition of CLA, as Noone
et al. (65) used 50 : 50 or 80 : 20 mixtures of 9,11-ct and 10,12-tc isomers and
Benito et al.s (75) preparation contained 11.4% 9,11-ct, 10.8% 8,10-tc, 15.3% 11,13-ct,
and 14.7% 10,12-tc, with 6.7% c,c and 5.9% tt isomers of CLA. To establish the effects
of CLA on cardiovascular risk factors in humans, more research is needed using
pure isomers or standardized mixture of CLA isomers.
3.5. CLA and Immune Function
In animal studies, CLA was reported to enhance immune response and attenuate
allergic reactions (76, 77). CLA has also been shown to prevent age associated
reductions in immune function (78). In Guinea pigs, feeding CLA was shown to
reduce the release of histamine and PGE
2
from isolated trachea challenged with
antigens (79), suggesting a strong antiallergic action. In mice, feeding CLA dose
dependently increased splenic lymphocyte proliferation in response to phytohemag-
glutinin but not to lipopolyssaccharide or conclavin A, suggesting that CLA has
selective actions on immune function. CLA stimulated IL-2 production (80) and
also increased the basal and mitogen stimulated natural killer cell activity of splenic
lymphocytes in mice that was associated with increased number of NK-cells but no
change in the ratio of NK-cells to total splenic lymphocytes (81). CLA has also
been shown to reduce the release of proinammatory mediators, including PGE
2
,
IL-1, and IL-6, from macrophages stimulated by interferon gamma (IFN-g) (82).
These actions appeared to be mediated through stimulation of PPAR-g pathway.
When fed to pigs, CLA enhanced cellular immunity by modulating white blood
cell types that control adaptive and innate immunity (83, 84). Feeding CLA to preg-
nant and lactating pigs caused an increase in IgG levels of colostrums without
affecting the serum IgG levels (85). In the same study, feeding CLA to suckling
piglets resulted in increased serum levels of IgG and lysosomes. These results indi-
cate enhancement of immune function in piglets. Following the feeding of CLA to
young healthy women, no effects were observed on several measures of immune
function including delayed type hypersensitivity response and numbers of circulat-
ing white cells and antibodies to vaccine, although an increase in CLA content of
mononuclear cells was observed (86, 87). Differences in immune responses may be
due to the selection of a young healthy population with optimal immune function,
species differences, or dietary CLA isomer composition.
CLA has recently been studied for its actions on peroxisome proliferator-
activated receptors (PPARs), most notably of the PPARg. As PPARg plays a role
in macrophage activity, Yu et al. (82) observed a stimulation of PPARg in
RAW264.7 mouse macrophage (RAW) cells by various CLA isomers. CLA also
decreased the production of PGE
2
, TNF-a, nitric oxide (NO), IL-1b, and IL-6 in
RAW cells treated with interferon-g (IFNg) (82). The inhibition of production of
these inammatory mediators was associated with a reduced expression of
mRNA for cyclo-oxygenase 2 (COX2), inducible NOS (iNOS), and tumor necrosis
factor alpha (TNF-a). Cheng et al. (88) observed similar inhibition of COX-2 and
NOS mRNA in lipopolysaccharide (LPS) stimulated macrophage cell, which was
associated with an inhibition of LPS-induced protein expression of the cytoplasmic
phosphorylated inhibitor kappaBalpha (IkBa) and nuclear p65 as well as NF-
kappaB nuclear protein-DNA binding afnity. This observation indicate a role of
NF-kappaB in regulation of anti-inamaotry actions of CLA.
4. STRATEGIES TO INCREASE DIETARY INTAKE OF CLA
As the interest in benecial effects of CLA is increasing, so are the efforts to
increase its dietary intake. Various strategies are being employed, which include
increasing the content of CLA in eggs, milk, and meat. In 1951, hens were shown
to incorporate CLA into egg lipids following dietary administration (5), but the egg
production was signicantly reduced. Recently, strategies to reduce the population
of problem birds, based on feeding CLA to the females to reduce the egg hatchabil-
ity, have been patented (89). Feeding 0.5% CLA in the diet of hens was shown to
increase the CLA content of egg lipids, which was associated with a signicant
increase in saturated fatty acids and a reduction in monounsaturated fatty acids
in the egg lipids. These changes in the egg composition also changed the properties
of the egg yolk in that it became hard when stored at cool temperatures (refrigera-
tor). This observation suggests that feeding CLA to poultry may not be an attractive
strategy to increase the CLA content of eggs as poultry breeders cannot bear the
economic burden of signicantly reduced fertility. Strategies are needed to
increase the content of CLA without affecting hatchability of eggs. Recently,
methods to increase the content of CLA in eggs have been patented (90, 91). These
strategies include incorporating CLA along with monounsaturated (91) or mono-
and polyunsaturated fatty acids (90) in the diet. Similarly, feeding CLA in a
diet to cattle increased CLA content in milk and meat and simultaneously reduced
total milkfat content (92) and increased milkfat saturated fatty acids. Other
strategies include feeding 11-trans-octadecaenoic (C18:1) acid (93) and other
sources of polyunsaturated fatty acids to cattle. Feeding either fresh forage or
supplementing the cattle diet with high linoleic acid meals/oils is another effective
way of increasing the content of CLA in milkfat (9497) and muscle. Most
common sources of polyunsaturated fatty acids include sunower oil, linseed
(axseed oil), safower oil, sh oil, or marine algae (98101). When CLA is fed
to cattle, it is better to protect it from rumen hydrogenation by converting to
calcium salts.
5. COMMERCIAL PRODUCTION OF CLA
Industrial conjugated linoleic acid (CLA) is a poorly dened blend of compounds
(102). Early commercial syntheses focused on maximizing total CLA content.
Many early products were rich in CLA but contained a number of positional iso-
mers. Market demand has now shifted for a product that contains two predominant
isomers, specically 9,11-c,t-octadecadienoic acid and 10,12-tc-octadecadienoic
acid. It is not surprising that alkali isomerization produced some undesirable posi-
tional isomers of CLA. In 1970, Mounts and Dutton (103) had shown unequivocally
that when potassium t-butoxide was used, at least four positional isomers of CLA
were produced. It was not until 1997, after the use of CLA as a dietary supplement
began, that Christie et al. (102) elegantly demonstrated that commercial CLAwas a
blend of positional isomers. In response to this discovery, new commercial CLA
products have been introduced that have comparatively high levels of the preferred
isomers. In spite of the improvements, all current available commercial CLA
products contain some level of the less desirable isomers and other components,
which may or may not be desirable.
Commercial processes for the synthesis of any compound of economic value is
normally proprietary information and the commercial methods of CLA production
are no exception. The process by which each brand of commercial CLA is synthe-
sized is not known by the authors of this review. Therefore, this review is directed
at the patent literature on CLA synthesis, major problems encountered in CLA
synthesis, and analysis of CLA from commercial suppliers.
5.1. CLA Production Raw Materials
The raw material for CLA production must be a material that is rich in linoleic
acid. This product could be in the triacylglycerol form, fatty acids or fatty acid
esters. The concentration of CLA in the nal product is directly dependent on
the level of linoleic acid in the starting material. The highest level of linoleic
acid available from botanical sources is not available in commercial products.
Extraction and rening equipment would be required to obtain oils with the highest
linoleic acid levels. Table 1 lists the commercial and noncommercial sources of oil
and fatty acids that are known to be rich in linoleic acid and their availability as
TAGs and fatty acids.
If commercial CLA is to be synthesized from a fatty acid, it must be recognized
that commercial fatty acids are generally not intended for use in the production of
CLA. Commercial fatty acids are usually produced by the reaction of water (steam)
and TAG oil at high temperatures in a continuous reaction (Reaction 1).
TABLE 1. Linoleic and Linolenic Acid Contents of Some Vegetable Oils (104).
% Linoleic % Linolenic Commercial Oil Commercial
Oil Source Acid Acid Available Fatty Acid Available
Corn 57 0 Yes
Cottonseed 53 0 Yes
Cucumber 72 0 No
Grapeseed 70 0 Yes
Linola
TM
Flaxseed 72 3 Yes
Poppy 77 0 Yes
Safower 75 0 Yes
Sunower 64 0 Yes Yes
Soybean 51 8 Yes Yes
Squash (pumpkin) 60 0 Yes
Walnut 62 12 Yes
COMMERCIAL PRODUCTION OF CLA 9
Reaction 1: Alkali or acid hydrolysis of acylglycerols.
R O
O
O
O
R
O
R
O
H
+
or
H
2
O
OH
HO
OH
OH
R OH
O
+ 3 HO
OH
OH
R O
O
+ 3
This reaction is accelerated through the use of a solid phase acid catalyst, which
is readily separated from the fatty acid and glycerol products after hydrolysis (105).
The disadvantage of this hydrolysis process is that the reaction is reversible and
products generated by this process contain appreciable amounts of mono- and dia-
cylglycerols, which may have undesirable side reactions in CLA synthesis. Fatty
acids may also be produced by the hydrolysis of TAGs in a pressurized reactor
at 200
C without the addition of a catalyst (105). This reaction may be catalyzed

at lower temperatures using zinc oxide in a batch reactor (105). The product of these
batch reactions also contains substantial amounts of mono- and diacylglycerols.
Hydrolysis of TAGs is possible using water and strong base to produce soaps
(Reaction 1). This reaction proceeds to completion and can be conducted at the
modest temperatures required to maintain the reaction mixture as a uid. More
than three moles of potassium hydroxide or sodium hydroxide are required to
hydrolyze one mole of TAG oil. As the caustic alkali cannot catalyze the reverse
reaction, this process can produce soaps that are virtually free of acylglycerols in
a single step (105). The soaps from alkali hydrolysis of TAGs are readily converted
to fatty acids by acidication with the addition of citric acid or strong mineral acids,
which include HCl, H
2
SO
4
, or H
3
PO
4
. Regardless of the method chosen for produc-
tion of fatty acids, the acids should be dried under vacuum after washing with brine
or by a combination of other acceptable methods (105).
There are commercially available fatty acids suitable for use in CLA production.
For example, Henkel Corporation (106) sells a series of fat products including those
shown in Table 2. However, none of the products listed in Table 2 would be pre-
ferred as starting materials for CLA production for reasons that will be discussed.
5.2. Enrichment of Linoleic Acid
A commercial interest may wish to produce CLA at concentrations greater than can
be obtained by modifying high linoleic acid plant oils. Several methods exist that
will improve the starting material by increasing the concentration of linoleic acid,
but only a few methods are used in industrial settings. Industrial separation of fatty
acids has been reviewed by others (104, 107). A limited discussion of these
methods will be presented.
Crystallization is used to separate saturated fats and oleic acid from linoleic acid.
If a highly concentrated product is required, the linoleic acid may be crystallized
once or repeatedly as the last step in purication. Crystallization is a mild procedure
but usually requires the use of a solvent (108) such as acetone or methanol. The use
of low boiling point and ammable solvents raises concerns over plant safety, gov-
ernment regulations on manufacturing, and market acceptance of the product.
Furthermore, the removal of oleic acid by crystallization in solvent is only possible
by lowering the temperature of the liquor to below 40
C (108). To crystallize
linoleic acid, the temperature must be reduced to 75
C.
Dry or solvent free crystallization is also possible; but these methods often
require the addition of crystal modiers that become incorporated into the product
(108). Losses during crystallization can be very high as the crystals entrain large
amounts of fatty acid. However, these losses may be reduced by physically pressing
the crystals to remove the entrained solution (109). Linoleic acid-rich products of
dry crystallization would be preferred starting materials for CLA production over
those of solvent crystallized products; but the losses incurred in dry crystallization
may prohibit this method of manufacture. Crystal modiers may be selected so that
they do not adversely affect the quality or acceptance of the nal product.
Specic fatty acids may be concentrated by sequentially removing contaminat-
ing fatty acids as urea adducts and forming the urea adduct of the desired fatty acid.
This process requires dissolving the fatty acids or esters in urea and hot methanol
(or other alcohol) and cooling to effect adduct formation. The adduct is ltered
from the liquor and, if conditions are carefully controlled, the adducts can be
used to sequentially crystallize saturates, monounsaturates, diunsaturates, and triun-
saturates. A urea adduct rich in linoleic acid could be produced by rst removing
adducts of saturates and monounsaturates from a suitable oil and then forming the
desired adduct. Once formed, the adduct may then be decomposed by the addition
of water to the solid phase. Enriched linoleic acid could be recovered by solvent
extraction of the urea : water solution with a nonpolar solvent such as hexane.
All problems associated with crystallization in solvent mentioned previously also
occur in formation of urea adducts, with the exception of the requirement for
TABLE 2. Henkel Products (106).
Product 14:0 (%) 16:0 (%) 18:0 (%) 16:1 (%) 18:1 (%) 18:2 (%) 18:3 (%)
Soya fatty acids 0.5 16 4 1 25.5 48 5
(Emersol 610)
Linoleic acid 0.5 3.5 0.5 Trace 19.5 65.5 10.5
(Emersol 315)
Methyl Linoleate 0.5 3.5 0.5 Trace 19.5 65.5 10.5
(Emery 2221)
very low temperatures. Typically, urea adducts form between room temperature and
0
o
C (108).
Fatty acids may also be enriched by the use of various absorption media. Mole-
cular sieves can separate saturated fatty acids from unsaturated fatty acids dissolved
in acetone (110). Oleic acid and linoleic acid dissolved in blends of solvents,
including acetonitrile, tetrahydrofuran, water, and formamide, may be separated
using cross-linked polystyrene polymers such as Amberlite
TM
XAD-2 or XAD-4.
Selective extraction methods using two-phase solvent systems may also be used
to enrich fatty acids. Solvent systems such as dimethyl formamide, hexane, and
ethylene glycol can form a two-phase system that effectively partitions sunower
oil TAGs rich in linoleic acid from those depleted in linoleic acid (111). TAGs
partitioned in this way may contain up to 84.7% linoleic acid. This method would
not likely be used in industry because the magnitude of the losses are usually
unacceptable.
5.3. Approaches to CLA Production
CLA has been produced by the reaction of soaps with strong alkali bases in alcohol,
ethylene glycol, and glycerol (112114) (Reaction 2).
Reaction 2: Isomerization of cis,cis-9,12-octadecadienoyl soaps.
COO
COO
base
+
COO
The CLA product is generated by acidication of the soap solution with a strong
acid (sulfuric or hydrochloric acid) and repeatedly washing the product with brine
or an aqueous CaCl
2
solution.
CLA has been synthesized from fatty acid and soap blends using SO
2
in the
presence of a substoichiometric amount of soap forming base (115). This reaction
produced predominantly the all trans-conguration of CLA.
Of these methods, alkali isomerization of soaps is the least expensive process for
bulk preparation of CLA isomers; however, the use of either monohydric or poly-
hydric alcohols in alkali isomerization of CLA can be problematic. Lower alcohols
are readily removed from the CLA product, but they require that the production
facility be constructed to support the use of ammable solvents. Higher molecular
weight alcohols and polyhydric alcohols are considerably more difcult to remove
from the product and residual levels of these alcohols (e.g., ethylene glycol) may
not be acceptable in the CLA product.
Water may be substituted for the alcohols in the production of CLA by alkali
isomerization of soaps (116, 117). When water is used in this reaction, it is neces-
sary to perform the reaction in a pressure vessel, whether in a batch (116) or con-
tinuous mode of operation (117). The process for synthesis of CLA from soaps
dissolved in water still requires a complex series of reaction steps. Bradley and
Richardson (118) were able to produce CLA directly from TAGs by mixing sodium
hydroxide, water, and oil in a pressure vessel. Their method eliminated the need to
synthesize fatty acids followed by soap formation prior to the isomerization reac-
tion. However, the authors reported that they were able to produce an oil with only
40% CLA. Quantitative conversion of the linoleic acid in soybean oil to CLAwould
have produced a fatty acid mixture with approximately 51% CLA.
5.4. Reaction Kinetics and Production of Positional Isomers
The kinetics of conversion of linoleyl soaps to conjugated linoleyl soaps has been
well described by rst-order reaction kinetics (119). Total conjugation is readily
measured by simple methods such as increases of UV absorbance at 231.5 nm. In
industry, it is necessary to allow the reaction to proceed until most of the linolyl
soaps are conjugated but desirable to stop the reaction as soon as possible after
that point. The reaction is between 97% and 98.5% complete after 5 half-lives
and 6 half-lives respectively. There is little advantage in continuing the reaction
longer than this time and, as will be discussed below, undesired reactions may occur
with longer reaction times. The reaction constant is readily determined early in the
reaction when changes in the level of conjugation are large. The task of determining
the rate constant in a large reactor is complicated by the mass of the reactor and its
contents. A large batch reactor often requires several hours to reach the optimum
heat of reaction, at which time the reaction may be almost complete. Similarly,
cooling the contents of a large reactor as a means of stopping a reaction is usually
impractical. A reaction that nears 99% completion in 2 hours in a laboratory has a
half life of less than 20 minutes. Control of a batch reaction in a commercial opera-
tion may use analytical data from periodic sampling, but the analytical method must
be very rapid to be an effective tool for decision making.
The authors have found that the half-life of the reaction may vary by approxi-
mately 10% per 1
C. It follows that precise reactor temperature control is essential

to standardize quality control. A reaction planned to continue for 6 half-lives could
vary from 5 half-lives to 7 half-lives if the reactor temperature control is 2
C.
The isomerization reaction that leads to the production of positional isomers
(Reaction 3) has similar rst-order kinetics. Using this assumption, we have mod-
eled the sequential conversion of linoleyl soaps to a mixture of 9,11-cis-, trans-
octadecadienoyl and 10,12-trans-, cis-octadecadienoyl soaps (Reaction 2) and nal-
ly, the conversion of these two soaps to 8,10-octadecadienoyl and 11,13-octadeca-
dienoyl soaps. Reaction 3 shows the mechanism of one of these two isomerization
processes.
Reaction 3: Isomerization of cis-,trans-9,11-octadecadienoyl soap to trans-,cis-
8,10-octadecadienoyl soap via sigmatropic rearrangement.
H R R
1
11 9
R R
1
H R R
1
10
8
H
R = (CH
2
)
6
COOH
R
1
= (CH
2
)
5
CH
3
In an earlier review of commercial CLA production, Reaney et al. (120) devel-
oped a model of the base catalyzed sequential conversion of unconjugated linoleic
acid to a mixture of two isomers, 9,11-c,t- and 10,12-t,c-CLA, by rst-order
kinetics. After the formation of the two primary isomers, two additional isomers,
8,10-t,c-CLA and 11,13-c,t-CLA, were produced by a sequential reaction. It was
later reported by Saebo (121) that the second reaction producing the additional
isomers was actually a thermal sigmatropic rearrangement as shown in reaction 3.
The intramolecular rearrangement is independent of catalyst concentration and,
thus, the accumulation of additional isomers is a function of the reaction tempera-
ture and the duration of reaction. Saebo (121) reports that prolonged heating results
in the accumulation of isomers but that reaction solvent that allows for low-
temperature reactions may be used to prevent sigmatropic rearrangement and the
consequent formation of isomers.
5.5. Solvents Used in Production of CLA by Alkali Catalysts
Research reports describe the use of at least eight solvent systems for the produc-
tion of CLA using alkali catalysts (112116) (Table 3). The choice of solvent
greatly affects the reaction conditions of CLA production. The choice of solvent
by the manufacturer is determined by a number of considerations. Many markets
will not accept low levels of ethylene glycol, ethylene glycol monomethylether,
t-butanol, dimethyl sulfoxide (DMSO), or dimethyl formamaide (DMF) in the nal
product. This limitation could restrict the choice of solvents to only molten alkali,
glycerol, propylene glycol, water, and ethanol. The reaction in water and ethanol
only proceeds above the boiling temperature of these solvents and, therefore, a
pressure reactor would be required to operate using these solvents. Glycerol is
expensive, but it could be recovered from a commercial operation that produces its
own fatty acids. The quality of glycerol necessary to produce high-quality CLA has
not been investigated, but rening this glycerol stream to remove the salt might
prove difcult to a small operation. Recovery would have to be very efcient as
fatty acid production only generates 10% of the weight of the oil as glycerol.
5.6. Catalyst Selection
Numerous catalysts have been used in the production of CLA. We have found that
hydroxides of lithium, sodium, and potassium are all capable of generating CLA in
various solvents. As fatty acids neutralize the catalyst, it is necessary to add at least
one mole of catalyst for every mole of fatty acid in the reaction to ensure soap is
generated. We have found that, on a molar basis, potassium hydroxide has proven to
be a more effective catalyst than sodium hydroxide, with lithium hydroxide the
least effective and not suited for industrial CLA production. On a weight basis,
sodium and potassium hydroxide have similar efciency of conversion. Although
sodium hydroxide is much less expensive than potassium hydroxide, the disposal
costs for the waste neutralized alkali should also be considered. Potassium salts
are easily used as fertilizer and can be applied to elds, whereas sodium salts
cannot be disposed of in a similar fashion.
The effective form of the catalyst is not necessarily determined by the added
catalyst itself but rather by the solvent used. When water is used as the solvent
and sodium ethoxide is the catalyst, the effective form of the catalyst is likely
the hydroxide ion. If t-butanol is used as the solvent and sodium methoxide is added
TABLE 3. Summary of Solvents Used in CLA Production.
Solvent Mol B.P. B.P. Temp Time % Phase
(reference) wt. (760) (Vac) (
C) (h) Reaction Sep

n
{ Color z Toxicity Other
Ethylene
glycol (148) 62.07 198.00 93
13
180 2.0 100.0 yes Poor yes
Glycerol (148) 92.11 290.00 182
20
180 0.75 100.0 yes Good none viscous
Propylene
glycol (149) 76.11 189.00 97
21
170 2.5 99.1 yes Good minimal
t-butyl
alcohol (103) 74.18 82.41 Low 90 4.0 98.5 no unknown yes High
Pressure
Water (118) 18 100.00 Low 225 2.5 40.0 yes Good none High
Pressure
DMSO (149) 78.14 189.00 83
17
30 1.5 78.0 no unknown yes fp
95
DMF (149) 73.09 153.00 76
39
no unknown yes fp
67
{Phase Separation Yes if a two-phase system is formed after acidication of the soap.
zColor As described in references.
fp ash point.
Superscript numbers refer to Vacuum in mm of Hg.

DMF, Dimethylformamide; DMSO, Dimethylsulfoxide.
to catalyze the reaction at 90
C, the reaction mixture will quickly release methanol

vapours and t-butoxide ion will become the effective catalyst.
Water consumes alkoxide catalysts and, in industrial production, maintaining
alkoxide catalysts in a water-free environment is difcult. Water is produced by
the neutralization of fatty acids with alkali hydroxide. As many alkoxide catalysts
contain some alkali hydroxide, it is not uncommon for the catalyst to be consumed
by this reaction.
5.7. Reaction Vessels
Alkali isomerization of linoleyl soaps requires a containment vessel that is both tol-
erant of heat and caustic. When a low boiling point solvent such as ethanol or water
is used, the vessel must also be capable of maintaining the reaction under pressure.
There are a limited number of materials that will meet these criteria. Polytetrauor-
oethylene and other uoropolymers are capable of withstanding both the heat of the
reaction and the caustic environment, but they cannot withstand pressure and are
poor heat conductors (122). Fluoropolymer coated parts and nickel and nickel
alloys such as Monel may be used in the construction of reaction vessels for pro-
duction of CLA. The high cost of these materials rules out their use in construction
of large batch reactors. Furthermore, none of these materials has sufcient strength
for use in pressure reactors if a reaction in water or alcohol is planned. The pre-
ferred choice for reactor construction is nickel-plated steel, which has the desired
strength, heat transfer, and chemical properties for conducting reactions in strong
caustic solutions. A coated vessel of this design requires regular inspection, as a
aw in the coating could lead to vessel failure.
5.8. Microbial Production of CLA
Pariza and Yang (123) have recently described the microbial production of 9,11-c,t-
CLA from linoleic acid using cultures of Lactobacillus sp. In their patented method,
early stationary phase Lactobacillus cultures were incubated with linoleic acid dis-
solved in propylene glycol. A total CLA level of 7 mg/g cells was produced, which
was over 96% 9,11-c,t-CLA. This type of conversion may lead to improved CLA
products in the future.
5.9. Synthesis of CLA by Dehydration of Ricinoleic Acid
(12-Hydroxy-cis-9-Octadecadienoic Acid)
The most attractive method for production of pure 9,11-c,t-CLA is through the
dehydration of ricinoleic acid. Synthesis from this relatively inexpensive starting
material has proven elusive as it is difcult to control the formation of dehydration
products (124). Synthesis of 9,11-c,t-CLA from ricinoleic acid has been reported
(125), which, although an efcient reaction, uses expensive elimination reagents
such as 1,8-diazobicyclo-(5,4,0)-undecene. For most applications, the high cost
of the elimination reagent increases the production cost beyond the level at which
commercial production of CLA is economically viable.
5.10. The Quality of Commercial CLA Products
The fate of other fatty acids and minor components during processing has not been
investigated. The conditions used to conjugate linoleic acid have little or no effect
on either monounsaturated or saturated fatty acids, however, any polyunsaturated
fatty acids may be conjugated. The products of the reaction of alkali catalysts on
these fatty acids are more complex than that discussed for linoleic acid (Reaction 4)
and will not be discussed except to note that these reactions may produce undesir-
able products.
Reaction 4: Isomerization of cis-,cis-,cis-9,12,15-octadecatrienoyl soap to isomers.
COO
COO
COO
COO
COO
base
+
+
+
+
cis-,cis-,cis-9,12,15
cis-,trans-,cis-9,11,15
trans-,cis-,cis-10,12,15
cis-,trans-,cis-9,13,15
cis-,cis-,trans-9,12,14
From our observations, glycerol does not form undesirable compounds under the
conditions of alkali isomerization of linoleic acid. However, we have found that a
number of commercial fatty acids and CLA preparations contain appreciable levels
of monoacylglycerols (MAGs). MAGs themselves are not toxic, but it is possible
that toxic compounds (such as ethylene glycol) may be incorporated into the nal
product through alcoholysis of the MAG by the low volatility alcohols used as a
reaction medium (Reaction 5).
Reaction 5: Alcoholysis of MAG with ethylene glycol.
O
OH
O
OH
OH
OH
O
O
HO
NMR and liquid chromatographic analysis of CLA samples from all sources
indicated that, although CLAwas the predominant compound, CLA esters and other
unknown compounds may also have been formed.
The minor components of vegetable oil, such as tocopherol, sterol, or squalene,
are stable to heat a strong alkali. However, tocopherol and other components pre-
sent in vegetable oil readily react with oxygen in the presence of metals. Tocopherol
stabilizes vegetable oils against oxidation and tocopherol loss through processing or
high metal content may lead to a decreased shelf-life of the CLA product. Three
commercial CLA products were analyzed for metal content using ICP. The metal
contents are given in Table 4.
The CLA samples tested were free of potentially toxic metals at the levels tested,
with the exception of a trace level of barium in product C. Products A and B had
very low total metal contents, whereas product C had appreciable levels of calcium.
The soaps probably derived from a CaCl
2
wash in a late stage of processing.
Product C also contained iron and chromium suggesting the use of a stainless-steel
reactor for processing. Even these small levels of metals may contribute to rapid
oxidation of product C, and it probably has a shortened shelf-life when compared
with the other products.
Three commercial samples of CLAwere subjected to a series of tests, which are
reported in Table 5. Viscosity was measured using ASTM test D445. It was pre-
sumed that increased viscosity might be related to increased oxidation if it had
occurred. Large differences in viscosity were not found. Color of the three samples
is reported, but it is only a subjective statement regarding the apparent quality of the
three materials. Included in the color analysis is the absorbance maximum observed
for CLA dissolved in hexane (1:1000 CLA:hexane) using a Cary UV/Vis spectro-
meter. The UV spectra indicated that conjugated dienes were the major source of
absorption of the oil samples.
Proton and carbon 13 NMR spectra were obtained from the three commercial
samples. A series of unknown spectral components were observed in product A
at 3.68 ppm, 3.98 ppm, 4.08 ppm, 4.15 ppm, 5.05 ppm, and 5.17 ppm. Comparison
of the unknown peaks with the spectra of MAGs indicated that most of the observed
peaks correlated with those present in the spectra of MAGs. An exact assignment of
the spectra was not attempted as many forms of MAGs are possible. Observation of
the spectral region of the olenic protons revealed that the three samples were
TABLE 4. Metal Contents of Three Commercial CLA Products (ppm).
Sample Na K Fe Cr B P Ca Ba Mg Li Total
A 3 3 1 0 5 2 5 0 1 0 20
B 2 28 0 0 3 8 1 0 1 0 43
C 4 6 11 8 3 3 917 1 2 1 956
Not detected Si, Pb, Cu, Sn, Al, Ni, Ag, Ti, Zn, Mo, V, Sb, Be.
TABLE 5. Summary of Analyses of Three Commercial CLA Products.
Viscosity
1
H-NMR Size Isomer
Sample cSt (40
C) Color (400 MHz) Exclusion RP-LC GC-FID Mix

A 28.9 Clear Acylglycerols Acylglycerols Polar Contamination Good
l 231.5 contamination 95%
A 0.720
B 27.9 Light No No Polar OK Poor
yellow acylglycerols acylglycerols contamination 70%
l 231.5
A 0.667
C 29.7 Brown No No Non-Polar OK Good
l 231.5 acylglycerols acylglycerols contamination 90%
A 0.676
predominantly cis-, trans- or trans-, cis-fatty acids (126). We have found that a
convenient measure of the CLA content of an oil can be obtained by comparing
the integrated values of the protons at 6.0 ppm and 6.3 ppm with the integrated
value of alpha methylene group (adjacent to the carbonyl) at 2.3 ppm. The olenic
protons were chosen as they do not overlap with oleic acid olenic protons or
olenic protons from conjugated linoleic acid with trans-, trans-congurations.
The ratio of cis-, trans-olen to alpha methylene protons is a useful measure of
CLA purity.
Carbon-13 olenic carbons were observed at 400 MHz. The carbon-13 spectrum
clearly demonstrates the formation of positional CLA isomers. As pure standards
were not available, it was not possible to unequivocally assign the spectra of
8,10- or 11,13- CLA, but it is clear that these isomers are predominantly cis-, trans-
or trans-, cis-fatty compounds.
Size exclusion chromatography was performed with a Waters GPC-Styragel
TM
HR 0.5 column (7.8 300 mm) at 35
C using tetrahydrofuran as a solvent owing

at 1.0 mL/min and detecting compounds with both UVabsorbance and evaporative
light scattering detection (ELSD, 40
C, 4.2 L/min gas ow). The goal of this ana-

lysis was to observe polymerization of the CLA products or the occurrence of acyl-
glycerols. Under these conditions, 18-carbon fatty acids had a retention time of 6.6
minutes and MAGs had a retention time of 5.9 minutes. All three samples had two
peaks observed by ELSD, one at the position expected for 18-carbon fatty acids and
the other as expected for their respective MAGs. Product A had the largest peak at
the position expected for MAGs. Observations made using UV absorbance at
233 nm reected the observations made with the ELSD.
Reversed phase liquid chromatography was performed on a 150 mm3.0 mm
Waters Symmetry
TM
C-8 column at 30
C and a ow rate of 1.0 mL/min. The

solvent phase was acetonitrile:tetrahydrofuran : 0.1% aqueous phosphoric acid
(50.4:21.6:28v/v/v). Under these operating conditions, most of the UV absorbance
occurred as a peak at 3.83 minutes for all samples. Chromatograms of all samples
had some small peaks (presumably the more polar compounds) eluting prior to the
major peak. Product C presented a small but signicant UV absorbing peak that
eluted after the peak at 3.83 minutes.
5.11. Rapid Analytical Methods
Industrial CLA syntheses must be controlled to both maximize the content of
preferred isomers such as 9,11-cis-, trans-octadecadienoic acid and minimize the
formation of undesirable isomers. For the analysis to be useful, the results of the
analysis should be available online or as quickly as possible. The authors are not
aware of any existing online tests for the quality of CLA preparations, but several
methods may have promise for use ofine.
The potential ofine analytical methods include UV, FTIR, proton NMR, car-
bon-13 NMR, gas chromatography, and capillary electrophoresis. With the excep-
tion of capillary electrophoresis and UV absorbance, none of these methods can
effectively analyze soap solutions and, therefore, for most analytical methods, neu-
tralization of soaps would be necessary.
A reaction medium that contains 40% soaps by weight can usually be dissolved
in ethanol. We have found that 100 mg of reaction mixture will totally dissolve in
10 mL of 95% ethanol when glycerol, water, or ethylene glycol are the reaction sol-
vent and alkali hydroxides are used as the catalyst. It is then possible to dilute the
reaction mixture solution 1000-fold to determine the UV absorbance at 231.5 nm.
When there is no other interfering UVabsorbance, this method is an excellent indi-
cation of the total conjugated double bonds. This method is also sensitive to the
presence of conjugated linolenic acids derived from linolenic acid, which is indi-
cated by a UV absorbance at 268 nm. None of the samples observed show three
conjugated double bonds.
To obtain more detailed information regarding the composition of fatty acids
requires additional analytical methods, some of which require extensive and
time-consuming sample preparation. For these methods, we have found that it is
possible to rapidly prepare a free fatty acid fraction. Alkali soaps from most reac-
tion mixtures are readily dissolved in a mixture of hexane and ethanol (1 : 1v/v) or
ethanol alone. When the soaps are neutralized by the addition of hydrochloric acid
and water, a two-phase system is evolved. The fatty acids remain in the nonpolar
phase while the polar solvent used in the reaction medium dissolves in the water.
The solution of dissolved fatty acids can be directly injected onto a GC column
specically designed for separation of free fatty acids, such as the DB-FFAP col-
umn, or a suitable nonpolar GC column, such as the HP-5 column. Analysis by
chromatography without derivitization affords the potential for rapid analytical
feedback. We have found that a 30 m, DB-FFAP column (0.32 mm id, 0.25 m
lm thickness) gave baseline resolution of most fatty acids without derivitization
(program 50
C for 1 min, 50200
C @ 25
C/min, 200220
C @ 2
C/min,
hold 20 min, He carrier 3 ml/min). The nonpolar HP-5 column (0.32 ID, 0.25
lm) gave poorer resolution of underivatized fatty acids with some tailing (program
50
C for 1 min, 50150
C @ 25
C/min, 150290
C @ 10
C/min, hold 6 min, He

carrier 2 ml/min). Both columns also partially separated isomers of CLA. It was
possible to observe the formation of the all trans-isomers, but detailed analysis
of positional isomers was not possible without additional effort.
6. ANALYSIS OF CONJUGATED LINOLEIC ACIDS
An analyst needs to recognize three major variables before the selection of a sui-
table method of analysis of CLA. CLA preparations can differ in degree of conju-
gation, position of double bonds, and conguration of double bonds. The most basic
variable is the degree of conjugation of the double bonds. Not all of the isolated
double bonds may become conjugated either in biological or chemical conversions.
The second issue to be addressed is the position of the double bonds in conjugated
linoleic acids. Initially, in linoleic acid, the double bonds begin at the ninth and
twelfth carbon atoms. After alkali isomerization, the predominant positional isomers
ANALYSIS OF CONJUGATED LINOLEIC ACIDS 21
are 9,11 and 10,12 dienes. However, minor amounts of other positional isomers are
also reported (127). In biological samples, the number of positional isomers is more
varied with 16 isomers being separated by silver-ion chromatography (128). The
nal consideration is the geometric isomers present in CLA. Whereas the double
bonds in linoleic acid are cis-, cis-, the isomerized product contains a predominance
of cis-, trans- and trans-, cis-isomers plus lesser amounts of cis-, cis- and trans-,
trans-isomers. Thus, the task for the analyst is to decide what information is
required and then select a method or methods that will provide that information.
6.1. Ultraviolet Spectroscopy
The early observation of an increase in the ultraviolet absorption (UV) near 233 nm
was recognition that the composition of milkfat was not consistent throughout the
year (129). This increase in absorption was not initially attributed to the increase in
conjugated dienes present in milk fatty acids; however, it is now accepted that this
is the case. There are subtle differences in the absorption maxima for the geometric
isomers. Czauderna (130) reported a maximum of 231.9 nm for the trans-, trans-
isomer, 234.3 nm for the cis-, trans- (or trans-, cis-) isomer and 235.4 nm for the
cis-, cis-isomer. These small differences would not likely be discernable in isomer
mixtures typically found in either biologically or chemically produced CLA. The
UV measurement provides no information on the position of the double bonds.
The advantage of using UV is the comparatively low cost of the spectrophotometer
and the fact that it can be performed on either intact acylglycerols, fatty acids, or
esters. In Figure 1, the UV spectra of linoleic acid and a commercial CLA product
are shown.
200 250 300 350 400 450
1.2
1.0
0.8
0.6
0.4
0.2
0
9,11, c,t,CLA + 10,12,t,CLA
Linoleic Acid
UV-Vis Spectra of Linoleic Acid and CLA
Wavelength (nm)
A
b
s
o
r
b
a
n
c
e
Figure 1. The ultraviolet spectra of linoleic acid and conjugated linoleic acid (CLA).
6.2. Infrared Spectroscopy
Infrared spectroscopy (IR) initially used double beam optics, photocells, and alkali
halide sample cells. These features at times tended to reduce the sensitivity of the
instrument and to decrease the signal to noise ratio. Newer instruments have
improved the signal to noise ratio by using Fourier Transform (FT) methodology
and modern electronics. The application of infrared spectroscopy to the problem
of identication of geometric isomers was rst reported about 50 years ago. It
was recognized that cis-, trans- (or trans-, cis-) dienes had a characteristic doublet
at 948 cm
1
and 982 cm
1
(131). The corresponding trans-, trans-diene had a
strong absorption band at 988 cm
1
. Using FTIR and a direct deposit interface,
Mossaba (132) reported the cis-, trans-doublet to occur at 949 cm
1
and
988 cm
1
and the trans-, trans-singlet at 993 cm
1
for 4,4-dimethyloxazoline
(DMOX) derivatives of CLA. An example of the spectra of linoleic acid and two
CLA positional isomers obtained with a FTIR using a synthetic sample disc are
shown in Figure 2A. In addition to these absorptions, there are other characteristic
frequencies attributed to carbon-hydrogen stretching. In one report of 4,4-
dimethyoxazoline derivatives (DMOX) of CLA isomers, it was possible to attribute
unique differences among geometric isomers (133). The cis-, trans-isomers had
characteristic bands at 3020 cm
1
and 3002 cm
1
(see Figure 2B); the cis-, cis-iso-
mers had bands at 3007 cm
1
and 3005 cm
1
; and the trans-, trans-isomer had a
single band at 3017 cm
1
. The use of these bands to determine geometric composi-
tion may be complicated by the much more intense absorption bands from other
carbon-hydrogen bands present in the same region of the spectrum. Although it
is theoretically possible to obtain a quantitative measurement of the amount of a
substance present by IR, it is not often done. In the case of CLA, the bands that
needed to be measured are minor bands compared with other bands, making the
determination imprecise and difcult.
100
80
60
40
20
0
1000 980 960 940 920 900 880
Wavelength (nm)
Linoleic Acid
9,11,c,t,CLA
10,12,t,c,CLA
Linoleic Acid
9,11,c,t,CLA
10,12,t,c,CLA
%

T
r
a
n
s
m
i
t
t
a
n
c
e
100
80
60
40
20
0
3040 3020 3000 2980 2960 2940
Wavelength (nm)
%

T
r
a
n
s
m
i
t
t
a
n
c
e
a
b
Figure 2. Infrared spectra of linoleic acid and 2 cis-, trans-, isomers of conjugated linoleic acid
(A) 1000 to 900 nm of the
CH vibration band and, (B) 3050 to 2950 nm of the CH stretching

bands.
6.3. Thin Layer Chromatography
Thin layer chromatography (TLC) has proven very useful in the separation of a vast
array of chemicals both synthetic and naturally occurring. When it comes to separa-
tion of closely related diene isomers, it is a difcult task with unmodied adsor-
bents such as silica gel. Silver nitrate-modied thin layer chromatography (Ag
-
TLC) has been used to separate CLA isomers. Some cis-, trans-isomers of CLA
have been separated as their methyl esters using hexane/diethyl ether, benzene,
or toluene (102). The initial solvent mixture caused the cis-, trans-isomers to
migrate faster than the cis-monoenes. Either of the later solvents resulted in the
cis-, trans-isomer migrating between the cis- and trans-monoenes. Ackman (134)
reported that the cis-, trans-isomer had a relative retention (R
f
) of 0. 61, whereas the
trans-, trans-isomer had a R
f
of 0.65. Although the difference in the R
f
values is not
great, it is possible to separate the isomers to either collect larger quantities or
investigate possible metabolites.
6.4. Gas Chromatography
Gas chromatography (GC), initially using packed columns and later using capillary
columns, provides another method for the analysis of fatty acids and esters or other
derivatives. As binding of the fatty acids to the column was problematic, fatty acid
methyl esters (FAME) were traditionally used. The method of forming the methyl
esters can be divided into three procedures: acid-catalyzed, base-catalyzed, and dia-
zomethane alkylation (135). No one method is suitable for all situations and all suf-
fer some deciency.
Acid-catalyzed procedures typically are either boron triuoride/methanol (BF
3
/
CH
3
OH) or hydrochloric acid/methanol (HCl/CH
3
OH). Many analysts use sulfuric
acid instead of hydrochloric acid. Acid-catalyzed procedures are used for free fatty
acids, phospholipids, or triacylglycerols, often at elevated temperatures. Although
the procedures are relatively efcient at production of methyl esters, there will be
some isomerization of some cis-, trans-isomers to the trans-/trans-isomers (136).
This isomerization can be reduced by using lower temperatures, for instance
60
C, for HCl/CH
3
OH or room temperature for BF
3
/CH
3
OH (137). However,
under these milder conditions, some phospholipids may not be esteried (137). In addi-
tion, methoxy adducts may be formed and hydroxy fatty acids may produce artifacts.
A base, such as sodium methoxide (NaOCH
3
), is useful in esterifying lipids as
found in acylglycerols, sterol esters, and phospholipids (138). Free fatty acid and N-
acyl lipids in sphingolipids will not be methylated. The NaOCH
3
method does not
apparently change the cis-, trans-isomer composition or form methoxy artifacts.
However, some artifacts that could interfere with shorter chain fatty acids were
observed.
Diazomethane is effective in esterifying free acids to their corresponding methyl
esters under mild conditions and is fast. It will not produce methyl esters from acyl-
glycerols, cholesterol esters, or phospholipids. Many researchers are concerned
about the potential hazard of the diazomethane, its precursors, and the actual
preparation of the reagent. Trimethylsilyl diazomethane is commercially available
and can be used as a source of diazomethane. However, some artifacts (trimethyl-
silyl CLA esters) and other trimethylsilyl impurities may interfere with analysis
(139). As there are limitations to each method of making methyl esters, the
analyst will need to select reagents and conditions that are most appropriate for
the substrate. For intact acylglycerols, sterol esters, and phospholipids, the
NaOCH3 method would be appropriate. If only free fatty acids are present, the dia-
zomethane method would be appropriate, particularly if double bond isomerization
is of concern.
The earliest of GC analyses were performed on columns packed with a solid sup-
port coated with a nonvolatile liquid phase. Packed columns are not frequently used
today as they have been replaced by capillary columns where the liquid phase is
immobilized on the internal surface of the capillary. As there are numerous liquid
phases available, it is now possible to obtain commercial columns that will separate
not only the methyl esters but also the underivatized fatty acids. This advancement
obviates the need for derivatization and the associated problems. A typical chroma-
togram of free fatty acids is displayed in Figure 3. Individual isomers of CLA
are now available to aid in the identication of isomers in the chromatogram.
Gas chromatography can provide quantitative information on the degree of
conjugation, positional, and geometric isomer distribution when suitable standards
are available.
6.5. High-Performance Liquid Chromatography
High-performance liquid chromatography (or less common, high-pressure liquid
chromatography, HPLC) is a preferred method of analysis for many compounds
because it does not require the high temperatures used in gas chromatography.
Separations in HPLC can be based on either a size exclusion or on an adsorption
principle. The size exclusion mode is useful for separating fatty acids from
0 5 10 15 20 25 30 35
0
10
20
30
40
50
60
70
80
90
pA
Palmitic
1
5
.
2
3
6
2
0
.
5
2
2
2
5
.
2
1
8
2
5
.
7
2
2
2
6
.
6
3
4
2
1
.
9
4
4
1
9
.
5
7
7
2
7
.
8
8
0
Stearic
Retention Time (min)
Linoleic
Oleic
cis-9,trans-11 CLA
trans-10,cis-12 CLA
trans-10,trans-12 CLA
trans-9,trans-11 CLA
Figure 3. Gas chromatogram of free fatty acids on a 30-m DBFFAP column (0.32 mm id,
0.25-um lm thickness) using a temperature gradient of 50
C for 1 min; 50
C150
C @ 25
C/
min, 150
C220
C @ 3
C/min; hold at 220
C for 3 min; He carrier gas @ 2 mL/min.

acylglycerols and has been applied to CLA analysis. Reaney et al. (120) have used a
Waters GPC-StyragelHR
TM
column for the purpose of observing possible polymer-
ization of CLA or the presence of acylglycerols. In three commercial samples
of CLA, the 18-carbon fatty acids could be separated from the MAGs. Both UV
detection at 233 nm and evaporative light scattering detection (ELSD) were used.
There was no reported separation of individual fatty acids or CLA isomers. Use
of a reverse phase column (Waters C-8 Symmetry
TM
column) for separation of
commercial CLA resulted in a major peak being observed that would correspond
to the free fatty acids (120). As with the size exclusion column, there was no
separation of either isomeric conjugated fatty acids or acids of different chain
length. A preferred technique is silver ion-modied high-performance liquid
chromatography (Ag
-HPLC). Using this modication, several groups have

reported successful separation of both positional and geometric isomers of CLA
(14, 140).
6.6. Mass Spectrometry
Mass spectrometry (MS) is a method to determine the mass of either an intact
ionized molecule or an ionized fragment. When MS is combined with the separa-
tion power of gas or liquid chromatography, much valuable information can be
obtained for structural determination. Although GC-MS or HPLC-MS may seem
to be an ideal tool for CLA analysis, it suffers a major problem as originally prac-
ticed. In order to obtain a mass spectrum, it is necessary to produce an ionized spe-
cies. With CLA, as well as other molecules, some ionization conditions cause the
double bonds to migrate to new positions. If the double bonds migrate, it is only
possible to obtain information on chain length and number of double bonds. To
allow for less harsh ionization conditions, it is possible to make derivatives at either
the carboxylic acid (remote site) or at the site of the conjugated double bonds (on
site). The derivatives have been selected to allow for easier ionization. In the case of
the on-site derivatives, the position of the diene is xed at one location. At the car-
boxylic acid site pyrrolidide, picolinyl ester and 4,4-dimethyloxazoline (DMOX)
derivatives are used. The fragmentation of the remote site derivative basically pro-
duces ions that are 14 mass units lighter than the previous ion for each methylene
(CH
2
) group. When a double bond is encountered, the decrease observed is 12 mass
units. A review on this subject has been compiled (141).
Two different approaches have been reported for on-site or double bond site deri-
vatization of fatty acids or other conjugated dienes. One method involves the com-
plete hydroxylation of the double bonds with osmium tetroxide (health caution)
followed by trimethylsilylation (142). The second method is based on the well
known Diels-Alder cyclo addition. MTAD (4-methyl-1,2,4-triazoline-3-5-dione)
has been shown to be a useful reagent for adduct formulation with FAME conju-
gated dienes. Fragmentation patterns of adducts are usually dominated by cleavage
fragments that include the ring formed during the cyclo addition plus either of the
residual carbon chains. In Figure 4 of the MTAD derivative of methyl cis-, trans-
9,11-octadecadienoate, these fragments occur at 322 m/z and 250 m/z, indicating
that the starting FAME was a 9,11 diene (143). Methyl cis-, trans-9,11-octadeca-
dienoate and methyl trans-, trans-9-11-octacadienoate readily formed adducts with
similar mass spectra but with different retention times when analyzed by GC-MS
(143). Methyl cis-, cis-9,11-octadecadienoate reacted more slowly to produce two
products with similar fragmentation patterns but with different retention times.
Unfortunately, it was demonstrated that the cis-, cis- and cis-, trans-isomers pro-
duced the same adduct, limiting usefulness when studying geometric isomers of
CLA products.
6.7. Nuclear Magnetic Resonance
Nuclear magnetic resonance (NMR) is a powerful technique for CLA analysis
(144, 145). NMR spectroscopy can provide information on the environments of both
the proton (
1
H) and the carbon-13 (
13
C) atoms in CLA and also provide correlation
between the two atoms. In one of the most basic analyses, it is possible to observe
the disappearance of the signals as a result of the isolated olenic protons
(5.35 ppm, Figure 5A) and the isolated methylene group (2.75 ppm) linoleic acid
as well as the appearance of new signals associated with the conjugated diene
(5.26.5 ppm, Figure 5B). Reaney et al. (120) reported that a convenient measure-
ment of CLA content can be obtaining by comparing the integrated areas of the
olenic protons at 6.0 ppm and 6.3 ppm with the area of the methylene protons
adjacent to the carbonyl (2.3 ppm). The NMR
13
C spectra for both linoleic and
cis-, trans-9,11-octadecadienoic acids are presented in Figure 5C and 5D, respec-
tively. The complete assignment for this and other isomers of CLA has been
reported (146, 147). A notable feature for CLA is the appearance of four well sepa-
rated signals between 129135 ppm (Figure 5D) compared with the two pairs of
0
50 100 150 200 250 300 350 400 450
10
20
30
40
50
60
70
250
290
322
376 407
M
+
m/z
A
b
u
n
d
a
n
c
e

(
t
h
o
u
s
a
n
d
s
)
250 m/z
322 m/z
N N
N
O O
CH
3
(CH
2
)
7
COOCH
3
CH
3
(CH
2
)
5
Figure 4. The mass spectrum of the adduct of methyl cis-, trans-9,11-octadecadienoate and 4-
methyl-1,2,4-triazoline-3,5-dione (MTAD). Reproduced by permission of AOCS Press (143).
narrowly separated doublets observed in linoleic acid spectrum (Figure 5C).
Detailed
1
H and
13
C and NMR spectroscopy have the potential to provide
information on both positional and geometric isomers of CLA and can provide
semiquantitative information on CLA concentration.
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
ppm
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
ppm
190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20
ppm
190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20
ppm
a
b
c
d
Figure 5. (A) The
1
H spectrum of linoleic acid; (B) the
1
H spectrum of cis-, trans-9,11-
octadecadienoic acid; (C) the
13
C spectrum of linoleic acid; and, (D) the
13
C spectrum of cis,
trans-9,11-octadecadienoic acid.
7. CONCLUSIONS
The research on CLA in growing animals is consistently showing effect on modu-
lation of body mass and fat, however, the effect in humans is not consistent. More
research is needed to delineate the effect of CLA and isomers on body composition
in humans. Major research emphasis, at present, is focused on the effects of CLA
and its isomers on body composition and carcinogenesis. Other areas that are
attracting attention include the effects of CLA and isomers on cardiovascular, meta-
bolic, and immune functions and the strategies to increase the content of CLA iso-
mers in meat and dairy products.
At the same time, research is still needed to improve the commercial production
of CLA. Although the content of desirable isomers in commercial CLA products
has improved, there is still a demand for highly enriched or pure 9,11-cis-, trans-
octadecadienoic acid products. The kinetic control of CLA synthesis will allow the
development of CLA products that are virtually free of isomers other than 9,11-ct
and 10,12-tc. Kinetic control of reactions requires exceedingly rapid analytical
techniques that can be applied inexpensively and online or virtually online.
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1

C without the addition of a catalyst (105). This reaction may be catalyzed

C. It follows that precise reactor temperature control is essential

C) (h) Reaction Sep

Superscript numbers refer to Vacuum in mm of Hg.

C, the reaction mixture will quickly release methanol

C) Color (400 MHz) Exclusion RP-LC GC-FID Mix

C using tetrahydrofuran as a solvent owing

C, 4.2 L/min gas ow). The goal of this ana-

C and a ow rate of 1.0 mL/min. The

C for 1 min, 50200

C for 1 min, 50150

C/min, hold 6 min, He

CH vibration band and, (B) 3050 to 2950 nm of the CH stretching

C/min; hold at 220

C for 3 min; He carrier gas @ 2 mL/min.

-HPLC). Using this modication, several groups have

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