Lipids (2016) 51:377397

DOI 10.1007/s11745-016-4135-z

REVIEW

Sources andBioactive Properties ofConjugated Dietary Fatty

Acids
AlanA.Hennessy1,2 PaulR.Ross2,3 GeraldF.Fitzgerald2,3
CatherineStanton1,2

Received: 15 January 2016 / Accepted: 17 February 2016 / Published online: 11 March 2016
AOCS 2016

Abstract The group of conjugated fatty acids known as this review, we will also highlight the common sources
conjugated linoleic acid (CLA) isomers have been exten- of these conjugated fatty acids, including plants, algae,
sively studied with regard to their bioactive potential in microbes and chemosynthesis.
treating some of the most prominent human health malig-
nancies. However, CLA isomers are not the only group Keywords Conjugated linoleic acid (CLA)
of potentially bioactive conjugated fatty acids currently Docosahexaenoic acid Lipid biochemistry Lipid
undergoing study. In this regard, isomers of conjugated metabolism Metabolism, fatty acids Polyunsaturated
-linolenic acid, conjugated nonadecadienoic acid and fatty acids (PUFA)
conjugated eicosapentaenoic acid, to name but a few,
have undergone experimental assessment. These studies Abbreviations
have indicated many of these conjugated fatty acid iso- apoB Apolipoprotein B
mers commonly possess anti-carcinogenic, anti-adipo- c  cis
genic, anti-inflammatory and immune modulating proper- CLNA Conjugated -linolenic acids
ties, a number of which will be discussed in this review. CDHA Conjugated docosahexaenoic acids
The mechanisms through which these bioactivities are CEPA Conjugated eicosapentaenoic acids
mediated have not yet been fully elucidated. However, CGLA Conjugated -linolenic acid
existing evidence indicates that these fatty acids may CLA Conjugated linoleic acid
play a role in modulating the expression of several onco- CNA Conjugated nonadecadienoic acid
genes, cell cycle regulators, and genes associated with CSA Conjugated stearidonic acid
energy metabolism. Despite such bioactive potential, COX Cyclooxygenase
interest in these conjugated fatty acids has remained low DHA Docosahexaenoic acid
relative to the CLA isomers. This may be partly attrib- EPA Eicosapentaenoic acid
uted to the relatively recent emergence of these fatty FAS Fatty acid synthase
acids as bioactives, but also due to a lack of awareness GIT Gastrointestinal tract
regarding sources from which they can be produced. In N2KO Nescient basic helix-loop-helix 2 knockout
PUFA Polyunsaturated fatty acids
PAI  Propionibacterium acnes isomerase
* Catherine Stanton PGE2 Prostaglandin E2
catherine.stanton@teagasc.ie
t  trans
1
Teagasc Food Research Centre, Moorepark, Fermoy, Co. TG Triacylglycerols
Cork, Ireland Ucp Uncoupling proteins
2
APC Microbiome Institute, University College Cork, Cork, VEGF Vascular endothelial growth factor
Ireland WAT White adipose tissue
3
School ofScience, Engineering andFood Science, University
College Cork, Cork, Ireland

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378
Introduction
Lipids may be defined as fatty acids, their derivatives, and
substances related biosynthetically or functionally to these
compounds [1]. These compounds are most commonly
found in the tissues of microorganisms, plants, animals,
and insects, and include important components for life
such as phospholipids, cholesterol, triacylglycerols (TAG),
glycolipids, and signalling molecules [25]. Central to the
composition of each lipid are fatty acids, which in their
simplest form consist of a straight chain of carbons, usually
even in number, terminated by a carboxyl group at one end
[6]. These molecules may be saturated, containing no dou-
ble bonds, or unsaturated, containing one or more double
bonds in either the cis (c) or trans (t) conformation [6]. It
is the highly reactive nature of the carboxyl group found
in fatty acids which predominantly permits the formation
of linkages with other molecules giving rise to the various
classes of lipid described previously.
Conjugated fatty acids are the positional and geomet-
ric isomers of unsaturated fatty acids, containing one or
more non-methylene interrupted double bonds in either
cis or trans conformation. Initial interest in conjugated
fatty acids as bioactive molecules arose following reports
of an anti-carcinogenic component found within ground
beef, later identified to be conjugated linoleic acid (CLA)
[79]. Since then, CLA isomers have been associated with
a range of bioactivities including anti-microbial, anti-
diabetogenic, anti-obesogenic, and anti-atherosclerotic
effects, along with reports of growth promotion, and of the
modulation of immune and inflammatory responses [10
12]. Indeed, to date CLA isomers remain the most widely
studied of all the conjugated fatty acids, despite growing
evidence that other both naturally derived and synthetic
conjugated fatty acids may also possess potent bioactive
properties [1315].
In this literature review, we will report the bioactivity
attributed to a range of conjugated fatty acids other than
the CLA isomers. Additionally, we will discuss current
knowledge regarding the sources of these conjugated fatty
acids and the scientific avenues being investigated for their
production.
Conjugated Linolenic Acids (CLNA)
Apart from the CLA isomers, the conjugated isomers
of -linolenic acid, known collectively by the acronym
CLNA, are the second most extensively studied of the
conjugated fatty acids [13]. This may be in part due to the
prevalence of these isomers within certain seed oils, etc.
(Table 1) [1634], but also due to the extensive range of
bioactivities associated with these isomers. Indeed, isomers
13
Lipids (2016) 51:377397
of CLNA have been associated with anti-adipogenic and
anti-carcinogenic properties, along with the modulation of
immune function and inflammatory response [13, 35].
AntiAdipogenic Properties
Anti-adipogenic effects of CLNA isomers have been
observed in a number of studies involving murine and por-
cine models [36, 37]. These studies have utilized a diverse
range of CLNA sources including a chemically derived
CLNA preparation [36], pomegranate seed oil [37, 38], a
genetically modified rapeseed oil [23], and calendic acid
[39], with the anti-adipogenic effects characterized by
reductions in body fat [38, 39], omental white adipose tis-
sue (WAT) [37] and in epididymal and perirenal adipose
tissue [23, 36]. Interestingly, not all animal studies have
reported the anti-adipogenic effect of CLNA isomers,
with investigations in a neonatal pig model reporting that
feeding animals 1% dietary CLNA (c9, t11, c15 and c9,
t13, c15) for twoweeks did not change adiposity or blood
lipid profiles [40]. Attempts have been made to elucidate
the mechanisms behind any anti-adipogenic effects, with
decreases in leptin [23, 36] and stearyl CoA desaturase [37,
41], and increases in PPAR [57], cAMP-activated protein
kinase activation [58] and increased cellular -oxidation
[36] mooted as possible contributing factors.
AntiCarcinogenic Properties
Conjugated fatty acids have historically been associated
with a range of anti-carcinogenic properties and in this
regard, the CLNA isomers are no exception. Oils derived
from pomegranate seed, bitter gourd seed, tung seed,
catalpa seed and pot marigold, or their primary constitu-
ent CLNA isomers have all demonstrated anti-carcinogenic
properties [13, 35, 59]. Indeed, pomegranate seed oil, rich
in the c9, t11, c13 CLNA isomer, has been assessed for
activity against cancer of the colon, skin, mammary tis-
sue, and prostate with some positive results (Table2). The
mechanisms behind the anti-carcinogenic activity remain
to be fully elucidated, however, tissue specific reductions
in cancer cell proliferation, invasiveness, and angiogenesis
along with increased cellular lipid peroxidation, alterations
in oncogene expression, and modulation of inflammatory
processes have been reported (Table2) [6063]. Similar to
pomegranate seed oil, bitter gourd seed oil and its primary
CLNA isomer, known as -eleostearic acid (c9, t11, t13),
have both exhibited anti-carcinogenic properties in stud-
ies conducted using colonic (HT-29, Caco-2), mammary
(MDA-wt, MDA-ER 7, MCF-7), and human leukemia
cells (HL60) [6468]. In these in vitro studies, exposure
to -eleostearic acid has been associated with increased
cellular lipid peroxidation, inhibition of DNA synthesis,
Lipids (2016) 51:377397 379

Table1Sources of conjugated fatty acids

Isomer Source Conc. (%) References Additional information

CLNA isomer
c9, t11, c13 Pomegranate seed oil 83 [16]
Fevillea trilobata oil 30 [17]
Momordica balsamina seed oil 50 [18, 19]
Snake gourd seed oil 40 [20, 21]
Ecballium elaterium seed oil 22 [20]
Milk fat 0.03 [22] Canadian milk fat
Rapeseed oil 2.50 [23] GMO rapeseed
c9, t11,t13 Tung seed oil 67.7 [16]
Bitter gourd seed oil 56.2 [16]
Snake gourd seed oil 3050 [24]
White mahlab 38.32 [25]
Parwal seed oil 3050 [24]
Sugihiratake mushroom NS [26] Edible Japanese mushroom
Parinarium species seed oil 2269 [27, 28]
Prunus yedoensis seed oil 35 [28]
Chrysobalanus icaco seed oil 22 [27]
t9, t11, c13 Catalpa seed oil 42.3 [16]
t9, t11, t13 Chemosynthesized >97 [29] Larodan Fine Chemicals, Sweden
c8, t10, c12 Jacaranda seed oil 31 [30]
t8, t10, c12 Pot marigold seed oil 5062 [16]
t8, t10, t12 Pot marigold seed oil 4.7 [31]
c9, t11, c15 Lactobacillus plantarum AKU 1009a 67 [32] -Linolenic acid conc. of 63mg/ml
Milk fat 0.03 [22] Canadian milk fat
9, 11, 15 Strains of Bifidobacterium breve 182.2 [33, 34] -Linolenic acid conc. of 0.450.5mg/ml
Strains of Bifidobacterium longum 0.1 [33] -Linolenic acid conc. of 0.45mg/ml
Bifidobacterium bifidum LMG10645 78.40 [34] -Linolenic acid conc. of 0.5mg/ml
Strains of Propionibacterium 150 [33] -Linolenic acid conc. of 0.45mg/ml
t9, t11, c15 Lactobacillus plantarum AKU 1009a 33 [32] -Linolenic acid conc. of 63mg/ml
CGLA isomer
c6, c9, t11 Lactobacillus plantarum AKU 1009a 27.20 [42] By washed cells from 13mg/ml -linolenic acid
c6, t9, t11 Lactobacillus plantarum AKU 1009a 40.80 [42] By washed cells from 13mg/ml -linolenic acid
6, 9, 11 Strains of Bifidobacterium breve 125 [33] -Linolenic acid conc. of 0.45mg/ml
Strains of Bifidobacterium longum 0.61.1 [33] -Linolenic acid conc. of 0.45mg/ml
Strains of Propionibacterium freudenreichii 0.1 [33] -Linolenic acid conc. of 0.45mg/ml
c6, t8, t10 Enzyme derived from Ptilota filicina NS [43] From -linolenic acid
CSA isomer
6, 9, 11, 15 Strains of Bifidobacterium breve 926 [33] Stearidonic acid conc. of 0.15mg/ml
Strains of Bifidobacterium longum 319 [33] Stearidonic acid conc. of 0.15mg/ml
Strains of Propionibacterium freudenreichii 13.6 [33] Stearidonic acid conc. of 0.15mg/ml
c9, t11, t13, c15 Parinarium laurinum seed oil 62 [27]
Chrysobalanus icaco seed oil 10 [27]
Sebastiania brasiliensis seed oil 39 [44]
Impatiens edgeworthii seed oil 48 [45]
Clavulina cristata NS [46]
c9, t11, t13, c15 Ulva fasciata Delile 14.65 [47] Fatty acid composition of the green algae
Chemosynthesized and purified >96 [48] Via bromination of -linolenic acid
t9, t11, t13, t15 Chemosynthesized and purified >96 [48] Via bromination of -linolenic acid

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380 Lipids (2016) 51:377397

Table1continued
Isomer Source Conc. (%) References Additional information

c9, t11, t13, t15 Chemosynthesized and purified >96 [48] Via bromination of -linolenic acid
5, 7, 11, 15 Ruminal biohydrogenation NS [49] From dietary stearidonic acid
5, 8, 10, 15 Ruminal biohydrogenation NS [49] From dietary stearidonic acid
CNA isomers
c10, t12 Chemosynthesized 43.60 Alkali isomerization of nonadecadienoic acid.
t11, c13 Chemosynthesized 43.70 Major isomers represented
CEPA isomers
c5, t7, t9, c14, c17 Acanthophora spicifera NS [50]
Ptilota filicina NS [43]
Enzymatic preparation 69.60 [50] Via fatty acid isomerase from Ptilota pectinata
Chemosynthesized 99.00 [51] One-pot double-Wittig protocol
t5, t7, t9, c14, c17 Acanthophora spicifera NS [50]
Enzymatic preparation 20.70 [50] Via fatty acid isomerase from Ptilota pectinata
Chemosynthesized 99.00 [51] One-pot double-Wittig protocol
c5, c8, t10, t12, c14 Bossiella orbigniana NS [52] Produced via an enzyme extract derived from the
algae
Chemosynthesized 16 [53]
Mixture Chemosynthesized 90 [54] Alkali isomerization of EPA
CDHA isomers
Mixture Chemosynthesized 65 [55, 56] Alkali isomerization of DHA

and the induction of apoptosis and cell cycle arrest. The
latter of these has been associated with up-regulation of
PPAR, GADD45, p21, Bax, p53, and caspase-3 expres-
sion [66, 68]. Tung seed oil, rich in -eleostearic acid, has
also exhibited anti-carcinogenic properties, with exposure
proving cytotoxic to a range of cancer cells as a result of
increased cellular lipid peroxidation, while increased
DNA fragmentation may also indicate apoptotic activity
(Table 2) [6971]. Interestingly, not all studies have dem-
onstrated anti-carcinogenic activity of -eleostearic acid,
with the isomer proving ineffective in preventing chemi-
cally induced carcinogenesis in Sprague-Dawley rats [72].
Catalpa seed oil, and more specifically the t9, t11, c13
CLNA isomer of which the oil is a rich source, has been
shown to possess anti-carcinogenic properties based on in
vitro and in vivo studies (Table2). These have included
reports of increased cytotoxicity in a human leukemia cell
line, and reductions in the incidence of chemically induced
cancers in rats [70, 73].
CLNA isomers with 9, 11, 13-C18:3 bond conforma-
tions are not alone in displaying potential anti-carcino-
genic activity. The8, 10, 12-C18:3 CLNA isomers such
as -calendic acid (t8, t10, c12) and -calendic acid (t8,
t10, t12) derived from Calendula officinalis seed or jacaric
acid (c8, t10, c12) derived from jacaranda seed have also
exhibited such properties (Table2). Indeed, in vitro stud-
ies have indicated that exposure to either -calendic acid
or -calendic acid can induce apoptosis of U-937 human
monocytic leukaemia cells, Caco-2 colon cancer cells and
JEG-3 human choriocarcinoma cells potentially as a direct
consequence of increased cellular lipid peroxidation [29,
70, 74]. In the most recent of these studies, Li et al. [74]
observed that exposure of JEG-3 cells to either calendic
acid isomer initiated increased cellular oxidative stress
and activation of p38-mitogen-activated protein kinases
(MAPK), a group of proteins involved in cellular differ-
entiation, apoptosis and autophagy. Moreover, the authors
observed that the presence of a selective inhibitor of p38-
MAPK, known as SB20350, nullified the apoptotic effect
of the calendic acid isomers. Exposure of human leuke-
mia HL-60 cells to either jacaric acid or jacaranda seed
oil resulted in increased cell death [30]. Following further
investigation, it was concluded the jacaric acid exposure
resulted in increased DNA fragmentation and lipid peroxi-
dation culminating in HL-60 cell apoptosis [30].
Given the anti-carcinogenic properties attributed to
seed oil-derived CLNA isomers, much research has been
directed towards the discovery of other novel sources of
CLNA isomers with some success, including dairy prod-
ucts and microbial strains of dairy and intestinal origin
[9295] (Table1). These investigations have revealed the
presence of the c9, t11, c15 CLNA isomer within the meat
and milk of ruminants [22, 96, 97], along with the capacity
of strains of Lactobacillus, Bifidobacterium and Propioni-
bacterium to produce both the c9, t11, c15 and t9, t11, c15
CLNA from free -linolenic acid [3234]. One such strain,

13
 Table2In vitro and in vivo bioactive properties of conjugated fatty acids
Model
CLNA isomers
(1) Transformed SV-T2
mouse fibroblast cells. (2)
U-937 human monocytic
leukaemia
F344 rats with azoxym-
ethane induced colonic
aberrant crypt foci
(1) MCF-7 breast cancer.
(2) MDA-MB-435 breast
cancer
(1) MCF-7 breast cancer,
(2) MCF-10A immortal-
ized breast epithelial cells,
(3) MDA-MB-231 breast
cancer
Female CD1 mice
LNCaP, PC-3 and DU145 Pomegranate seed oil (c9,
human prostate cancer cell t11, c13 CLNA) (NS)
lines
13
PC-3 prostate cancer
Isomer
c9, t11, c13 CLNA (99%) (1) 10M, (2) 5M.
Pomegranate seed oil (c9,
t11, c13 CLNA) (>70%)
Pomegranate seed oil (c9,
t11, c13 CLNA) (NS)
Pomegranate seed oil (c9,
t11, c13 CLNA) (NS)
Pomegranate seed oil (c9,
t11, c13 CLNA) (NS)
Pomegranate seed oil (c9,
t11, c13 CLNA) (NS)
Dose and duration Function Potential mechanism of Compared to References
action
Cytotoxic Lipid peroxidation, Similar cytotoxicity to tung [70]
24h supported by the in seed oil, substantially
cytotoxicity on addition of more cytotoxic than
Lipids (2016) 51:377397
BHT and high suscepti- catalpa seed oil or pot
bility of the oil to lipid marigold
peroxidation
0.011.00% wt diet, Incidence (3856%) and c9, t11 CLA conc. and 1% CLA had no effect on [75]
32weeks multiplicity (0.500.73 PPAR expression in the incidence and multi-
0.880.96) of azoxym- the non-lesional colonic plicity of azoxymethane
ethane induced colonic mucosa induced colonic aberrant
aberrant crypt foci crypt foci
(control diet: 81% and
1.881.54, respectively)
(1a) 100g/ml, (1b) 10g/ (1a) 90% in proliferation, NS NS [76]
ml. (2) 50g/ml super- (1b) 75% in invasive-
critical fluid pomegranate ness. (2) Induced 54%
seed oil. apoptosis
(1) >100g/ml, (2) Angiogenesis (1) and (2) Down regula- NS [77]
10g/ml, (3) 10g/ tion of the angiogenic
ml supercritical fluid promoter vascular
pomegranate seed oil. 24h endothelial growth factor.
(3) Up regulation of the
angiogenic suppressors
migration inhibitory
factor
5.0% wt diet pomegranate Incidence and multiplicity 17% Reduction in NS [78]
seed oil, 20weeks of 7,12-dimethylbenzan- 12-O-tetradecanoylphor-
thracene induced skin bol 13-acetate induced
tumor ornithine decarboxylase
activity
Variable concentrations and Proliferation of prostate In G2/M cells from 11 to NS [79]
duration cancer cell lines, in 22% (2.30.001-fold)
PC-3 invasion up regulation of cyclin-
dependent kinase inhibitor
p21 and (0.60.14-fold)
down-regulation of c-myc,
in the DU145 cell line
4g/ml, 72h Invasion of PC-3 prostate NS NS [80]
cancer
381


Table2continued
382

Model Isomer Dose and duration Function Potential mechanism of Compared to References
action

13
DLD-1 colorectal cancer, Tung seed oil (c9, t11, t13 25M, 24h Cytotoxic NS CLA up to a conc. of [69]
HepG2 hepatoma, A549 CLNA) (80%) 100M was not cytotoxic
lung cancer, MCF-7 breast towards the cancer cell
cancer, and MKN-7 stom- lines assayed
ach cancer
Male F344 rats with azox- Bitter gourd seed oil (c9, 0.01% wt diet, 32weeks Incidence (47%) and c9, t11 CLA conc. and NS [81]
ymethane induced colonic t11, t13 CLNA) (60.2%) multiplicity (64%) of PPAR expression in
aberrant crypt foci azoxymethane induced the non-lesional colonic
colonic aberrant crypt foci mucosa
Nude mice with transplanted Tung seed oil (c9, t11, t13 50mg every 48h, 32days Cell apoptosis Lipid peroxidation, Supplementation with c9, [71]
DLD-1 colorectal cancer CALA) (79.7%) DNA fragmentation t11 and t10, c12 CLA
resulted in lower DNA
fragmentation than tung
seed oil
Caco-2 colon cancer Bitter gourd seed oil (c9, 25M, 24h Cell apoptosis Expression of GADD45, Supplementation with c9, [66]
t11, t13 CLNA) (NS) p53 and PPAR. expres- t11 CLA resulted in lower
sion of Bcl-2 DNA fragmentation and
higher cell viability than
bitter gourd seed oil or
pure c9, t11, t13 CLNA
Caco-2 colon cancer c9, t11, t13 CLNA (>98%) >10M, 2448h Cell apoptosis Expression of Bcl-2, NS [29]
DNA fragmentation,
lipid peroxidation, Activ-
ity lost on addition of
5M -tocopherol
(1) Transformed SV-T2 c9, t11, t13 CLNA (99%) (1) 10M, (2) 7M, Cytotoxic Lipid peroxidation, Similar cytotoxicity to [70]
mouse fibroblast cells. (2) 24h supported by the in pomegranate seed oil, sub-
U-937 human monocytic cytotoxicity on addition of stantially more cytotoxic
leukaemia BHT and high suscepti- than catalpa seed oil or
bility of the oil to lipid pot marigold
peroxidation
Sprague-Dawley rats with c9, t11, t13 CLNA (70.7%) 0.011.0% wt diet, Small non-significant in NS NS [72]
DMBA induced mammary 34weeks the incidence, multiplicity
and colon cancers of tumors
(1) Normal A31 mouse Catalpa seed oil (t9, t11, (1) 210M, (2) 25M, Cytotoxic Lipid peroxidation, Less cytotoxic than tung [70]
fibroblast cells. (2) Trans- c13 CLNA) (99%) (3) 10M supported by the in seed oil or pomegranate
formed SV-T2 mouse cytotoxicity on addition of seed oil, substantially
fibroblast cells. (3) Human BHT and high suscepti- more cytotoxic than pot
monocytic leukaemia cell bility of the oil to lipid marigold
line U-937 peroxidation
Lipids (2016) 51:377397
 Table2continued
Model Isomer Dose and duration Function Potential mechanism of Compared to References
action

Male F344 rats with azox- Catalpa seed oil (t9, t11, (1) 0.1%, (2) 1.0% wt diet, (1) Incidence of azoxym- t9, t11 CLA conc. in the NS [73]
ymethane induced colonic c13 CLNA) (40.2%) 4weeks ethane induced colonic liver and non-lesional
aberrant crypt foci aberrant crypt foci in colonic mucosa, expres-
F344 rats from 9928 in sion of COX-2
Lipids (2016) 51:377397

the control to 3518, (2)

significantly indices of
apoptosis and indices of
proliferation
Caco-2 colon cancer t9, t11, t13 CLNA (>97%) >10M, 2448h Apoptosis of Caco-2 Expression of Bcl-2, Compared to the c9, t11, [29]
colon cancer cells expression of Bax, DNA t13 CLNA isomer, this
fragmentation, lipid isomer retained activity
peroxidation at -tocopherol conc.
5M
Human monocytic leukae- t8, t10, c12 CLNA (99%) >20M, 24h Cytotoxic Lipid peroxidation, Less cytotoxic than tung [70]
mia cell line U-937 supported by the in seed oil, pomegranate
cytotoxicity on addition of seed oil, or catalpa seed
BHT and high suscepti- oil
bility of the oil to lipid
peroxidation
Caco-2 colon cancer t8, t10, c12 CLNA (>98%) >6.25M, 48h Apoptosis DNA fragmentation, Higher DNA fragmentation [29]
lipid peroxidation, Activ- than either the c9, t11,
ity lost on addition of t13 or t9, t11, t13 CLNA
50M -tocopherol isomers
Caco-2 colon cancer t8, t10, t12 CLNA (>97%) >6.25M, 48h Apoptosis DNA fragmentation, Compared to the t8, t10, [29]
lipid peroxidation c12 CLNA isomer, this
isomer retained activity
at -tocopherol conc.
50Mm
CGLA isomers
SW480 colon cancer c6, c9, t11 CGLA (>95%) 200M, 72h Cytotoxic Lipid peroxidation Lower cytotoxicity to a [82]
normal human colonic
cell line
CSA isomers
SW480 colon cancer 6, 9, 11, 15 CSA (>95%) 25200M, 24h Cytotoxic Lipid peroxidation, Greater cytotoxicity than [82]
ratio of n-6 to n-3 PUFA, stearidonic acid. Lower
expression of Bcl-2 cytotoxicity to a normal
human colonic cell line

13
(1) 36B10 glioma. (2) C6 c9, t11, t13, c15 (NS) 12M, 24h Cytotoxic Lipid peroxidation Did not display toxicity [83]
rat glioma. (3) A172 towards foetal rat astro-
human glioma. (4) Human cytes
monocytic leukaemia cell
line U-937
383


Table2continued
384

Model Isomer Dose and duration Function Potential mechanism of Compared to References
action

13
(1) THP-1 monocytic leu- c9, t11, t13, c15 (NS) <5M Cytotoxic Lipid peroxidation Greater cytotoxicity than [84]
kaemia. (2) HL-60 human oleic, linolenic and
promyelocytic leukaemia. monocytic acids. Caco-2,
(3) Y-79 human retinoblas- fibroblast and endothelial
toma cells. (4) U-937 cells cells less susceptible
36B10 glioma cells c9, t11, t13, c15 (NS) 14M, 24h Cytotoxic Activation of c-Jun Actions not observed in [85]
N-terminal protein kinase, normal astrocytes
inactivation of forkhead
transcription factor-3a,
mitochondrial superoxide
dismutase activity
CEPA isomers
HepG2, DLD-1, A549 and Mixture of isomers 25100M, 24h Cytotoxic Lipid peroxidation, con- NS [14]
MKN-7 cancer cell lines densation and fragmenta-
tion of DNA suggestive of
induced apoptosis
DLD-1 colorectal adenocar- c5, t7, t9, c14, c17 (99%) 520M, 24h Cytotoxic, apoptosis Morphological changes in Also effective against [51]
cinoma cells, condensation and HL-60 acute promyelo-
fragmentation of DNA cytic leukaemia cells
DLD-1 colorectal adenocar- t5, t7, t9, c14, c17 (99%) 520M, 24h Cytotoxic, apoptosis Morphological changes in Also effective against [51]
cinoma cells, condensation and HL-60 acute promyelo-
fragmentation of DNA cytic leukaemia cells
DLD-1 colorectal adenocar- Mixture of isomers 10M, 24h Apoptosis Up-regulation of expression NS [86]
cinoma of genes induced by p53,
activation of the mitochon-
drial apoptosis pathway
via Bax and the death
pathway via TRAIL
HCT116 colorectal adeno- Mixture of isomers Variable Cell cycle arrest Inhibition of polymerases , NS [87]
carcinoma , , and topoisomerases
I and II resulting in cells
becoming locked in the
G1/S phase
VEGF stimulated HUVEC Mixture of isomers 5mol/l, 10days VEGF mediated angio- Down regulation of VEGF- Significantly more effective [88]
cells genesis and migration induced MMP2 and than EPA
MMP9 secretion
Male BALB/c mice CEPA enriched oil 20g/100g of dietary FA, 70% in the weight of DNA fragmentation More effective than CLA [89]
4weeks tumors within tumor cells, lipid and EPA fed mice
peroxidation
Lipids (2016) 51:377397
 Table2continued
Model Isomer Dose and duration Function Potential mechanism of Compared to References
action

CDHA isomers
HepG2, DLD-1, A549 and
MKN-7 cancer cell lines
Mixture of isomers 25100M, 24h Cytotoxic Lipid peroxidation, con- NS [14]
densation and fragmenta-
tion of DNA suggestive of
Lipids (2016) 51:377397

induced apoptosis
KPL-1 mammary cancer Mixture of isomers (65% IC50=97mol/l after 72h Cell cycle arrest and Expression of p53 and IC50 for EPA=270mol/l [56]
cells of total FA) apoptosis p21, cyclin D1 and after 72h
Bcl-2
Colo 201 colonic cancer cell Mixture of isomers (65% IC50=31.6M after 72h Cell cycle arrest and p21 and cyclin D1, IC50 for DHA=46.8M [55]
of total FA) apoptosis cyclin E and PCNA. cel- after 72h
lular Bcl-xL and Bcl-2
BAEC bovine aortic Mixture of isomers 1020mol/l, 72h Tube formation, and Cell apoptosis More effective than equiva- [90]
endothelial cells BAEC proliferation and lent conc. of DHA
migration
Female Sprague-Dawley Mixture of isomers 0.2 or 1.0% diet, Tumor size, incidence and NS Provision prior to 7weeks [91]
rats with MNU induced 740weeks multiplicity
mammary carcinogenesis
on day 49
Female BALB/c-nu/nu mice Mixture of isomers (CDHA 1.0%, Tumor volume Potentially via cell cycle NS [56]
with tumors initiated by 65% of total FA) arrest and apoptosis
KPL-1 mammary cancer
cell transplant
Male BALB/c-nu/nu mice Mixture of isomers (CDHA 1.0%, 26days Tumor volume Potentially via cell cycle NS [55]
with tumors initiated by 65% of total FA) arrest and apoptosis
Colo 201 colonic cancer
cell transplant
ICR Mice with tumors initi- Mixture of isomers 510mg, 7days Vessel formation in area Cell apoptosis Animals not fed CDHA [90]
ated by DLD-1 colonic surrounding tumor
cancer cell transplant

13
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386
namely Bifidobacterium breve NCIMB 702258, displayed
the capacity to bioconvert 79.1% of substrate -linolenic
acid (0.41mg/ml) to the c9, t11, c15 CLNA isomer [98].
When SW480 colon cancer cells were exposed to an
oil rich in this CLNA isomer (55.6%) for 5days, a dose
dependent decrease in SW480 cell viability was observed,
that was significantly greater than the control (-linolenic
acid enriched oil) [98]. As a follow up from this investi-
gation, our group assessed the anti-carcinogenic proper-
ties of the c9, t11, c15 CLNA produced by the strain B.
breve DPC6330 following isolation of the isomer from a
fermented oil (>95% purity) [82]. The study showed that
exposure of SW480 cells to 0200M of the CLNA iso-
mer resulted in a dose dependent decrease in cell viability
over the 72h of exposure. Nonetheless, the reduction in
SW480 cell viability observed with the CLNA isomer did
not differ significantly from that of equivalent concentra-
tions of -linolenic acid (respective IC50: 1804.0 and
178 3.4M). It was, however, noted that the c9, t11, AntiCarcinogenic Properties
c15 CLNA isomer displayed significantly greater inhibi-
tory activity against the SW480 cell line than a normal
human fetal colonic (FHC) cell line after 24h of exposure,
suggesting a level of selective anti-carcinogenic activity.
Mechanistic studies have indicated that increased lipid per-
oxidation, changes in the ratio of -6 to -3 polyunsatu-
rated fatty acids (PUFA) within the cellular phospholipids
and reductions in the cellular concentration of the anti-
apoptotic oncogene Bcl-2 may all have played a role in the
inhibitory activity of the c9, t11, c15 CLNA isomer [82].
AntiInflammatory andImmune Modulating Properties
There is increasing evidence of anti-inflammatory and
immune modulating properties of CLNA isomers and in
particular CLNA rich oils [13]. In this regard, both pome-
granate seed oil and bitter gourd seed oil have yielded
positive results. Pomegranate seed oil is rich in the CLNA
isomer known as punicic acid (c9, t11, c13), and has been
shown to enhance production of both IgG and IgM by B
cells when included in the diet of C57BL/6N mice [99].
Additionally, further studies have indicated that punicic
acid may positively modulate the immune and inflamma-
tory responses in the gut via PPAR, PPAR and TNF-
dependent mechanisms [100, 101]. Dietary administration
of punicic acid resulted in decreased neutrophil activation
and reactive oxygen species mediated tissue damage in rats
with trinitrobenzenesulfonic acid induced colonic inflam-
mation, further substantiating its potential role in the con-
trol of inflammatory gut diseases [100]. Bitter gourd seed
oil, rich in c9, t11, t13 CLNA, is an effective stimulator
of IFN- production in mice treated with heat inactivated
Propionibacterium acnes [102]. It has also been reported
that ingestion of bitter gourd seed oil resulted in reduction
13
Lipids (2016) 51:377397
of the inflammatory response in allergen mediated asthma
[103]. This action was characterized by reductions in the
level of inflammatory eicosanoids and white blood cells
detected in the lung, along with a reduction in IgE and
increase in IgG [103].
Conjugated Linolenic Acid (CGLA)
Unlike the CLNA isomers previously discussed, conju-
gated -linolenic acid (CGLA) isomers are not commonly
found in the seed oils of plants but can be produced via the
microbial isomerisation of free -linolenic acid by a num-
ber of microbial strains (Table1) [33, 42, 43]. Indeed, stud-
ies have reported the production of the c6, c9, t11 and c6,
t9, t11 CGLA isomers by strains of Lactobacillus, Bifido-
bacterium and Propionibacterium [33, 42, 104].
Although little investigative work has been conducted into
the potential bioactive properties of the CGLA isomers, our
group have found that the isomer exhibits anti-carcinogenic
properties in vitro [82]. In this investigation, both SW480
colon cancer cells and normal human fetal colon FHC
cells were exposed to the c6, c9, t11 CGLA isomer (>95%
purity) at concentrations ranging from 12.5 to 200M
over 72h. The results indicated that the CGLA isomer dis-
played greater inhibitory activity against SW480 cells than
equivalent concentrations of -linolenic acid. Moreover, at
a concentration of 200M, CGLA displayed significantly
greater inhibitory activity against the SW480 cells than the
FHC cell line. Mechanistic investigations indicated that
inclusion of -tocopherol in the medium during exposure
of cells to 200M CGLA resulted in a 5983% reduction
in inhibitory effect. This observation indicates a prominent
role for lipid peroxidation in the anti-carcinogenic activity
of CGLA.
Conjugated Stearidonic Acid (CSA)
Stearidonic acid is an n-3 fatty acid and a direct precur-
sor in mammals of the long-chain PUFA, eicosapentaenoic
acid (EPA) and docosahexaenoic acid (DHA). Studies have
demonstrated that via the activity of the microbial enzyme,
linoleic acid isomerase (LAI), stearidonic acid can be con-
verted to its conjugated 6, 9, 11, 15-CSA isomers. Indeed,
some strains of Lactobacillus, Bifidobacterium and Propi-
onibacterium have been identified that possess this ability
[33, 105]. Furthermore, in vitro ruminal studies assessing
the biohydrogenation of stearidonic acid have detected a
number of CSA isomers including 5, 7, 11, 15-CSA and at
Lipids (2016) 51:377397 387
least three 5, 8, 10, 15-CSA isomers suggesting substantial
potential for CSA production among the ruminal microbi-
ota [49] (Table1).
AntiCarcinogenic Properties
While there are a lack of data on the bioactive properties
of CSA isomers, our group have reported that a purified
fraction (>95%) of microbially produced 6, 9, 11, 15-CSA
exhibited anti-carcinogenic activity [82] in vitro. Indeed,
exposure of SW480 colon cancer cells to the CSA isomer
at concentrations of 25 and 200M for 24h resulted in a
dose dependent reduction in cell viability. Additionally, this
study reported that the IC50 of the CSA isomer after 24h
was lower than that of the parent compound stearidonic
acid (1042.9 and 1623.7M, respectively), whilst (FAS), adipocyte lipid binding protein and hormone-
it was also more inhibitory to SW480 cells than to nor-
mal human colonic cells (FHC) [82]. Mechanisms of the
inhibitory activity of the CSA isomer were found to include
increased lipid peroxidation, reductions in the ratio of n-6
to n-3 PUFA within cellular phospholipids, and/or cellular
reductions in the anti-apoptotic oncogene Bcl-2.
A conjugated C18:4 fatty acid known as -parinaric acid
can also be found in the oils of certain plant seeds and algae
(Table 1). This c9, t11, t13, c1518:4 conjugated isomer
along with its t9, t11, t13, t1518:4 counterpart (-parinaric
acid) have previously been utilized as probes to character-
ize lipidlipid and lipidprotein interactions [106109],
and as sensitive markers to characterize lipid peroxidation
in eukaryotic systems [110113]. There is also evidence to
indicate that -parinaric acid may possess cytotoxic prop-
erties towards cancer cells, including human monocytic
leukaemia cells and malignant gliomas, mediated through
increased cellular lipid peroxidation [83, 84]. -Parinaric
acid has been shown to be capable of binding PPAR
[114], to activate c-Jun N-terminal protein kinase, to inac-
tivate forkhead transcription factor-3a, and to reduce the
activity of mitochondrial superoxide dismutase in malig-
nant rat astrocytoma cells (36B-10 cells) [85, 115]. These
data indicate that -parinaric acid likely impairs the cells
ability to respond to increased oxidative stress, and modu-
lates changes in cellular autophagy and apoptosis.
Conjugated Nonadecadienoic Acid (CNA)
Unlike conjugated C18 fatty acids, conjugated C19 fatty
acids are not regularly found in nature but rather chemi-
cally synthesized [15, 116] (Table1). Nonetheless, they
have been assessed for bioactive properties and in this
regard have shown potential as modulators of adiposity,
energy expenditure and inflammatory response.
AntiAdipogenic Properties
Initial studies into the effect of a dietary mixture of CNA
isomers (0.3% of diet) on body composition of mice indi-
cated that the intake of the isomer for twoweeks resulted
in a substantial reduction in body fat [117]. Moreover, in
vitro investigations by the same group using 3T3-L1 adi-
pocytes indicated that exposure to this mixture of CNA
isomers inhibited heparin-releasable lipoprotein lipase, an
important enzyme involved in TAG metabolism. CNA has
also been attributed with the inhibition of the differentia-
tion of pre-adipocytes to adipocytes [118, 119]. This was
characterized by decreased intracellular TAG accumula-
tion and changes in the expression of proteins involved
in fatty acid metabolism, including fatty acid synthase
sensitive lipase [118, 120122]. CNA also reduced pro-
tein levels of adipocyte transcription factors, PPAR and
CCAAT/enhancer binding protein [118]. Uncoupling
proteins (Ucp) are key proteins involved in the regulation
of energy homeostasis and potentially adiposity. An in
vitro study using the 3T3-L1 cell line has implicated CNA
isomers with up-regulating the expression of Ucp-2 and
Ucp-3, potentially increasing the use of lipid as an energy
source [120, 123].
Apolipoprotein B (apoB) is required for the hepatic
assembly and secretion of very low density lipoprotein and
elevated concentrations have been associated with hyper-
lipidemia in vivo. CNA isomers were shown to reduce
apoB secretion by HepG2 cells, potentially indicating ben-
efits towards cardiovascular disease [124].
Furthermore, dietary CNA has proved an effective
modulator of adiposity and energy metabolism in murine
models (Table2). The first of these in vivo studies in
mice indicated that 0.1% dietary CNA was as effective
as 0.5% dietary CLA in reducing body fat via increased
energy expenditure and beta-oxidation of fatty acids [122].
Moreover, dietary CNA positively altered the expres-
sion of hormone-sensitive lipase in WAT, an attribute not
seen with the CLA isomer. Further comparisons of CNA
(0.1%) with CLA (0.5%) in ICR mice over 412weeks
revealed that CNA significantly reduced body weight and
fat mass [15], which were attributed to increased energy
expenditure via improved voluntary movement along with
reductions in serum leptin and TNF-, general decreases
in the expression of FAS and PPAR in adipose tissue, and
increases in Ucp-2 and hormone sensitive lipase in WAT.
Kim et al. [125] reported that CNA may also be effective
in the control of adult onset obesity. Using nescient basic
helix-loop-helix 2 knockout (N2KO) mice, it was demon-
strated that relative to the control diet, mice in receipt of
0.1% (w/w) CNA (prior to the development of obesity)
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388
displayed lower reductions in voluntary movement and
weight gain. These results were further complimented by
comparison of CNA fed N2KO and wild type mice, with
the CNA fed N2KO mice displaying significant improve-
ments in the expression of genes involved in lipid metab-
olism and energy expenditure. Indeed, CNA feeding in
N2KO mice was observed to reduce hepatic FAS expres-
sion as well as leptin levels in WAT. It was also found
in this study that wild type animals fed CNA displayed
increased Ucp-2 expression in muscle, potentially reflect-
ing increased use of lipid as an energy source in this tissue.
Inflammatory Response
CLA isomers have previously proven to be potent modu-
lators of inflammatory response, prompting similar inves-
tigations into CNA isomers [10]. In this regard, CNA
preparations have proven to be comparable to CLA iso-
mers with regard to their impact on the cyclooxygenase
(COX) enzymes, COX-1 and COX-2 that are involved in
the conversion of arachidonic acid to prostacyclins and
thromboxane (Table2). COX-2 is the inducible form of the
enzyme, activated primarily in response to stimuli such as
lipopolysaccharide or inflammatory cytokines [126]. Using
the RAW 264.7 macrophage cell line, it was demonstrated
that CNA exposure was effective in reducing cellular
COX-2 concentrations as well as prostaglandin E2 (PGE2),
in whose production COX-2 plays an important role [127,
128]. Interestingly, as PGE2 can stimulate cancer progres-
sion, COX-2 inhibition by CNA may also impart some
anti-carcinogenic activity [129]. COX-1 is constitutively
expressed in the body, particularly in the gastric mucosa
and kidneys. Although multifaceted in action, COX-1 plays
an important role in platelet aggregation, in particular via
the action of its metabolite thromboxane A2, and it is with
regard to this activity that CNA has been implicated. In the
study of Li et al. [116], a CNA preparation was shown to
reduce the activity of free ovine COX-1 as well as COX-1
activity within RAW 264.7 cells. Moreover, it was dem-
onstrated that CNA exposure inhibited collagen induced
aggregation of platelets in the blood of guinea pigs under
in vitro conditions potentially via reduced COX-1 mediated
thromboxane A2 release [116].
Conjugated Eicosapentaenoic Acids (CEPA)
The n-3 fatty acid EPA has long been recognized for its
bioactive properties and as a result it is not surprising that
its conjugated isomers also exhibit bioactive potential.
Conjugated EPA (CEPA) isomers occur naturally in marine
algae [43, 50, 52] but are most commonly synthesized for
experimental purposes (Table1).
13
Lipids (2016) 51:377397
AntiAdipogenic Properties
Like many other conjugated fatty acids, CEPA isomers
have been investigated for their ability to modulate adipos-
ity and energy expenditure in murine models. In the first
of these studies, a small group (n =8) of male Sprague-
Dawley rats were administered CEPA enriched oil [at
0.81.0% (w/w) of their diet] for 4weeks [54]. During
this time, it was noted that the CEPA supplemented group
had a significantly lower body weight and epididymal adi-
pose tissue weight compared with animals supplemented
with an equivalent concentration of EPA. Additionally,
animals on the CEPA supplemented diet displayed similar
or improved hepatic and plasma lipid profiles compared
with animals receiving EPA, whilst the CEPA group also
had markedly improved concentrations of plasma TNF-.
Concentrations of the hepatic enzymes malic enzyme and
FAS (both involved in lipogenesis) were also notably lower
in the CEPA group, whilst the activity of acyl-CoA oxi-
dase was significantly enhanced, reflecting greater hepatic
-oxidation of lipid. A subsequent study into the impact of
dietary CEPA in Wister rats (n=7) also found that CEPA
supplementation led to significantly reduced WAT/body
weight ratio of animals compared to those in receipt of
other lipid supplemented diets [130]. However, no other
significant improvements in adiposity or lipid profile were
noted.
AntiCarcinogenic Properties
A number of studies have assessed the anti-carcinogenic
potential of CEPA experimentally both in vitro and in
vivo (Table2). These studies revealed that CEPA expo-
sure reduces the viability of HepG2, DLD-1, A549 and
MKN-7 cancer cell lines [14, 51]. The apparent mecha-
nism for this reduction in viability in the DLD-1 cell line
was identified as increased cellular lipid peroxidation,
culminating in the induction of apoptosis. This apoptotic
activity was characterized by morphological changes in
cells, along with increased condensation and fragmenta-
tion of the DNA, which could subsequently be significantly
reduced by the inclusion of the anti-oxidant -tocopherol
in the assay medium. The pro-apoptotic activity of CEPA
against cancers cells via increased lipid peroxidation was
also observed in vivo by Tsuzuki et al., who reported on
the effectiveness of CEPA in reducing tumor growth in
nude mice (initiated via DLD-1 cancer cell transplanta-
tion). Indeed, mice in receipt of 20mg CEPA/100mg total
dietary fat displayed reduced tumor mass and higher body
mass than mice in receipt of the control or other lipid sup-
plemented diets [89]. Similar to the existing in vitro evi-
dence, tumor cells within the CEPA supplemented group
displayed increased condensation and fragmentation of
Lipids (2016) 51:377397 389
the DNA along with increased cellular lipid peroxidation.
Some work has been performed addressing the mechanism
by which CEPA might mediate its pro-apoptotic effects in
the DLD-1 cell line as a consequence of lipid peroxidation.
In one study, it was observed that CEPA exposure up-regu-
lated the expression of 14 genes associated with apoptosis
[86]. Six of these genes, DR4, FADD, p21, GADD45, Bid
and FAS, are induced by p53, the cell cycle regulator and
tumor suppressor, with the remaining eight under the sub-
sequent control of these six. Further investigation by this
group demonstrated that the up-regulation of these genes
corresponded with actual cellular increases in the concen-
tration of p53 and p21, and of the pro-apoptotic oncogene
Bax, along with a decrease in the anti-apoptotic oncogene
Bcl-2 [86]. Furthermore, the study found that CEPA expo-
sure significantly increased the cellular activity of caspase
enzymes involved in p53 mediated apoptosis. Combined,
these data indicate that increased lipid peroxidation as a
result of the exposure of DLD-1 cells to CEPA initiates cel-
lular apoptosis via p53 activation [86].
In addition to the induction of apoptosis via increased
cellular lipid peroxidation, CEPA exposure has also been
implicated in inhibition of the activity of DNA polymer-
ases and DNA topoisomerases [131, 132]. During DNA
replication and repair, these enzymes play important roles
in catalyzing the addition of deoxyribonucleotides to the 3
terminus of DNA, as well as during the cutting and rejoin-
ing of DNA, thus, their inhibition detrimentally impairs this
activity. In studies conducted using leukaemia (HL-60) and
colonic (HCT116) cell lines, CEPA preparations were dem-
onstrated to inhibit the activity of five mammalian poly-
merases (, , , , ) and the human topoisomerases (I, II) docosahexaenoic acid (CDHA) and isolated CDHA iso-
via direct interaction with the enzyme [87, 131]. A more
comprehensive study has indicated that CEPA irreversibly
binds to DNA topoisomerases and to polymerases in par-
ticular, resulting in an accumulation of single strand DNA
stabilized by replication protein A [132]. The elevated con-
centration of stable single strand DNA results in the activa-
tion of ataxia telangiectasia and Rad3 related protein kinase
which phosphorylates checkpoint kinase 1 and 2. Phospho-
rylation of checkpoint kinases causing their activation nor-
mally results in the initiation of cell cycle checkpoints and
ultimately cell cycle arrest, DNA repair or cell death via
p21 or p53 mediated events. Of these activities, exposure
of cancer cells to CEPA was observed to increase cell cycle
arrest, characterized by a higher proportion of cells locked
in the G1/S phase of their cell cycle than cells in the control
samples and by the increased expression of cyclins A and
E, which play a role in maintaining the cells in the G1/S
phase [87, 132]. Furthermore, investigators have assessed
the impact of CEPA as an adjunct to radiation in the sup-
pression of HCT 116 colon cancer cell growth in vitro
and it was found that post-irradiation, addition of CEPA
significantly enhanced the sensitivity of HCT 116 cells to
the effects of irradiation as a result of their inhibitory activ-
ity against at least three polymerases (, , ) [133].
Visual observations by Tsuzuki et al. [89], during their
investigation into the effect of CEPA on transplanted tumor
growth in nude mice suggested that CEPA might exert some
anti-angiogenic properties in tumors under in vivo condi-
tions prompting further in vitro studies. As the development
of blood vessels via angiogenesis is critical to sustained
tumor growth, any inhibitory activity could be important,
particularly in light of the other anti-carcinogenic proper-
ties exhibited by CEPA preparations. To achieve this, a co-
culture of HUVEC cells and human fibroblasts, stimulated
to produce blood vessels by vascular endothelial growth
factor (VEGF) was assessed for vessel formation after
11days of growth in the presence or absence of CEPA or
EPA [88]. The results indicate the exposure to the fatty
acids significantly reduced vessel formation, whilst CEPA
exposure also suppressed the migration (denudation injury
model) and proliferation (WST-1 assay) of HUVEC cells.
Preliminary investigations into the reason for this activity
revealed that in the VEGF stimulated HUVEC cells, CEPA
exposure down regulated the expression of at least two
proteins involved in angiogenesis, namely matrix metallo-
peptidase-2, and -9.
Conjugated Docosahexaenoic Acids (CDHA)
Although not available in nature, a number of investiga-
tions have assessed synthetic preparations of conjugated
mers for potential bioactive properties [55, 56]. Ultimately,
these investigations have yielded some evidence indicat-
ing that CDHA enriched preparations, like CEPA enriched
preparations, may possess anti-adipogenic and anti-carci-
nogenic properties.
AntiAdipogenic Properties
In the earliest study to report the anti-adipogenic properties
of CDHA, a 200mg dose of a chemically derived CDHA
preparation was given to rats by oral gavage for fourweeks
[130]. The results indicated that rats in receipt of dietary
CDHA displayed significantly reduced WAT weight, and
an improved lipid profile within the liver and plasma (total
cholesterol and TAG). Okada et al. [130], assessed the
effect of the inclusion of CDHA (as 2% of diet) in the diet
of Wister rats versus the control group fed an equivalent
concentration of soybean oil. Animals in receipt of dietary
CDHA exhibited lower mean WAT weight compared to the
control; however, these differences did not reach signifi-
cance. Improvements in plasma lipid profile in the CDHA
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390
group were observed with significant reductions in total
triglyceride, total lipid and total cholesterol compared with
the control.
AntiCarcinogenic Properties
In parallel to their in vitro investigation into the cytotoxicity
of CEPA towards a range of cancer cell types, Igarashi and
Miyazawa [14], assessed the potential cytotoxic and apop-
tosis inducing potential of a chemically produced mixture
of CDHA isomers. CDHA exposure was effective in reduc-
ing the viability of HepG2 (hepatic), DLD-1 (colonic),
A549 (lung) and MKN-7 (stomach) cell lines, however, the
MCF-7 (mammary) cell line did display some resistance.
Further investigation using the DLD-1 cell line indicated
that CDHA exposure increased cellular lipid peroxidation
and initiated condensation and fragmentation of the cellular
DNA, both characteristic of apoptotic cell death.
The potential anti-carcinogenic activity of CDHA iso-
mers was also indicated in an early in vivo study in which
Sprague-Dawley rats were exposed to the carcinogen
N-methyl-N-nitrosourea in order to induce mammary tumor
development [91]. In this study, animals were provided
with dietary CDHA prior to or post-carcinogen exposure
and the number and size of the mammary tumors assessed.
The results showed that rats receiving CDHA post-expo-
sure had significantly lower tumor size and incidence,
whilst pre-exposure to CDHA was ineffective. CDHA has
also proven to be effective in subsequent animal studies,
reducing the incidence and size of tumors in BALB/c-nu/
nu mice (KPL-1 mammary and Colo 201 colonic cancer
cell transplants, respectively) [55, 56]. Studies have also
attempted to extrapolate the mechanisms behind the activ-
ity of CDHA via flow cytometry to assess the impact on
cell cycle and Western blotting to assess the expression of
key regulators of apoptosis [55, 56]. The results showed
that following 72h exposure, both KPL-1 and Colo-1 cells
exposed to CDHA became locked in a G1/S phase, similar
to that previously described for CEPA, with the emergence
of a sub-G1 fraction characteristic of apoptosis. Western
blotting indicated that in KPL-1 cells, CDHA exposure
increased the cellular concentration of the cell cycle regula-
tors p53 and p21, and resulted in a reduction in cyclin D1
(involved in cell cycle progression) and Bcl-2 (anti-apop-
totic oncogene). These results complimented the earlier
data of Danbara et al. [55], where Colo 201 cells exposed
to CDHA displayed increases in pro-apoptotic oncogenes
(Bak and Bcl-xs) and decreases in anti-apoptotic onco-
genes (Bcl-XL and Bcl-2), in addition to changes in the
cellular concentration of p21 (increased) and the cyclins
D1 and E (decreased). Overall, the results of these studies
indicate that CDHA exposure in vitro can arrest cell cycle
progression and initiate an apoptotic process in Colo 201
13
Lipids (2016) 51:377397
and KPL-1 cells, which can result in reductions in the num-
ber and size of tumors in mice transplanted with these can-
cer cell types.
In addition to its apparent cell cycle arrest and pro-
apoptotic properties, CDHA exposure has also displayed
anti-angiogenic attributes similar to CEPA in cancer cells.
In this in vitro study, BAEC endothelial cells exposed to
10mol/l CDHA displayed significantly reduced vessel
formation compared with unexposed cells [90]. Addition-
ally, CDHA exerted anti-proliferative activity (WST-1),
reduced cell migration (denudation injury model) and dis-
played pro-apoptotic properties (morphological changes
and DNA fragmentation). A subsequent in vivo study also
indicated that in male ICR mice, dietary CDHA (510mg/
days) inhibited angiogenesis of chamber implanted DLD-1
colorectal cancer cells.
Natural Strategies forConjugated Fatty Acid
Production
The chemical production of conjugated fatty acids is often
a cost effective and efficient method for the production of
many novel conjugated fatty acids. This can also be said
of many naturally occurring conjugated fatty acid isomers
of which there are ample natural sources that can easily
be extracted (Table1) [134]. However, many other conju-
gated fatty acids are not as common in nature or if found,
are present in low quantities (<3% of total fatty acid con-
tent). This has prompted research into the identification of
novel sources or strategies by which their concentration can
be increased both by chemosynthesis and through natural
strategies, the latter of which we will discuss [33, 92, 135].
CLA isomers occur naturally in the meat and milk fat of
ruminants as a result of microbial biohydrogenation of die-
tary linoleic and linolenic acids or endogenous conversion
of ruminally produced vaccenic acid in the mammary gland
and adipose tissue [92, 136]. Under normal circumstances,
the CLA content of milk and meat can be relatively low, yet
it has been demonstrated that through manipulation of the
ruminant diet, significant elevations in the CLA content can
be achieved [137139]. Moreover, CLA enriched milk can
be utilized to produce secondary value added products such
as cheese, ghee, butter and yoghurt that contain further ele-
vations in CLA content, and which are suitable for incor-
poration into the human diet [135, 140, 141]. The presence
of CLA isomers in dairy products owes much to the rumen
microbiota, including strains of Butyrivibrio fibrisolvens,
Megasphaera elsdenii, Propionibacterium acnes and
Clostridium proteoclasticum, as well as rumen protozoa,
which possess the capacity to convert dietary linoleic acid
and linolenic acids to CLA isomers [142145]. Indeed,
such is the complexity of ruminant CLA production that up
Lipids (2016) 51:377397 391
to 17 different CLA isomers have been detected in rumi-
nal digesta, in addition to other various CLNA isomers [22,
146148].
Given the high isomerization capacity of the ruminal
microbiota, researchers have focused attention on min-
ing the microbiota of similar environmental niches for
cultures with the capacity to conjugate unsaturated fatty
acids. These investigations have identified a plethora of
CLA producing strains of intestinal, cutaneous and dairy
origin including bifidobacteria, lactobacilli, Lactococcus,
Leuconostoc, Enterococcus, Clostridia, Propionibacteria,
Pediococcus and Streptococcus [92, 149151]. Identifying
and characterizing the mechanisms by which these strains
catalyze the conversion of linoleic acid to CLA has been
the subject of much study. This has resulted in the identi-
fication of an isomerase enzyme known as Propionibacte-
rium acnes isomerase (PAI) whose activity is characterized
by the bioconversion of linoleic acid to t10, c12 CLA [152,
153], and a multi-component enzymatic isomerase system
characterized by the bioconversion of linoleic acid to c9,
t11 and t9, t11 CLA referred to as LAI [154156].
In addition to the bioconversion of linoleic acid to c9,
t11 and t9, t11 CLA, LAI has undergone a number of sub-
strate studies. These have indicated that the enzymatic
activity is associated with the cell membrane and has a
specificity for conjugating C18 unsaturated fatty acids
which contain a double bond in the 9, 12 positions [33,
156]. In this regard, the enzyme can be successfully used
to conjugate linoleic, -linolenic, -linolenic and steari-
donic acids, whilst similar substrates like nonadecadie-
noic acid and eicosadienoic acid remain unchanged [33].
In studies conducted using the strain Lb. plantarum AKU
1009a, a strain exhibiting LAI activity, the production of
c9, t11 and t9, t11 CLA from linoleic acid was observed to
consist of two distinct steps with 10-hydroxy-12-octadece-
noic acids produced as intermediates [104, 105]. Moreo-
ver, this two-step system of CLA production has also been
observed in the production of c9, t11, c15 and t9, t11, c15
CLNA by bacteria [32, 105].
PAI has not previously exhibited the same level of sub-
strate specificity as LAI and has been associated with the
conversion of a number of unsaturated fatty acids to their
conjugated isomers [157]. Interestingly, the enzyme is not
capable of conjugating all fatty acids and whilst expo-
sure of strains exhibiting PAI activity to -linolenic acid
resulted in CGLA production, exposure to -linolenic acid
did not yield a conjugated fatty acid, suggesting a level of
substrate preference possibly dictated by bond position.
Furthermore, studies investigating the activity of PAI have
not reported the detection of any intermediary products,
an observation substantiated by genetic studies in which
the genes encoding PAI were cloned into Escherichia coli
[158160].
The fatty acid isomerization capacity of microbes
derived from various environmental niches offers huge
potential for naturally increasing the content and variety
of potentially bioactive conjugated fatty acids to which
humans are exposed. Indeed, strains of dairy origin can be
successfully utilized to increase the conjugated fatty acid
content of yoghurts, cheeses and other fermented products,
whilst potentially probiotic strains could also be exploited
for the in situ production of conjugated fatty acids in
the gastrointestinal tract (GIT) from dietary fatty acids
[161164].
Humans consume a large and diverse range of fatty
acids in the diet, primarily as TAG, from which the human
GIT has developed the capacity to release a large quantity
as free fatty acids [165]. While the majority of this lipid
is absorbed by the intestinal lining (~95%), a proportion
remains unabsorbed and as such is free to interact with
the intestinal microbiota [166]. Indeed, it has been esti-
mated that 68g of lipid will enter the colon daily [166].
Some research has been conducted to investigate whether
this lipid could be utilized as a substrate for in vivo con-
jugated fatty acid production by intestinally derived con-
jugated fatty acid producers when these cultures are used
as probiotics. In one of the first of such studies, 129/SvEv
mice were fed a probiotic mix of CLA producers (termed
VSL3) and fecal pellets from thewere animals subse-
quently collected [167]. The recovered fecal pellets were
resuspended in a linoleic acid containing medium and ex
vivo CLA production monitored following incubation. The
results demonstrated that mice produced 100-fold more
CLA post-probiotic supplementation than beforehand,
suggesting supplementation with CLA producing cultures
does have the potential to increase endogenous CLA pro-
duction provided there is access to suitable substrate. The
capacity of the VSL3 probiotic mix to increase c9, t11 CLA
production has also been observed in vivo when C57BL6
mice received the culture mix by oral gavage [168]. The
results of this study revealed that VSL3 supplementation
resulted in a significant increase in colonic CLA concen-
trations relative to the control, but that these increases did
not transfer through to the plasma. Additional in vivo stud-
ies in which the murine diet was supplemented with indi-
vidual strains of both t10, c12 and c9, t11 CLA producing
bacteria were conducted with some success [169171]. Lee
et al. [169, 170] observed the anti-adipogenic impact of
feeding the t10, c12 CLA producing strains Lb. rhamnosus
PL60 or Lb. plantarum PL62 on diet-induced obese mice
as a result of in vivo CLA production. Additionally, Wall
et al. [171] observed increases in c9, t11 CLA content of
the livers of BALB/c mice after consumption of a linoleic
acid supplemented diet in tandem with the conjugated fatty
acid producing strain B. breve NCIMB 702258. Such in
vivo studies indicate that the intake of conjugated fatty acid
13

392
producing probiotics is capable of instigating increases in
endogenously produced CLA to the extent that a physio-
logical benefit may be observed. A human study has also
demonstrated the efficacy of ingesting CLA producing
probiotics in initiating an increase in systemic CLA con-
tent [172]. Human subjects were fed Lb. rhamnosus PL60
and serum CLA concentrations monitored and the results
demonstrated that Lb. rhamnosus PL60 supplementation
increased the CLA content in the serum of subjects.
Although the aforementioned animal and human trials
demonstrate that significant increases in endogenously pro-
duced CLA can be achieved via supplementation with con-
jugated fatty acid producing probiotics, there are questions
as to whether microbially produced conjugated fatty acid
could be absorbed efficiently enough to impart systemic
benefits. These questions arise as a result of the fact that
although the upper intestine is responsible for the major-
ity of fatty acid absorption, it is the location with the low-
est microbial population (103104cfu/g), whilst the dis-
tal intestine contains the highest proportion of microbes
(10111012cfu/g), but has relatively low levels of fatty acid
absorption [173175]. Indeed, a study has indicated that
when mice were fed a linoleic acid rich or -linolenic acid
rich diet, marked increases in the respective concentrations
of CLA and CLNA in the intestinal contents of the caecum
and colon could be detected along with substantially lower
increases in the jejunum and ileum [176]. However, more
detailed analysis demonstrated that although increases
in the conjugated fatty acid content were detected in the
caecal tissue, these increases did not result in increased
plasma conjugated fatty acid concentrations. Thus, it would
appear that while the intestinal microbiota may be capa-
ble of metabolizing dietary fatty acids to their conjugated
derivatives, these increases are likely to be localized to the
intestine.
Conclusion
CLA isomers are likely to retain their place as the most
extensively studied of all the conjugated fatty acids cur-
rently under review. However, there are a number of addi-
tional natural and chemically derived conjugated fatty acids
which offer significant bioactive potential including those
which possess anti-carcinogenic, anti-adipogenic, anti-
inflammatory and immune-modulating properties. In many
instances, these bioactive conjugated fatty acids can be effi-
ciently and cost effectively extracted from natural sources
or chemosynthesized from unsaturated fatty acid substrates.
There are occasions where other natural strategies of conju-
gated fatty acid production can be employed including the
use of microbes demonstrating LAI or PAI activity. Indeed,
such microbes could potentially be utilized either to enrich
13
Lipids (2016) 51:377397
fermented foods in these bioactivefatty acids or as probi-
otic cultures that could produce conjugated fatty acids from
dietary substrates within the human GIT. However, such
strategies are not without their hurdles as questions remain
over the efficiency and form in which these conjugated
fatty acids can be absorbed in the intestine. Thus, although
it is evident that conjugated fatty acids have significant
potential as bioactive nutrients, further research is required
to determine if these attributes can be systemic in humans
rather than localized to the GIT.
Acknowledgments This review was funded in partby EU project
QLK1-2001-02362 and by the APC Microbiome Institute supported
by Science Foundation Ireland.
Compliance with ethical standards
Conflict of interest The authors have declared no conflict of interest.
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