Linoleic acid esters of hydroxy linoleic acids are anti-inflammatory lipids found in plants and mammals

Article abstract of DOI:10.1074/jbc.RA118.006956

Fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids. Here we identify the linoleic acid ester of 13-hydroxy linoleic acid (13-LAHLA) as an anti-inflammatory lipid. An oat oil fraction and FAHFA-enriched extract from this fraction showed anti-inflammatory activity in a lipopolysaccharide-induced cytokine secretion assay. Structural studies identified three LAHLA isomers (15-, 13-, and 9-LAHLA) as being the most abundant FAHFAs in the oat oil fraction. Of these LAHLAs, 13-LAHLA is the most abundant LAHLA isomer in human serum after ingestion of liposomes made of fractionated oat oil, and it is also the most abundant endogenous LAHLA in mouse and human adipose tissue. As a result, we chemically synthesized 13-LAHLA for biological assays. 13-LAHLA suppresses lipopolysaccharide-stimulated secretion of cytokines and expression of pro-inflammatory genes. These studies identify LAHLAs as an evolutionarily conserved lipid with anti-inflammatory activity in mammalian cells.

Full text of DOI:10.1074/jbc.RA118.006956

JBC Papers in Press. Published on May 31, 2019 as Manuscript RA118.006956
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.006956
Linoleic acid esters of hydroxy linoleic acids are anti-inflammatory lipids found in plants and
mammals
1
, #
2
1
2
2
3
Matthew J. Kolar , Srihari Konduri , Tina Chang , Huijing Wang Clare McNerlin , Lena Ohlsson ,
4
2, *
1, *
Magnus Härröd , Dionicio Siegel , Alan Saghatelian
Affiliations:
1
From the The Salk Institute for Biological Studies, Clayton Foundation Laboratories for Peptide Biology,
2
1
0010 N. Torrey Pines Rd, La Jolla, CA, 92037; Skaggs School of Pharmacy and Pharmaceutical
Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0934;
3
Department of Clinical Sciences, Division of Experimental Vascular Research, Lund University, Lund,
4
Sweden; Härröd Research, Frans Persons väg 6, 40229 Gothenburg, Sweden
Running title: LAHLAs are anti-inflammatory lipids
#
*
Present address: UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA, 92093
To whom correspondence should be addressed: Dionicio Siegel: Skaggs School of Pharmacy and
Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-
934, drsiegel@ucsd.edu; Alan Saghatelian: The Salk Institute for Biological Studies, Clayton
0
Foundation Laboratories for Peptide Biology, 10010 N. Torrey Pines Rd, La Jolla, CA, 92037,
asaghatelian@salk.edu.
Keywords: oat oil, FAHFAs, polyunsaturated lipids, inflammation, lipid synthesis
Abstract
Fatty acid esters of hydroxy fatty acids (FAHFAs) are
Fatty acid esters of hydroxy fatty acids (FAHFAs) are
a recently discovered class of biologically active
lipids. Here, we identify the linoleic acid ester of 13-
hydroxy linoleic acid (13-LAHLA) as an anti-
inflammatory lipid. An oat oil fraction and FAHFA-
enriched extract from this fraction showed anti-
inflammatory activity in LPS-induced cytokine
secretion assay. Structural studies identified three
LAHLA isomers (15-, 13-, and 9-LAHLA) as being
the most abundant FAHFAs in the oat oil fraction. Of
these LAHLAs, 13-LAHLA is the most abundant
LAHLA isomer in human serum after ingestion of
liposomes made of fractionated oat oil, and it is also
the most abundant endogenous LAHLA in mouse and
human adipose tissue. As a result, we chemically
synthesized 13-LAHLA for biological assays. 13-
LAHLA suppresses LPS-stimulated secretion of
cytokines and expression of pro-inflammatory genes.
These studies identify LAHLAs as an evolutionarily
conserved lipid with anti-inflammatory activity in
mammalian cells.
a recently discovered class of lipids with anti-diabetic
and anti-inflammatory activity (1). Because there are
numerous FAHFAs, they are classified into families
based on the composition of fatty acid and hydroxy
fatty acid. For example, palmitic acid esters of
hydroxy stearic acids (PAHSAs) and oleic acid esters
of hydroxy stearic acids (OAHSAs) are two FAHFA
families. Furthermore, within a FAHFA family, there
are multiple regioisomers that differ in the position of
the ester linkage (e.g., 5-PAHSA and 9-PAHSA) (1).
Biological testing of 5- and 9-PAHSA revealed
potent anti-diabetic and anti-inflammatory activity
(1-3). Mechanistic studies revealed that FAHFAs
regulate several cellular and physiological pathways,
with at least some of the biology being attributable to
agonism of GPR120 and GPR40, two G-protein
coupled receptors (GPCRs) (1,3). Other ligands for
these GPCRs include the saturated and
polyunsaturated fatty acids (4,5). GPR120 is the
endogenous receptor for omega-3 fatty acids, and it
mediates the anti-inflammatory effects of these lipids
(5).
Introduction
The anti-inflammatory activity of FAHFAs have
been reported in vitro and in vivo (1,2). Initially,
1

cellular experiments with bone marrow derived
dendritic cells (BMDCs) showed that treatment of
cells with 9-PAHSA reduced the amplitude of
cytokine secretion and expression of cellular
inflammation markers. In addition, administration of
Testing the anti-inflammatory activity of oat oil
fractions
Previous reports have identified anti-inflammatory
activity for colloidal oatmeal (7) and beneficial
metabolic effects upon consumption of liposomes
made of fractionated oat oil rich in polar lipids (8,12).
Crude oat oil, obtained by ethanol extraction of oats,
can be fractionated using different concentrations of
ethanol, water and sugar (12). The resulting fractions
are rich in neutral lipids (called T1 fraction) or rich in
polar lipids (called T2 fraction). We measured the
anti-inflammatory effects of oat oil fraction T1 by
measuring interleukin 6 (IL-6) release from the RAW
264.7 cells upon treatment with lipopolysaccharide
(LPS). This assay was previously used to measure
anti-inflammatory activities of 9-PAHSA and 13-
DHAHLA (6). The T1 fraction exhibited a dose-
dependent anti-inflammatory activity with significant
suppression of LPS-stimulated IL-6 secretion starting
at 10 ppm (~15% inhibition) (Fig. 1b).
Solid-phase extraction (SPE) of the T1 fraction
using a protocol for FAHFA enrichment (13),
resulted in fraction (SPE-T1 fraction) with more
potent anti-inflammatory activity than T1. For
example, the SPE-T1 fraction resulted in ~50% IL-6
inhibition at 10 ppm and > 90% IL-6 inhibition at 100
ppm (Fig. 1c). To show that these effects were due to
activity and not cytotoxicity, we performed an MTT
viability assay, and there was no observed cellular
death at the tested concentrations of the T1 oat oil
fraction and SPE-T1 fraction (Fig. S1).
9
-PAHSA to mice on a high-fat diet reduced
inflammation in adipose tissue of treated mice (1).
-PAHSA also showed robust anti-inflammatory
activity in a mouse colitis model. Administration of
-PAHSA to mice undergoing chemically induced
colitis improved clinical and molecular inflammation
2). Moreover, an analysis of the impact of 9-PAHSA
9
9
(
in the immune system revealed effects on the innate
and adaptive immune system (2). And most recently,
Kuda
docosahexaenoic acid of 13-hydroxy linoleic acid
13-DHAHLA), a novel FAHFA, inhibits LPS-
and
co-workers
demonstrated
that
(
induced cytokine secretion in the mouse macrophage
cell line RAW 264.7 (6). Furthermore, 13-DHAHLA
is more potent than 9-PAHSA, highlighting the need
to find more biologically-active FAHFAs to
determine structure-activity relationships.
Including all the regioisomers, there are at least
80 known FAHFAs, yet only three of these have been
tested for their biological activity (1,6). To expedite
the discovery of additional bioactive FAHFAs, we
wondered whether there existed other natural sources
of biologically active lipids that would lead to
bioactive FAHFAs. Oat oil emerged as a potential
source of bioactive FAHFAs because colloidal
oatmeal has anti-inflammatory activity (7), and
physiological studies with liposomes made of
fractionated oat oil revealed beneficial metabolic
effects (8). Furthermore, a digalactosyldiacylglycerol
The SPE-T1 fraction contains Linoleic acid esters
of hydroxy linoleic acid (LAHLAs)
(
(
DGDG) lipid found in oat oil contains a FAHFA
also referred to as an estolide), the linoleic acid ester
We measured FAHFAs levels in the SPE-T1
fraction by LC-MS. These measurements were
designed to detect different FAHFA families
including PAHSAs, OAHSAs, and LAHLAs (Fig.
2a). LAHLAs are the most abundant FAHFAs in the
SPE-T1 fraction (~300 pmol/mg), while PAHSAs
and OAHSAs are detected at much lower
concentrations (~1 pmol/mg) (Fig. 2a). We detected
three different LAHLA peaks in the LC-MS
chromatogram (Fig. 2b and Fig. S2) indicative of
three different LAHLA isomers. No other FAHFAs
were as abundant as the LAHLAs, and, therefore, we
focused on the structural and functional
characterization of the LAHLAs.
of 15-hydroxy linoleic acid (15-LAHLA) (Fig. 1a)
9-11).
We reasoned that the existence of a FAHFA-
(
containing DGDG meant that oat oil is a potential
source for additional biologically-active FAHFA
families. And we hypothesized that FAHFAs might
contribute the anti-inflammatory activity of oat
extracts. We identified free linoleic acid esters of
hydroxy linoleic acids (LAHLAs) in an oat oil
fraction rich in neutral lipids with anti-inflammatory
activity (Fig. 1b), and synthetic 13-LAHLA as
another anti-inflammatory FAHFA.
Results
LAHLA regioisomers
2

To be able to study these LAHLAs in greater detail,
we needed to know which regioisomers were present
and then synthesize pure version of these lipids for
biological assays. To identify the structures of these
LAHLA regioisomers, we applied a recently reported
strategy for the structural elucidation of the 13-
DHAHLA (6). This method utilizes multiple
fragmentation steps (MS3) to identify the position of
the hydroxy group (Fig. 3a). Since LAHLAs and
DHAHLAs both contain hydroxy linoleic acid (HLA)
the method should be directly transferable to identify
LAHLA regioisomers.
and 9-LAHLA increasing with time and 15-LAHLA
decreasing (Fig. S4). Overall, 13-LAHLA is the most
abundant of all the LAHLAs (Fig. 4c). The serum
concentrations of LAHLAs reached ~ 40 nM which is
45X higher than control subjects. These data identify
13-LAHLA as the primary serum LAHLA after
ingestion of liposomal oat oil. While 13-LAHLA
increases in serum correlate with the physiological
effects of oat oil in these individuals (8), we doubt
that serum 13-LAHLA at 40 nM can account for the
physiological response to oat oil becasuse we show
that 13-LAHLA has an IC of ~20 µM in a cellular
5
0
We did not have access to an instrument capable of
MS3, so we performed a pseudo-MS3 instead (13).
The source voltage of the mass spectrometer was
increased to induce in-source fragmentation of
LAHLAs to linoleic acid and HLA, and the HLA was
further fragmented in the mass spectrometer to reveal
the position of the hydroxy group (Fig. 3a). Analysis
of the oat oil samples using this approach led to HLA
fragments that co-elute with the different LAHLA
isomer peaks (Fig. 3b, c). Using this approach, we
identified the LAHLA isomers to contain 15-, 13-,
and 9-HLA (Fig. 3c). The 13- and 9-LAHLA product
ions are identical with reported fragments for 13- and
anti-inflammatory assay (see below). Thus,
Additional studies in animal models will be required
to determine what, if any, effects dietary LAHLAs
have in biology.
LAHLAs are endogenous mammalian lipids
We have previously shown that PAHSAs are
endogenous FAHFAs with the highest levels found in
adipose tissue (1). There exist multiple PAHSA
regioisomers with 9-PAHSA being the most abundant
PAHSA. Although PAHSAs are present in some
foods, they can be synthesized endogenously. We
explored if LAHLAs were also present in adipose
tissues. We examined subcutaneous white adipose
tissue (SQWAT) of wild-type (WT) mice fed ad
libitum for the presence of LAHLAs and observed
that there were multiple LAHLA regioisomers
present with 13-LAHLA being the most abundant
regioisomer (Fig. 5a, b). We also observed a similar
finding with 13-LAHLA being the most abundant
regioisomer in human white adipose tissue (Fig. 5c,
d). The discovery of 13-LAHLA as a naturally
occurring lipid in rodents, humans, and oat oil pointed
to 13-LAHLA as an ideal candidate for further
investigation of LAHLA biology.
9-DHALHAs (6) providing strong evidence for these
regioisomer assignments.
LAHLA levels after ingestion of liposomal oat oil
Liposomes made from fractionated oat oil rich in
polar lipids (fraction T2) were used as a dietary
supplement to look for beneficial postprandial
metabolic effects (8). Liposomal oat oil experiments
correlated with lower glucose levels and elevated
levels of beneficial hormones including peptide YY
(PYY), glucagon-like peptide 1 (GLP-1), glucagon-
like peptide 2 (GLP-2) and cholecystokinin (CCK).
We measured FAHFA levels in human serum,
samples acquired in a previous study (8), to determine
whether any LAHLAs from ingested oat oil could be
detected in the human serum.
Synthesis of 13-LAHLA
To determine if 13-LAHLA has direct effects on
inflammation, we first needed to synthesize 13-
LAHLA (Fig. 6a). Synthetic FAHFAs have
exclusively contained saturated HFA fragments due
to the early identification and relative ease of
synthesis of this class of FAHFAs (1,14-16). For the
laboratory synthesis of 13-LAHLA the challenge was
preparing large quantities of 13-hydroxylinoleic acid
(13-HLA is synonymous with 13-HODE and coriolic
acid) with the correct olefinic configuration. While
there have been several multistep syntheses of 13-
HLA (17-21), we utilized a concise approach
LAHLAs levels in subjects ingesting liposomal oat
oil were much higher than in control subjects,
indicating that oat oil was the source of plasma
LAHLA levels (Fig. 4a). Furthermore, we see no
change in PAHSAs or OAHSAs consistent with oat
oil having high LAHLA levels (Fig. 4b and Fig. S3).
In subjects that received liposomal oat oil, we
detected the 15-, 13-, and 9-LAHLAs (Fig. 4c). The
levels of the LAHLA isomers are dynamic with 13-
3

implementing a selective selenium dioxide oxidation
of the methyl ester of linoleic acid (22) (Fig. 6a).
While the oxidative transformation proceeded in low
yield the approach dramatically simplified the
synthesis. Importantly, standard phase column
chromatography could be applied to separating the
multiple products, including olefin isomers, with the
desired 13-HLA methyl ester produced as the major
product. This reaction enabled access to ample
quantities of 13-HLA methyl ester from readily
available methyl linoleate. From this intermediate
esterification of the secondary, allylic alcohol of 13-
HLA methyl ester with the acid chloride of linoleic
acid yielded the methyl ester of 13-LAHLA.
Selective saponification of the methyl ester in
preference to the fatty acid ester, due to steric effects,
generated synthetic 13-LAHLA for the first time.
Comprehensive NMR analysis confirmed the correct
olefinic isomer was present and the placement of the
9-PAHSA and 13-DHAHLA have been shown to
have anti-inflammatory effects. 9-PAHSA blocks
LPS-stimulated dendritic cell (1) and macrophage (6)
activation, and reduces inflammation in a mouse
colitis model (2). We analyzed the effects of 9-
PAHSA and 13-LAHLA on suppression of LPS-
stimulated cytokine expression in RAW 264.7
macrophages. Both 9-PAHSA and 13-LAHLA at 10
µM significantly suppressed mRNA levels of IL-6
and IL-1β, with 13-LAHLA having a stronger effect
(Fig. 7a, b). Administration of 13-LAHLA to RAW
264.7 cells in the absence of LPS had no impact on
control IL-6 levels (data not shown). In addition, 13-
LAHLA (10 µM) also suppressed mRNA levels of
iNOS and COX-2 in RAW 264.7 cells when
stimulated with LPS (Fig. 7c, d). The reduction in
expression of pro-inflammatory cytokines as well as
downstream regulators of inflammation iNOS and
COX-2 indicate that 13-LAHLA has broad anti-
inflammatory effects.
th
ester linkage was at the 13 carbon.
The characteristic precursor-to-product ion
transitions and the retention times were the same for
the synthetic and natural 13-LAHLA indicating a
structural match (Fig. S5). Using the synthetic 13-
LAHLA, we validated the structure of the LAHLA
isomers in oat oil and human plasma as 13-LAHLA
Three hydrolases—CEL, AIG1, and ADTRP—
have been identified in vitro as FAHFA-specific
lipases (23,24), and these enzymes led us to perform
additional experiments to ensure that 13-LAHLA and
not LAHLA hydrolysis products are responsible for
the activity we observe. First, we measured the
activity of 13-LAHLA and 13-HLA in this
macrophage-stimulation assay and found that 13-
LAHLA is significantly more active that 13-HLA
(Fig. 6b).
Oat oil contains both stereoisomers of 13-LAHLA as
determined by hydrolysis
(Fig. 8). Neither 13-LAHLA or 13-HLA was toxic
towards cells. Comparison of linoleic acid and 13-
HLA showed that they are equipotent in this assay
We set out to determine the stereochemistry of 13-
LAHLA as we previously determined that absolute
configuration of FAHFAs is important in their
regulation (15). With commercially available
enantiopure 13-HLA/HODE standards, we reasoned
that we could purify and hydrolyze LAHLAs to
determine their HLA backbone stereochemistry using
LC-MS. We developed a chiral separation method
that resolved the S-9-, R-9-, S-13-, and R-13-HLAs
(Fig. S7). These experiments demonstrate that 13-
LAHLA is more active than either breakdown
product, supporting that the LAHLA is the
biologically active species in these studies.
Discussion
Lipids are fundamental regulators in biology with
roles in membrane structure, sources of energy, and
signaling molecules. Lipids in the latter category are
often classified into families, such as prostaglandins
(Fig. S6). We then purified FAHFAs from oat oil and
subjected this sample to alkali hydrolysis and
analyzed this sample for the presence of HLAs (Fig.
S6). In this hydrolyzed sample we observed equal
amounts of S- and R-13-HLA. 9-HLA was below our
limit of detection under these conditions. Based on
the data we hypothesize that the formation of the 13-
LAHLAs is non-enzymatic because both the S and R
(25) or oxysterols (26), that share a structural feature
but with many distinct members. Individual members
in a family can have distinct activities or different
potencies for the same activity. For example, with
FAHFAs, 9-PAHSA is anti-inflammatory but 5-
PAHSA is not (1), and similar differences in
structure-activity relationships are observed with
other lipid classes (25). Analytical experiments
suggest that there are many FAHFA families
13-LAHLA stereoisomers in oat oil.
13-LAHLA is an anti-inflammatory lipid
4

(
1,6,27,28), but only a few of these have been
Testing of 13-LAHLA for its ability to inhibit LPS-
stimulated macrophage activity revealed that this
lipid is active with an IC50 of ~30 µM. While 9-
PAHSA is active in this assay, we find that it is much
less so than 13-LAHLA (Fig. 7).
functionally characterized (1,6). FAHFAs were
detected in foods, including vegetables (1), and recent
work has detected these lipids in rice and the plant
model organism Arabidopsis thaliana (29). We
hypothesized that there might be additional,
uncharacterized FAHFAs in bioactive lipid extracts
from plants.
The historical record reports the use of oats (Avena
sativa) to treat skin conditions thousands of years of
years ago. Today, oats, in particular colloidal oats, are
present in many commercial personal care products
We extended these measurements to include
iNOS and COX-2 as additional inflammatory
markers (Fig. 7c, d). Nitric oxide (NO) signaling is a
key trigger in the immune system to help rid infection,
but in some cases overactive NO signaling is thought
to damage healthy cells (36,37). There are three NO
producing enzymes endothelial-, neuronal-, and
inducible nitric oxide synthase (NOS) (eNOS, nNOS,
and iNOS) (38). iNOS is the primary regulator of NO
signaling in macrophages, and we measured changes
in the expression of iNOS in the presence and absence
of LPS. These experiments revealed modest
inhibition of iNOS expression (50%) at 10 µM of 13-
LAHLA. We also measured COX-2 levels to infer
changes in the prostaglandin pathway (39). We find
that administration 13-LAHLA (10 µM) inhibits
COX-2 expression by over 50%. Together these data
indicate that 13-LAHLA affects many branches of the
cellular inflammatory response including cytokine
production and secretion, nitric oxide signaling, and
prostaglandin production.
One challenge when testing endogenous lipids,
such as the LAHLAs, is that these metabolites are
susceptible to endogenous metabolism, and caution
must be taken to conclude that a specific metabolite
is biologically active. For instance, 13-DHAHLA was
shown to be produced upon administration of DHA to
cells and mice (6), suggesting that at least some of the
biological activity of DHA might come from 13-
DHAHLA (6). With 13-LAHLA, it is possible that
the breakdown products 13-HLA or LA are active in
these assays.
(e.g., soaps, shampoos, and shaving creams) (30,31).
Identification of active molecules in oats identified
avenanthramide alkaloids (32,33) which have anti-
oxidant and anti-inflammatory activity in vitro (34)
and in vivo (35). Our interest in oat oil was kindled
by a physiological study in humans that found
beneficial metabolic and hormonal effects of a
liposomal oat oil dispersion (8).
We hypothesized that oat oil might contain
biologically active FAHFAs because some of the
observed effects of liposomal oat oil in humans
mirrored that of FAHFAs in mice (1,3), and oats
contained a complex lipid, a DGDG, which contains
an esterified 15-LAHLA (Fig. 1a) (9). In this work
we have identified free FAHFAs in oat oil with
LAHLAs being the most pronounced. Our hypothesis
was bolstered by the observation that 13-LAHLA is
anti-inflammatory.
We also detected LAHLAs as endogenous lipids
and increased LAHLA concentrations upon ingestion
of liposomal oat oil (fraction T2) (Fig. 4a, c). Since
the in vitro biological activity has an IC50 of ~28 µM
in the cell based anti-inflammatory assay (Fig. 8), we
doubt that serum concentrations of 13-LAHLA at 40
nM is regulating any biology. Future studies that
utilize pure LAHLAs pharmacologically or perturb
LAHLA effects metabolically will be necessary to
understand what role, if any, LAHLAs have in
mammalian biology.
We measured the IC50s of 13-LAHLA and 13-
HLA on IL-6 secretion and found that 13-HLA is less
active than 13-LAHLA (~30 µM for 13-LAHLA vs
274 µM for 13-HLA) (Fig. 8). In a separate
This is the second observation of LAHLAs in
vivo, as a study that developed a higher resolution
method to measure FAHFA levels reported these
lipids (28).
experiment, we find that 13-HLA and LA have
similar IC50s (Fig. S8) to indicate that both of these
lipids are at least an order of magnitude less active
than the intact 13-LAHLA. The data argues that even
if 13-LAHLA is hydrolyzed, the products are less
active and therefore less likely to contribute to the
observed activity in our assays.
Recent work that characterized the function of
13-DHAHLA provided a guide (6) for the in vitro
characterization of 13-LAHLA. We suspected that
the 13-LAHLA and 13-DHALHA might have similar
activities due to their similar structures of a 13-HLA
core esterified with a polyunsaturated fatty acid.
In summary, our findings demonstrate that
FAHFAs are an evolutionary conserved class of
5

lipids, from plants to humans, with anti-inflammatory
activity. Moreover, the characterization of 13-
LAHLA here along with the reported activity of 13-
Experimental Procedures
Chemicals
DHAHLA begin to reveal
a
trend that
polyunsaturated FAHFAs are more active than the
saturated FAHFAs tested so far (i.e. 9-PAHSA).
Previous work on the stereochemistry of hydroxy
linoleic acids, specifically 15-HLA, was identified as
possessing R stereochemistry (9). The existence of a
stereocenter on this lipid indicates that the lipid is the
product of an enzymatic, and therefore stereo specific
pathway, instead of a non-enzymatic pathway. Future
efforts will look to synthesize LAHLAs enantiomers
so that we can determine if these lipids from oat oil
and tissues are enantiopure, and whether the different
stereoisomers vary in their biological activity.
The identification of anti-inflammatory activity
for a natural lipid that is non-toxic when consumed
and the ability to modify this lipid while retaining
activity indicate that we should continue to explore
the biology and functions of FAHFAs, especially 13-
LAHLA as we examine the functional roles in the
body and explore the therapeutic potential of this
pathway.
9-PAHSA and the enantiopure 9- and 13-HODEs
(synonymous with HLA) were purchased from
Cayman Chemical.
Oat oils
Ethanol extracted crude oat oil was produced in
large scale at Swedish Oat Fiber, Bua, Sweden. This
crude oat oil was fractionated using different mixtures
of water, ethanol and sugar resulting in the neutral
lipids rich T1 fraction, the polar lipid rich T2 fraction,
and an oat syrup containing sugar and ethanol soluble
oat protein (12). The main lipid classes in the T1 oil
were triglycerides (90 wt.%); slightly polar lipids like
free fatty acids, sterols, diglycerides and
monoglycerides (6 wt.%); galactolipids (2 wt.%); and
phospholipids (2 wt.%). The T2 fraction contains
triglycerides (40 wt.%); slightly polar lipids like free
fatty acids, sterols, diglycerides and monoglycerides
(3 wt.%); galactolipids (29 wt.%); and phospholipids
(
29 wt.%). The SPE-T1 fraction was enriched using a
previously reported solid-phase extraction method
40).
(
Mouse and human tissue for FAHFA
measurements
Wild-type C57BL/6J mice were purchased
from Jackson Laboratories. All animals were housed
in groups on a 14-h light, 10-h dark schedule at the
Salk. All animal care and experimental procedures
were in accordance with the standing committee on
the Use of Animals in Research and Teaching at Salk
and Institutional Animal Care and Use Committee
(IACUC), and the National Institutes of Health
Guidelines for the Humane Treatment of Laboratory
Animals. SQWAT was collected immediately after
euthanasia and snap frozen using liquid nitrogen.
Human adipose tissue from a 46-year-old African
American
female
was
purchased
(BioreclamationIVT, Hicksville, NY). Human
plasma samples were obtained and prepared as
previously described (8). All participants gave their
written informed consent prior to the study. The
studies were approved by the Regional Human Ethics
Committee of Lund-Malmö, Sweden (Registration
Number 2010/18, 2011/55). In that study initial blood
6

samples were taken after the subjects fasted
overnight. Subsequent blood samples were taken at 1,
Cell Proliferation using MTT Assay
3
, 5, and 7 hours post-ingestion of a controlled
After the media was removed for IL-6 measurements,
the remaining cells were assessed for their viability
using the MTT assay according to the manufacturer’s
breakfast supplemented with either liposomal oat oil
or control diet. The breakfasts contained 35g lipids,
i.e. the breakfast with oat oil contained 2mmol DGDG
with FAHFA. An IRB letter of exemption for the
deidentified human plasma samples used in this study
is on file at the Salk Institute for Biological Sciences.
protocol
(Calbiochem).
Briefly,
3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) was dissolved in sterile PBS (5
mg/mL) and filtered through a 0.2 µm Sterile Millex
Filter to prepare a 500 µg/mL solution in RPMI.
Prepared solution was added to adherent cells and
incubated at 37°C for 4 hours. Sterile DMSO was
then supplied upon removal of MTT solution.
Relative cell viabilities were quantified using a plate
reader at an absorbance of 570 nm.
Lipid extraction and SPE
Lipid extraction of samples and solid-phase
extraction was performed as previously described
(40).
Briefly, WAT (150 mg) was dounce
homogenized on ice in a mixture of PBS, methanol,
and chloroform (1.5 mL/1.5 mL/3 mL). Five pmol of
C -9-PAHSA was added to the chloroform prior to
4
qRT-PCR
1
3
lipid extraction as an internal standard. The mixture
was vortexed and then centrifuged at 2200 g for 5
min. The organic layer (bottom) was then transferred
to a new vial, dried down, and then stored at -80°C
for future use. The human plasma was prepared
similarly, except no dounce homogenization was
performed. SPE was performed at room temperature
using a Strata SI-1 silica cartridge (500 mg silica, 3
mL, Phenomenex). The column was washed using 6
mL of ethyl acetate followed by column equilibration
with 6 mL hexane. The lipid extract from the prior
step was then added to the equilibrated column and
neutral lipids were removed using 6 mL of 95:5
hexane:ethyl acetate followed by the elution of
FAHFAs with 4 mL ethyl acetate. This eluate was
dried down and then subjected to LC-MS analysis.
RAW 264.7 cells (6-well plate, 60-70% confluent)
were co-treated with FAHFA and LPS (100 ng/mL)
or the equivalent amount of DMSO in cell media for
20 hours. Total RNA was isolated using a Purelink
RNA Mini Kit (Thermo Fisher), according to the
manufacturer’s instructions. RNA from each replicate
(2 µg) was reverse transcribed to double-stranded
cDNA using the QuantiTect Reverse Transcription
Kit (Qiagen). qRT-PCR was performed using a
LightCycler 480 II (Roche), with SYBR Green qPCR
Master Mix (Bimake). qRT-PCR reaction conditions
were: 95 °C for 10 min; 35 x (95 °C for 15 s; 55 °C
for 30 s; 72 °C for 30 s). The relative quantification
-
ΔΔCt
of gene expression was calculated by using the 2
approximation method. The primers used in this study
are were GAPDH, iNOS1 (41), COX2 (41), IL-6 (6),
and IL-1B (6) (IL-1B and IL-6 primer sequences were
obtained through personal communication). GAPDH
forward 5’-AGG TCG GTG TGA ACG GAT TTG-
Cell culture methods
RAW264.7 cells were cultured in RPMI 1640,
supplemented with 10% FBS at 37 °C and 5% CO .
2
3’, GAPDH reverse 5’-TGT AGA CCA TGT AGT
TGA GGT CA-3’; COX2 forward 5’-GGA GAG
ACT ATC AAG ATA GT-3’, COX2 reverse: 5’-
GGA GAG ACT ATC AAG ATA GT-3’, iNOS
forward 5’-AAT GGC AAC ATC AGG TCG GCC
ATC ACT-3’, iNOS reverse 5’-GCT GTG TGT CAC
AGA AGT CTC GAA CTC-3’; IL-6 forward 5’-
AAC CAC GGC CTT CCC TAC TT-3’, IL-6 reverse
5’- GCC ATT GCA CAA CTC TTT TCT C-3’; IL-
1Β forward 5’- ACC TGG GCT GTC CTG ATG
AGA G-3’, IL-1B reverse 5’-CCA CGG GAA AGA
CAC AGG TAG C-3’.
IL-6 ELISA
RAW 264.7 cells were seeded using a 48-well plate
4
(
2.5 X 10 cells per well). The next day when cells
were ~50% confluent, cells were co-treated with LPS
100 ng/mL) and compound (oat oil extract, FAHFA,
3-HLA, linoleic acid) in a total volume of 200 µL of
(
1
media at 37°C. After 20 hours, the media was
collected and secreted IL-6 was quantified using the
mouse IL-6 ELISA MAX Deluxe Kits following
TM
the manufacturer’s protocol (BioLegend).
7

Targeted LC-MS analysis of FAHFAs
performed using a Thermo Scientific Q Exactive Plus
fitted with a heated electrospray ionization source. To
facilitate in-source fragmentation, the in-source CID
was set to 60.0 eV. To attain MS2 data of HLA, the
m/z of the negative HLA ion m/z 295.227 was added
to the inclusion list to of masses to be fragmented.
FAHFAs were measured on a TSQ Quantiva LC-MS
instrument using multiple reaction monitoring
(MRM) in negative ionization mode as previously
described (41). Resolution of FAHFAs was achieved
using an Acquity UPLC BEH C18 column (1.7 µm,
2
.1 mm X 100 mm, Waters) with a flow rate of 0.2
mL/min with a 93:7 (MeOH: H O) mobile phase with
mM ammonium acetate and 0.03% ammonium
Targeted LC-MS analysis of HLA enantiomers
2
5
HLA enantiomers were measured on a TSQ
hydroxide. LAHLAs were analyzed by monitoring
the following precursor-to-product ion transitions,
m/z 557.5 à 279.2 and m/z 557.5 à 295.2, which
correspond to the parent LAHLA to linoleic acid and
LAHLA to hydroxy linoleic acid, respectively.
Quantiva LC-MS instrument in negative ionization
mode. Resolution of HLAs was achieved using a
Lux Cellulose-3 chiral column (3 µm, 250 x 4.6 mm,
Phenomenex) with an isocratic flow rate of 0.2
mL/min of 75:25 MeOH:H O + 0.1% formic acid
2
solution at 35°C. HLAs were analyzed by pseudo-
MRM (collision energy of 9 V, RF lens set at 92),
monitoring the precursor-to-product ion transition
m/z 295.3 à 295.3.
Pseudo-MS3 for analysis of LAHLA regioisomers
Oat oil (5 µL) was dissolved in 2:1 CHCl3:MeOH
(
400 µL) and then 5 µL was subjected to LC-MS
analysis. LC separation was achieved using a Gemini
U C-18 column (Phenomenex). Resolution of
Conflict of Interest
5
LAHLAs was achieved using an Acquity UPLC BEH
C18 column (1.7 µm, 2.1 mm X 100 mm, Waters)
with a flow rate of 0.2 mL/min with a 93:7 (MeOH:
The authors declare that they have no conflicts of
interest with the contents of this article.
H
2
O) mobile phase with 5 mM ammonium acetate
and 0.03% ammonium hydroxide. MS analysis was
8

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Figure Legends
Figure 1. Anti-inflammatory activity of oat oil. a, the reported structure of a DGDG from oat seed. b,c activity
of oat oil fraction called T1 (b) and solid phase extracted sample of the T1 fraction (c) on IL-6 secretion from LPS-
stimulated RAW 264.7 cells. Data represent mean + S.E.M. for three biological replicates. **p, < 0.0001; *p, <
0.01; versus LPS by one-way ANOVA.
Figure 2. LAHLAs are primary oat oil FAHFAs. a, LAHLAs are more abundant than PAHSAs or OAHSAs in
oat oil. b, LC-MS chromatogram showing three LAHLA regioisomers.
Figure 3. Identification of LAHLA regioisomers in oat oil. a, Schematic of a pseudo-MS3 approach to identify
the LAHLA regioisomers. b, LC-MS chromatograms of FAHFA-enriched oat oil with a source energy of 80 eV
showing the presence of LAHLAs (m/z 557.4575, grey) and HLA fragments from the LAHLAs (m/z 295.2279,
red). c, MS/MS of HLAs from regioisomers 1-3 identify the LAHLAs as 15-LAHLA (1), 13-LAHLA (2), and 9-
LAHLA (3).
Figure 4. LAHLAs in plasma after ingestion of liposomal oat oil. a, Levels of plasma LAHLAs and b, PAHSAs
in subjects fed liposomal oat-oil versus control fed subjects over 7 hours. c, LC-MS chromatograms of LAHLA
regioisomers in human plasma over a 7-hour time period after liposomal oat oil ingestion. Time 0 hours represents
plasma FAHFAs after an overnight fast. Data represents mean + S.E.M. (n = 10-12). *p, < 0.05 by two-sided
Student’s t-test.
Figure 5. LAHLAs are present in mouse and human adipose tissue. a, LC-MS chromatograms of mouse
SQWAT LAHLAs (top) and the 13-LAHLA standard (bottom). b, 13-LAHLA and total LAHLA levels in mouse
SQWAT. c, LC-MS chromatogram of human WAT LAHLAs (top) and the 13-LAHLA standard (bottom). d, 13-
LAHLA and total LAHLA in human WAT. Data represents mean + S.E.M (n = 3).
Figure 6. Synthesis of 13-LAHLA and confirmation that this is the major isomer in human serum post
liposomal oat oil ingestion. a, The synthesis of 13-LAHLA was initiated by conversion of linoleic acid to methyl
linoleate under Fisher esterification conditions. Allylic oxidation of methyl linoleate with stoichiometric selenium
dioxide afforded methyl 13-hydroxy linoleate. Linoleoyl chloride, prepared from linoleic acid and oxalyl chloride,
was combined with methyl 13-hydroxy linoleate to generate the methyl ester of 13-LAHLA. Selective hydrolysis
of the methyl ester yielded 13-LAHLA in 17% overall yield starting from linoleic acid. b, Chromatogram of
LAHLA isomers in human plasma seven hours after liposomal oat oil ingestion (red) compared to the 13-LAHLA
synthetic standard (blue) provided confirmation of this regioisomer.
Figure 7. Anti-inflammatory effects of 13-LAHLA. a, b, 9-PAHSA (10 µM) and 13-LAHLA (10 µM) reduce
IL-6 and IL-1β mRNA levels in RAW 264.7 macrophages treated with LPS (100 ng/mL). c, d. Effects of 13-
LAHLA (10 µM) on iNOS and COX-2 mRNA levels in RAW 264.7 macrophages treated with LPS (100 ng/mL).
Data represents mean + S.E.M. (n = 3-4). *, p < 0.05; **, p < 0.001; ***, p < 0.0001 by two-sided Student’s t-test
for FAHFA treated versus LPS treated alone.
Figure 8. 13-LAHLA structure and activity. Activity of 13-LAHLA and 13-HLA reveal greater activity for 13-
LAHLA. Cells were viable over the entire dose range (data not shown).
12

Figures
Figure 1
a
DiGalactosyl DiacylGlycerol
DGDG)
OH
OH
OH
OH
(
HO
HO
O
O
O
O
OH
O
O
O
H
O
O
O
FAHFA
(15-LAHLA)
b
2500
2
1
1
000
500
000
*
**
5
00
0
**
0
2
10 100 400
Oat Oil [ppm]
c
2
2
1
1
500
000
500
000
**
**
5
00
0
**
**
0
2
10 100 400
Oat Oil FAHFA fraction [ppm]
13

Figure 2
a
b
1
4
3
2
1
00
00
00
2.0×10
7
.5×107
1
1
5
2
.0×107
00
4
.0×106
2
0
3
0.0
PAHSA OAHSA LAHLA
5
6
7
8
9
Retention time (min)
14

Figure 3
a
b
13-LAHLA
LAHLA (m/z 557.4575)
HLA (m/z 295.2279)
O
O
HO
O
1
1
5
.5×107
2
in source fragmentation
.0×107
1
13-HLA
OH
-
O
O
.0×106
MS/MS of HLA isomer
3
-
O
OH
0
.0
O
m/z 195.14
m/z 179.14
5
6
7
8
HLA Product Ions Distinguish
LAHLA Regioisomers
Retention time (min)
171.10
100
c
223.17
195.14
1
00
100
50
isomer 1
isomer 2
isomer 3
5
0
50
0
1
79.14
0
1
0
00
150
200
250 100
150
200
250 100
150
200
250
mass-to-charge ratio (m/z)
mass-to-charge ratio (m/z)
mass-to-charge ratio (m/z)
15-HLA
13-HLA
9-HLA
O
OH
O
O
OH
-
O
-
O
-
O
OH
MS/MS
MS/MS
O
MS/MS
OH
O
O
-
O
-
O
-
O
m/z 223.17
m/z 195.14
m/z 171.10
15

Figure 4
a
b
5
4
3
2
1
0
0
0
0
0
0
50
40
30
20
10
0
Oat Oil
Control
Oat Oil
Control
*
*
*
*
0
1
2
3
4
5
6
7
0
1
2
3
4
5
6
7
Time (hr)
Time (hr)
c
2
3
.2×105
2
1
8
.4×105
0
1
hr
hr
.6×105
1
3
3 hr
.0×104
5
7
hr
hr
0
.0
7
8
9
10
11
Retention time (min)
16

Figure 5
a
c
mouse SQ WAT
Human WAT
1
1
5
.5×105
6×103
4×103
2×103
0
.0×105
.0×104
0
.0
8
9
10 11 12 13
8
9
10 11 12 13
Retention time (min)
Retention time (min)
1
3-LAHLA standard
13-LAHLA standard
6
4
2
×105
×105
×105
0
8×105
6
4
2
×105
×105
×105
0
8
9
10 11 12 13
8
9
10 11 12 13
Retention time (min)
Retention time (min)
b
d
mouse SQ WAT
Human WAT
1
00
4
3
2
1
0
8
6
4
2
0
0
0
0
0
13-LAHLA Total LAHLA
13-LAHLA Total LAHLA
17

Figure 6
a
1
. H
2
SO
4
, CH
3
OH
O
O
O
2. SeO
2
HO
CH
3
H
3
CO
Cl
OH
22% 2-steps
CH
3
+
oxalyl chloride
9%
CH
3
9
O
O
O
1
2
. pyr
. LiOH
HO
CH
3
76% 2-steps
CH
3
13-LAHLA
b
3
2
1
8
.2×105
.4×105
.6×105
.0×104
Serum LAHLA Levels @ 7 hr
3-LAHLA Synthetic Standard
1
0
.0
7
8
9
10
11
Retention time (min)
18

Figure 7
a
b
IL-6 mRNA
IL-1β mRNA
6
4
2
00
00
00
0
150
100
50
**
***
***
***
0
LPS (100 ng/mL)
LPS (100 ng/mL)
c
d
iNOS mRNA
COX-2 mRNA
3
2
1
000
000
000
0
250
2
00
150
00
**
1
*
5
0
0
LPS (100 ng/mL)
LPS (100 ng/mL)
19

Figure 8
2
1
1
1
1
0000
1
3-LAHLA
3-HLA
7500
5000
2500
0000
1
13-LAHLA
IC50 = 28.3 µM
13-HLA
IC50 = 274 µM
7
5
2
500
000
500
0
-6
-5
-4
-3
Log (Concentration)
10
20

Linoleic acid esters of hydroxy linoleic acids are anti-inflammatory lipids found in
plants and mammals
Matthew J. Kolar, Srihari Konduri, Tina Chang, Huijing Wang, Clare McNerlin, Lena
Ohlsson, Magnus Härröd, Dionicio Siegel and Alan Saghatelian
J. Biol. Chem. published online May 31, 2019
Access the most updated version of this article at doi: 10.1074/jbc.RA118.006956
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