Rapid tin-mediated access to a lysophosphatidylethanolamine (LPE) library: Application to positional LC/MS analysis for hepatic LPEs in non-alcoholic steatohepatitis model mice

Article abstract of DOI:10.1016/j.chemphyslip.2016.09.003

Even though lysophospholipids have attracted much interest in recent years on account of their unique bioactivity, research related to lysophospholipids is usually hampered by problems associated with standard sample preparation and discrimination of regioisomers. Herein, we demonstrate a quick tin-chemistry-based synthetic route to lysophosphatidylethanolamines (LPEs) and its application in the positional analysis of hepatic LPEs in non-alcoholic steatohepatitis (NASH) model mice. We found that the preference of hepatic LPE regioisomer largely depends on the unsaturation of acyl chain in both control and NASH model mice. In addition, hepatic C18:2-LPE and C20:5-LPE levels were significantly lower in the NASH model mice than those in the control. The LC/MS technique based on the library of LPE regioisomers allows an accurate observation of hepatic LPE metabolism and might provide useful information to elucidate yet ambiguous pathogenesis of NASH.

Full text of DOI:10.1016/j.chemphyslip.2016.09.003

Chemistry and Physics of Lipids 200 (2016) 133–138
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Rapid tin-mediated access to a lysophosphatidylethanolamine (LPE)
library: Application to positional LC/MS analysis for hepatic LPEs in
non-alcoholic steatohepatitis model mice
Takayuki Furukawa, Hirotoshi Fuda, Satoshi Miyanaga, Chinatsu Watanabe,
*
Hitoshi Chiba, Shu-Ping Hui
Graduate School of Health Science, Hokkaido University, North 12, West 5, Kita-ku, Sapporo, 060-0812, Japan
A R T I C L E I N F O
A B S T R A C T
Article history:
Even though lysophospholipids have attracted much interest in recent years on account of their unique
bioactivity, research related to lysophospholipids is usually hampered by problems associated with
standard sample preparation and discrimination of regioisomers. Herein, we demonstrate a quick tin-
chemistry-based synthetic route to lysophosphatidylethanolamines (LPEs) and its application in the
positional analysis of hepatic LPEs in non-alcoholic steatohepatitis (NASH) model mice. We found that
the preference of hepatic LPE regioisomer largely depends on the unsaturation of acyl chain in both
control and NASH model mice. In addition, hepatic C18:2-LPE and C20:5-LPE levels were significantly
lower in the NASH model mice than those in the control. The LC/MS technique based on the library of LPE
regioisomers allows an accurate observation of hepatic LPE metabolism and might provide useful
information to elucidate yet ambiguous pathogenesis of NASH.
Received 20 June 2016
Received in revised form 27 September 2016
Accepted 28 September 2016
Available online 1 October 2016
Keywords:
Lysophosphatidylethanolamine
Tin
Regioisomer
Non-alcoholic steatohepatitis
LC/MS
ã 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
lysophosphatidylethanolamines (LPEs) still remain undiscovered,
which is mostly due to the limited commercial availability of LPEs.
Phospholipids (PLs) are fundamental biomolecules that consist
of a polar head group, a glycerol backbone, and fatty acids. Their
importance in membrane assembly and molecular signaling as e.g.
lipid mediators has been well documented (Aoki et al., 2015).
Lysophospholipids (LPLs) represent a PL subclass and contain only
one fatty acid on the glycerol backbone. Recent studies revealed
that these unique PLs, especially lysophosphatidic acids (LPAs),
contribute to an array of biological responses such as platelet
aggregation, smooth muscle contraction, cell proliferation, and cell
migration (Mills and Moolenaar, 2003) via binding to the
corresponding receptors. Compared to LPAs or lysophosphatidyl-
serines (LPSs) (Makide et al., 2014) the functions of
Another frequently encountered problem in LPE and LPL
research is 1,2-O-acyl migration (Fig. 1) (Plückthun and Dennis,
1982; Liu et al., 2014), as this equilibrium furnishes 1-LPE and 2-
LPE regioisomers, which exhibit different physico-chemical
(Okudaira et al., 2014) and biological (Xu et al., 2005) properties.
Unfortunately, suppression of this migration still remains difficult,
and accordingly the precise determination of the regioisomer ratio
prior to a use of the mixture is of paramount importance. NMR
spectroscopy can be a powerful tool for this purpose (Medina and
Sacchi, 1994), even though it requires relatively large sample
quantities and is thus not ideally suited for the analysis of
biological samples. Conversely, mass spectrometry (MS) is more
advantageous in terms of its detection limit, and has already been
applied to the characterization of various LPLs (Hsu and Turk,
2009). Nevertheless, their complete identification still requires the
use of standard samples.
Abbreviations: Tr, triphenylmethyl; DPPE, 1,2-dipalmitoyl-sn-glycero-3-phos-
phoethanolamine; PE, phosphatidylethanolamine; LPL, lysophospholipid; LPE,
lysophosphatidylethanolamine; LPC, lysophosphatidylcholine; LPI, lysophosphati-
dylinositol; LPA, lysophosphatidic acid; LPS, lysophosphatidylserine; PL, phospho-
lipid; HG, head group; iPrOH, isopropanol; DMF, dimethylformamide; Bu2SnO,
dibutyltinoxide; Teoc, 2-(trimethylsilyl)ethoxycarbonyl; TFA, trifluoroacetic acid;
FA, fatty acid; PUFA, polyunsaturated fatty acid; NASH, non-alcoholic steatohepa-
titis; NAFLD, non-alcoholic fatty liver disease.
Herein, we report a new short synthetic route to LPEs, which is
suitable for the construction of an LPE library. Moreover, we
applied this synthetic library into the positional analysis of LPEs in
murine liver tissues by using LC–MS platform. In this study, we
employed a non-alcoholic steatohepatitis (NASH) model mice
developed before (Yimin et al., 2011), and compared the hepatic
* Corresponding author.
E-mail address: keino@hs.hokudai.ac.jp (S.-P. Hui).
http://dx.doi.org/10.1016/j.chemphyslip.2016.09.003
0009-3084/ã 2016 Elsevier Ireland Ltd. All rights reserved.

134
T. Furukawa et al. / Chemistry and Physics of Lipids 200 (2016) 133–138
Fig. 1. 1,2-O-acyl migration in LPEs.
levels of LPE regioisomers between the NASH model and the
control mice. NASH is a progressive hepatic disease developed in
association with fatty liver and multiple risk factors such as
oxidative stress and proinflammatory factors. The pathogenesis of
NASH is yet ambiguous, and therefore, no specific diagnosis/
treatment/prevention method is available. This study aims to
obtain useful information for understanding of the pathogenic
processes of NASH from the viewpoint of hepatic LPE metabolism.
(ꢂ50 mg wet weight) using a conventional Folch method (Folch
et al., 1957). Animal experiments were conducted according to the
Regulations for the Care and Use of Laboratory Animals of
Hokkaido University. Our experimental protocol was approved
by the Institutional Animal Care and Use Committee (IACUC) at
Hokkaido University (Approval No. 10-0028).
2.3. LC-ESI-MS/MS conditions and procedures
2. Experimental
LPE regioisomers were separated on an HPLC with an Atlantis
T3 column (3
m
m ꢀ 2.1 mm ꢀ 150 mm; Waters) using an aqueous
2.1. Materials and methods
AcOH (0.1%)/acetonitrile gradient system (flow rate: 0.3 mL minꢃ1
;
for details, see: Supplementary information). The following ESI
conditions were applied: capillary voltage = 4000 V, desolvation
temperature = 350 ꢁC, sheath gas = 50 psi, and auxiliary gas = 10 psi.
LPE precursor ions and product ions were detected in positive ion
mode; for LPE precursor ions: FTMS (resolution: 60,000); for
product ions: ITMS (CID normalized collision energy = 35 V).
Obtained data were analyzed using the Qual browser (Thermo
Unless otherwise noted, all reagents were purchased from
Wako Pure Chemical Industries Ltd. (Tokyo, Japan) or Sigma-
Aldrich. TLC was performed on pre-coated plates (20 cm ꢀ 20 cm;
layer thickness: 0.25 mm; silica gel 60F254; Merck), and spots were
visualized by spraying with an ethanol solution of Ninhydrin,
followed by heating to 250 ꢁC for ꢂ0.5 min or by exposure to UV
light (
l
= 254 nm) when applicable. Column chromatography on
m; Kanto
Fisher Scientific). 10 mL of hepatic lipid extracts was injected, and
silica gel (N60; spherical type; particle size: 40–50
m
extracted ion chromatogram (EIC) was obtained on the basis of
theoretical molecular weight (10 ppm error range) for each LPEs.
Regioisomer ratios were calculated based on integrated peak areas.
All assays were done in five replications: livers from five different
NASH model and control mice were analyzed.
Chemical Industry) was carried out using the specified solvent
systems, whereby the solvent ratio is given in v/v. 1H and 13C NMR
spectra were recorded on a 400 MHz JNM-ECP400 spectrometer
(JEOL, Japan; 1H: 400 MHz, 13C: 100 MHz), and multiplicities are
given as singlet (s), broad (br), doublet (d), doublet of doublets
(dd), triplet of doublets (td), triplet (t), quintet (q), or multiplet (m).
Chemical shifts are expressed in ppm and referenced to TMS (dH
0.00), CHCl3 (dH 7.26), CH3OD (dH 3.31), CDCl3 (dC 77.16), or CD3OD
2.4. Data processing
Relative intensity was calculated by integrated peak area over
liver weight. Statistical differences were analyzed by two-tailed
Mann-Whitney U test (Prism 6, GraphPad Software Inc.) and a
value of P < 0.05 was regarded as significant.
(
dC 49.00) as the internal standard (Fulmer et al., 2010). Assign-
ments in the 1H NMR spectra were made by first-order analysis
using the ACD/NMR processing software (Advanced Chemistry
Development, Inc.). High-resolution electrospray ionization mass
spectra (HR-ESI-MS) and liquid chromatography mass spectra
(LC–MS) were recorded on an LTQ Orbitrap XL (Thermo Fisher
Scientific), equipped with a Shimadzu Prominence HPLC system.
3. Results and discussion
3.1. Chemical synthesis of an LPE library
2.2. Liver tissues and lipid extraction
Although the chemical synthesis of several LPEs has already
been reported (D’Arrigo, 2010), most of these examples represent a
Murine liver tissues were prepared as we reported previously
(Yimin et al., 2011). Briefly, NASH model mice were produced by
the combination of high-fat diet for 23 weeks and several
intravenous injections of oxidized low-density lipoproteins. This
model shows histhopathological and metabolic features similar to
human NASH (Yimin et al., 2011). Mice fed a regular diet were used
for control study. Hepatic lipids were extracted from a liver tissue
‘total synthesis’ approach. Even though such a strategy is
undoubtedly useful, it is not suitable for the construction of a
library, as it is simply too time consuming. We envisaged that a
semi-synthetic approach should be more favorable for the
construction of such a library, as it may potentially require
significantly less steps. It should also be noted here that the
chemical semi-synthesis of LPEs still remains unexplored.
Fig. 2. Synthetic strategy towards LPEs in this study; R, R’ = fatty acid chains; PG = protecting group.

T. Furukawa et al. / Chemistry and Physics of Lipids 200 (2016) 133–138
135
Table 1
Our “semi-synthesis” strategy is shown in Fig. 2. As a starting
Product yields for each step and selectivity values for the regioisomers.
point, we chose commercially available diacyl-phosphoethanol-
amine 1 (PE). The protection of the amino functionality and a
subsequent deacylation should afford intermediate 2. A regiose-
lective mono-acylation of this diol 2 with a variety of fatty acid
derivatives, followed by the deprotection of the amino group
should furnish the targeted LPE molecules in only two steps.
For the protection of the amino functionality in commercially
Entry RCOCl (R = )
Yield (%)
2-LPE:1-LPE
Acylation Deprotection
1
2
3
4
5
6
7
8
C10:0 (capric acid) 7
27
18
18
11
44
26
54
77
69
56
60
47
59
69
37
79
80
3:1
7:3
7:3
7:3
3:1
3:1
3:1
3:1
3:2
7:3
3:1
C16:0 (palmitic acid) 8
C18:0 (stearic acid) 9
C18:1 (oleic acid) 10
C18:2 (linoleic acid) 11
C18:3 (pinolenic acid) 12
available
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine
(DPPE, 4), we chose the trityl group, which have already
successfully employed in a previous PE synthesis (Aneja et al.,
1969; Plückthun et al., 1985). The protection of the amino group in
4 using tritylbromide and triethylamine proceeded quantitatively,
and the subsequent deacylation under Zemplén conditions
(Zemplén and Pacsu, 1929) afforded the desired amino-protected
diol 6 in 87% yield (Scheme 1).
C18:3 (a-linolenoic acid) 13 10
C18:3 (g-linolenoic acid) 14 26
9
10
11
C20:4 (arachidonic acid) 15 27
C20:5 (EPA) 16
C22:6 (DHA) 17
37
38
Morestericallyfavorablesn-1 OHreactspriortomore hinderedsn-2
OH (Roelens, 1996).
With ready access to intermediate 6, we proceeded to the
subsequent acylation step. Given that the target of this synthetic
route was to synthesize LPE in a highly regioselective manner, we
attempted a tin-mediated activation, given the proven track record
of this method in carbohydrate chemistry (Jäger and Minnaard,
2015). Moreover, this approach has already been successfully
applied to the synthesis of LPCs by Servi et al. (Fasoli et al., 2006).
When we applied this approach to diol 6, we obtained trityl-
protected LPEs 70–170 inyields that were merely moderate (18–44%)
compared to previously reported LPCs (Scheme 2) (Fasoli et al.,
2006; Niezgoda et al., 2013). The results of these experiments are
summarized in Table 1. During these experiments, we realized that
thisacylationstepisverysensitivetowater.Whensolventswerenot
driedenoughpriortouse, decreasedyieldswereobserved(entries 4
and7, Table1). AsolventscreeningrevealedthatDMFaffordedmore
reproducibility than conventional isopropanol (data not shown).
Even though trityl-protected LPEs 70–170 were soluble in CDCl3, we
were unable to obtain useful NMR spectra, and observed only broad
features. Therefore, we proceeded to the final deprotection step
without prior NMR analysis. A subsequent work-up under acidic
conditions afforded LPEs 7–17 in good yield (35–80%) (Table 1).
After deprotection, we observed regioselectivity ratios (2-LPE:1-
LPE) of 3:1–7:3, with the exception of 3:2 for 15 (entry 9, Table 1).
Even though these ratios are not as high as for the previously
reported LPCs (Fasoli et al., 2006; Niezgoda et al., 2013), they are
nevertheless acceptable, considering the aforementioned prob-
lematic acyl migration. Even if the acylation was highly regiose-
lective, the acidic work-up should promote a 1,2-O-acyl migration
and thus lower the selectivity. It is generally considered that the
regioselectivity of tin-mediated reaction is induced by steric effect:
We also tested the Teoc protecting group in order to shield the
amino functionality (Scheme 3), as Teoc can be removed under
milder conditions than the trityl group. Fortunately, this change
increased the yield in the acylation step to 72%. Unfortunately, the
deprotection was not successful at all. A fluoride-induced removal
did not proceed, and treatment with TFA afforded complex product
mixtures. This result indicates that the judicious choice of
protecting group for the amino functionality should be critical
for both acylation and deprotection.
3.2. Analysis of LPE regioisomers by LC/MS
So far, discrimination of regioisomers has been carried out
predominantly by two methods: NMR spectroscopy and mass
spectrometry. While NMR is able to provide a variety of information,
itrequiresrelativelyhighsamplequantities,anditsapplicabilitythus
dependsonthesolubilityoftheLPE,whichinturnlargelydependson
the fatty acid moiety. LPEs with PUFA chains tend to exhibit higher
solubility than those with saturated FA chains. For instance, LPE 17
with a DHA FA chain is soluble in methanol, while LPE 9 with a
stearic acid chain is virtually insoluble, even in chloroform/
methanol or dimethylsulfoxide (DMSO). This difference restricts
the versatility of the NMR approach in our study. Conversely, mass
spectrometry requires very small sample quantities, which renders
solubility virtually irrelevant. Especially liquid chromatography
tandem-mass spectrometry (LC–MS/MS) represents a powerful
approach to distinguish 1/2-LPEs (Han and Gross, 1996; Fang et al.,
2003; Lee et al., 2011).
Scheme 1. Synthesis of common intermediate 6.
Scheme 2. Tin-mediated acylation of 6 and subsequent deprotection of 70–170.

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T. Furukawa et al. / Chemistry and Physics of Lipids 200 (2016) 133–138
Scheme 3. Protecting the amino functionality with a Teoc group.
In order to understand pathogenic processes in NASH, we
Table 2
established a novel NASH murine model and characterized by
morphological, histological and biochemical means (Yimin et al.,
2011). This model was confirmed to have increased TG, free fatty
acid, and total cholesterol in the liver. However, no information
about other class of lipids in this model has been available so far.
Therefore, we performed further analysis of hepatic lipids in this
model, especially focused on LPEs, using LC/MS and a synthetic
library as references.
At first, we established an LC/MS condition to separate LPEs and
regioisomers (see Supplementary information). So as to charac-
terize LPEs in LC–MS, we measured retention times (Table 2) and
confirmed a fragmentation pattern in MS/MS spectra. Although
three olefinic isomers of C18:3 (12–14) could not be separable in
this condition, other LPEs were possible. Regioisomers can be
distinguished by the LC and MS/MS because similar fragmentation
patterns are observed as reported (Fang et al., 2003; Lee et al.,
2011). Briefly, 2-LPE series provided in general highly intense
[M+HꢃH2O]+ signals and moderately intense [M+HꢃHG]+. In
contrast, the 1-LPE series exhibited an opposite fragmentation
pattern, i.e. strong [M+HꢃHG]+ and very weak [M+HꢃH2O]+ signals
(Fig. 3 and Supplementary information).
LC–MS analysis of liver LPEs from control and NASH model mice. *: detected as a
overlapped signal, y: observed as a main peak and confirmed by MS/MS, N.D.: not
detected.
LPE
Retention Time (min)
Synthetic Reference
Control
NASH
1-LPE
2-LPE
C10:0 (7)
C16:0 (8)
C18:0 (9)
C18:1 (10)
C18:2 (11)
C18:3 (12–14)*
C20:4 (15)
C20:5 (16)
C22:6 (17)
14.48
18.65
20.29
19.11
14.73
18.98
20.69
19.40
18.42
N.D.
N.D.
18.63, 18.93y
20.64y
18.64, 18.95y
20.69y
19.11, 19.38y
18.14y, 18.39
17.4–17.8
18.21y, 18.43
17.47y
19.12, 19.41y
18.14y, 18.39
17.4–17.8
18.23y, 18.42
17.48y
18.16
17.4–17.8
18.23
17.49
18.44
17.69
18.41
18.22
18.18y, 18.37
18.18y, 18.36
Non-alcoholic fatty liver disease (NAFLD) is the most common
chronic liver disease in worldwide and might progress into non-
alcoholic steatohepatitis (NASH) known as more severe state of
disease (Musso et al., 2016). Many studies have investigated the
lipid content in a liver tissue of NASH or NAFLD (Byeon et al., 2016;
García-Cañaveras et al., 2011; Eisinger et al., 2014; Gorden et al.,
2015; Nam et al., 2015). Most of the previous studies on hepatic fat
focused on triglycerides (TG) metabolism, because TG is the major
constituent of hepatic fat. On the other hand, no statement
concerning LPE regioisomers can be found in the literature to our
best knowledge.
With diagnostic tools in hand, we began to analyze hepatic LPEs
using the same LC–MS condition. Results are summarized in
Table 2. Surprisingly, the positional preferences in hepatic LPEs are
largely dependent on the degree of unsaturation of acyl chain
(Fig. 4); C16:0 (8) and C18:0 (9) LPEs are found in exclusively 2-LPE
form, however, C20:4 (15), C20:5 (16), and C22:6 (17) LPEs are
observed in exclusively 1-LPE form. C18:1 (10) and C18:2 (11) LPEs
Fig. 3. Representative product ion pattern for 1-(top) and 2-LPEs (bottom).

T. Furukawa et al. / Chemistry and Physics of Lipids 200 (2016) 133–138
137
Fig. 4. Positional preferences of C16:0 (8), C18:1 (10), C18:2 (11) and C20:5 (16).
appear transitional; C18:1 (10) is 2-LPE rich whereas C18:2 (11) is
1-LPE rich. This remarkable difference can’t be explained by the
theory of chemical equilibrium: Thermodynamically more stable
2-LPE (1-Acyl) should be favorable than 1-LPE (2-Acyl) as reported
in previous studies (Plückthun and Dennis, 1982; Liu et al., 2014).
2015), our murine model showed closer outcome in human, so we
consider that our NASH model mice would be valuable to
understand pathogenic processes of NASH in human.
4. Conclusion
Thus,
a contribution of biological or enzymatic factors is
anticipated: for example, the different substrate specificity of
lipases in a liver tissue, such as LPA1 or LPA2 (Okudaira et al., 2014),
but this hypothesis needs to be proven in future. It is also
noteworthy that there is no clear difference between control and
NASH in this point.
Conversely, significant differences are observed in relative
abundance (Fig. 5). In C18:2 (10) and C20:5 (16) LPEs, decreased
intensities are detected in a NASH liver tissue. Although other LPE
species are not significant, diminishing trend is found in whole
series with the exception of C20:4 (15). As similar trend was
documented in the analysis of human liver tissues (Gorden et al.,
Despite the growing interest on LPLs as lipid mediators,
synthetic access and appropriate characterization tools still remain
limited. In this study, we focused on LPEs, whose functions are not
yet well understood. We developed a short novel method for the
synthesis of various LPEs, and applied into the analysis of hepatic
LPE extracted from NASH model and control mice.
We showed that the diol skeleton can be successfully
modulated by a tin-mediated acylation that has previously been
employed successfully in carbohydrate chemistry. This synthetic
route to LPEs requires in total only four steps and is thus
substantially less laborious than conventional routes. Using this
Fig. 5. Comparison of NASH and control liver tissues. C18:3 (12–14) were excluded for clarity. N = 5, ns: not significant, **: P < 0.01.

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T. Furukawa et al. / Chemistry and Physics of Lipids 200 (2016) 133–138
route, we constructed an LPE library including eleven FAs. We also
found that this tin-mediated acylation is highly sensitive to water
and that the selection of the protecting group for the amino moiety
should be important for further improvements. These results
should also be helpful for the synthesis of other LPE analogues or
other classes of LPLs.
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Acknowledgement
This study was financially supported by the Japanese Ministry
of Education, Culture, Sports, Science and Technology (MEXT) in
the context of the Regional Innovation Strategy Support Program
Sapporo Health Innovation “Smart-H”.
Appendix A. Supplementary data
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