November 2022

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

Short Communication

Quantiﬁcation of threonine-containing lysophospholipids using
ESI-LC-MS/MS in mouse tissues
1,2

2

3

4

Michiyo Okudaira , Kuniyuki Kano , Kumiko Makide , Daisuke Saigusa ,2

2

Tomohiko Ohwada , Junken Aoki *Faculty of Hospital Pharmacy, Teikyo University, 2‒11‒1 Kaga, Itabashi-ku, Tokyo 173‒8605, Japan
Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo,
7‒3‒1 Hongo, Bunkyo-ku, Tokyo 113‒0033, Japan
3
Laboratory of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University,
6‒3 Aoba, Aramaki, Aoba-ku, Sendai 980‒8578, Japan
4
Laboratory of Biomedical and Analytical Sciences, Faculty of Pharma-Sciences, Teikyo University,2‒11‒1 Kaga, Itabashi-ku, Tokyo 173‒8605, Japan
1

2

Abstract

Lysophosphatidylthreonine (LysoPT) is a potent inducer of mast cell degranulation that was previously identi-

ﬁed as an analog of a bioactive lysophospholipid, lysophoshatidylserine (LysoPS). However, it remains to be solved if LysoPT is present in vivo. In the present study, we tried to detect LysoPT in biological samples using liquid chromatography/
tandem mass spectrometry (LC-MS/MS) and examined its tissue distribution. In several mouse tissues, we detected LysoPT
as a mixture of LysoPT with several diﬀerent fatty acid chains, i.e., LysoPT species. C18:0-LysoPT was a major LysoPT
species in various tissues and plasma with the highest expression in the stomach. The fatty acid species and tissue distributions of LysoPS and LysoPT were similar, suggesting that the two lysophospholipids share a common synthetic pathway.
The present study indicates that LysoPT behaves as an endogenous lysophospholipid mediator.
Key words: lysophosphatidylthreonine, LysoPT, lysophosphatidylserine, LysoPS, LC-MS/MS, lysophospholipid mediator

Introduction

and in vitro

1‒4). However, we poorly understand the role ofMast cells are involved in developing immediate allergic

LysoPS in activating mast cells in pathophysiological con-reactions by releasing various inﬂammatory mediators suchas histamine and serotonin stored in their cytoplasmic gran-texts. We previously synthesized a series of LysoPS analogs

ules in response to an antigen. Administration of lysophos-

chain, or ester bond of LysoPS. We showed that one ofphatidylserine (LysoPS), a kind of lysophospholipids, hasbeen shown to potentiate the antigen-induced histamine

them, lysophosphatidylthreonine (LysoPT), which has amethyl group at the β -carbon of serine, was much morerelease (degranulation) from rodent mast cells both in vivo

potent in inducing mast cell degranulation than LysoPS*Corresponding author

LysoPT induced histamine release from mast cells in vitro
and in vivo at a concentration less than one-tenth of Lys-

with modiﬁcations in either the glycerol backbone, acyl

Junken Aoki.

oPS. Like LysoPS, LysoPT also induced hypothermia andDepartment of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7‒3‒1 Hongo,

anaphylactic shock in mice. Injection of LysoPT into micecaused transient decreases of body temperature at a concen-

Bunkyo-ku, Tokyo 113‒0033, Japan
Tel: ＋81‒3‒5841‒4720, Fax: ＋81‒3‒5841‒0280

tration 50-fold lower than that of LysoPS, raising the possi5)

bility that LysoPT functions as a lipid mediator .

E-mail: jaoki@mol.f.u-tokyo.ac.jp
Received: August 12, 2022.

5,6)

Despite its potent pharmacological activities to induce

Accepted: October 27, 2022.

Epub November 16, 2022.

mast cell degranulation, hypothermia, and anaphylactic

DOI: 10.24508/mms.2022.11.008

shock, little is known about the presence of LysoPT in vivo.
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Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

In addition, the synthetic pathway of LysoPT has been
unknown. Other glycero-lysophospholipids, such as lyso-

Sample preparation

phosphatidic acid (LPA) and LysoPS, are known to be pro-arinized capillary and centrifuged at 1,500×g for 10 min at

duced from their precursors, including phosphatidic acid(PA), phosphatidylserine (PS), and lysophosphatidylcholine

4°
C. The resulting supernatant was used as plasma. For
serum preparation, blood was collected using a non-coated. Interestingly,

capillary and was incubated for 1 h at 37°
C to induce blood

phosphatidylthreonine (PT), which is a plausible precursor

coagulation. Then it was kept at 4°
C for 4 h to retract theof LysoPT, has been shown to be present in tissues from10)
various species, including rat brain , hippocampal neu-

blood clots and was centrifuged at 1,500×g for 10 min atrons , and the endoplasmic reticulum of Toxoplasma gon-

A 10 μL of plasma and serum samples were mixed with
100 μL of CH3OH containing 100 nM C17:1-LysoPS in a7‒9)(LPC) by several kinds of phospholipases

For plasma preparation, blood was collected using a hep-

4°
C. The resulting supernatant was used as a serum.

11)

12)

dii . Various phospholipase A1 and A2 types are present,
indicating that LysoPT is present in vivo.

1.5 mL siliconized sample tube. It was sonicated in a waterbath-type sonicator to recover lipids in CH3OH. The

Our previous study developed a liquid chromatography/
tandem mass spectrometry (LC-MS/MS) method to analyze

extracts were sequentially centrifuged at 21,500×g, and theresulting supernatant was passed through an acetyl cellu-

13)lysophospholipids . However, due to the lower sensitivity,

lose ﬁlter (pore size 0.22 μm). A volume of 10 μL of samples was injected into LC-MS/MS.

we could not detect LysoPT in biological samples. In thepresent study, we changed the mass spectrometry (MS)For tissue samples, mice were rapidly perfused transcardially with cold PBS through the left ventricle, and subse-

instrument and optimized the method for LysoPT analysis.
The new method makes it possible to detect and quantify

quently, tissues were collected. 5‒10 mg tissues were transferred to 2.0 mL sample tubes, and 10-fold volume CH3OH

LysoPT concentrations as low as 500 pM. In addition, themethod is helpful for examining the tissue distribution of
LysoPS in mice. The present method may help to understand the pathophysiological roles of LysoPT and LysoPS

containing 100 nM C17:1-LysoPS and beads were added
for homogenization using an MS-100R beads cell disrupter.

as lipid mediators.

The extracts were sequentially centrifuged at 1,500×g and21,500×g, and the resulting supernatant was passed

Materials and Methods

through a ﬁlter. A volume of 10 μL of the sample wasinjected into LC-MS/MS.

Chemicals

LysoPS standards (C16:0-LysoPS, C18:0-LysoPS,
C18:1-LysoPS) and internal standard, C17:1-LysoPS, were

Liquid chromatography system and conditions

This study used the A NANO SPACE SI-2 LC system(Osaka-soda, Tokyo, Japan). The system consists of dual

purchased from Avanti Polar Lipids. C16:0-LysoPT,
C18:0-LysoPT, and C18:1-LysoPT were chemically synthe5)

sized previously and used as LysoPT standards. Methanol

pumps, an autosampler, a column oven, and an online(CH3OH) and acetonitrile (CH3CN) of LC/MS grade weredegasser. The chromatographic separation was performed
on CAPCELL PAK C18 ACR (250 mm×1.5 mm i.d., 3 μm
particle size) (Osaka-soda, Tokyo, Japan) analytical column

obtained from Kanto Chemical (Tokyo, Japan). Ammonium
formate (HCOONH4) of LC/MS grade was obtained from
Wako Pure Chemical Industries. Ultra grade water was pre-coupled with a CAPCELL PAK C18 ACR (10 mm×2.0 mm

pared with Milli-Q Advantage A 10 Ultrapure Water Puriﬁ-i.d., 3 μm particle size) guard column maintained at 40°
C.

cation System from Millipore.

The ﬂow rate was 150 μL min , and the mobile phases−1were formed using 5 mM HCOONH4 (H2O/CH3CN＝100/0,

Animalsv/v) as eluent A and 5 mM HCOONH4 (H2O/CH3CN＝5/95,

Male C57 BL/6 mice (8‒12 weeks) were used. Micev/v) as eluent B whereby the solutions were adjusted to pH
4.0 using HCOOH, respectively. The liquid chromatogra-

were purchased from Japan SLC and maintained according
to the Guidelines of Animal Experimentation of The Uni-phy (LC) analysis started with 50% B for 0.2 min, followed

versity of Tokyo. The animal protocol was approved by theInstitutional Animal Care and Use Committee at the Uni-

by a linear gradient from 50 to 100% B in 12.0 min andmaintenance of 100% B for 5 min. Subsequently, themobile phase was immediately returned to the initial condi-

versity of Tokyo.
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Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

divert valve was used to divert the LC eﬄuent to the waste

C17:1-LysoPS as internal standard. 10 μL of each standard
solution was injected into LC-MS/MS. The ratio between

during the ﬁrst 5 min and the last 2 min.

analytes and the internal standard peak area was calculated

tions and maintained for 3 min until the end of the run. A

for quantiﬁcation.

Mass spectrometry system and conditions

We determined the precision of the present method by

The MS system was a Thermo Fischer Scientiﬁc TSQ

analyzing six samples containing the same concentration of
LysoPS and LysoPT on the same day (intra-day analysis).

Quantiva triple quadrupole mass spectrometer with aheated electrospray ionization (HESI) source. The operat-This procedure was repeated for three days (inter-day anal-

ing condition was optimized for C18:1-LysoPS, dissolvedysis). The coeﬃcient of variation within one sample run(intra-day) and between sample runs (inter-day) was deter-with CH3OH (1 μM) by continuously infusing at a 5 μL
−1

min . The HESI spray voltage was 2,500 V, the heated ion
transfer tube temperature was 329°
C, the sheath gas pres-mined for various concentrations of LysoPS and LysoPT.
To determine the recovery, a mixture of LysoPT species(C16:0, C18:0, and C18:1) at 50 nM and 100 nM were pre-sure was 38 psi, the auxiliary gas pressure was 12 psi, andthe heated vaporizer temperature was 279°
C. Both thesheath gas and the auxiliary gas were nitrogen gas. The col-pared and added to the mouse tissue homogenates (brain,lision gas was argon at a pressure of 2.0 mTorr. HESI was

intestine, muscle and skin, ten mg each) or plasma (10 μL).

thymus, heart, lung, liver, spleen, kidney, stomach, small
Lipids were extracted by adding 90 μL CH3OH containing

performed in a negative ion mode. The optimized HESI ofseven LysoPS species (C16:0, C18:0, C18:1, C18:2, C20:4,

internal standard (C17:1-LysoPS). The samples for MS

C22:6, and C17:1 (used as internal standard)) and of three
LysoPT species (C16:0, C18:0, and C18:1) produced abun-

injection were prepared as described above. The extraction

dant [M−H] ions at m/z 496.3, 524.3, 522.3, 520.3, 544.3,

recovery was calculated by the following formula:[(detected concentration-endogenous concentration)/stan-

568.3, 508.3, 510.3, 538.3 and 536.3, respectively. Samples

dard concentration]×100 (%).

−

were analyzed by the selected reaction monitoring (SRM)−

Resultsmode, employing the transition of the [M−H] precursor
ions to their product ions. For the MS/MS analysis, the col-LC-MS/MS analysis of LysoPT

lision energies for collision-induced dissociation of LysoPS

Previously, we constructed a system to quantify various
lysophospholipids, including LysoPS, in biological samples
13)
using LC-MS/MS . However, using this method, we could

and LysoPT were 19 eV. In SRM mode, the transitions of
the precursor ion into the production were monitored: m/z
496.3 → 409.3 for C16:0-LysoPS, m/z 524.3 → 437.3 for

not detect LysoPT, possibly because it was not suﬃciently

C18:0-LysoPS, m/z 522.3 → 435.3 for C18:1-LysoPS, m/z
520.3 → 433.3 for C18:2-LysoPS, m/z 544.3 → 457.3 forsensitive. In the previous study, we used TSQ Quantum
Ultra mass spectrometer (Thermo Fischer Scientiﬁc) mass
system. Subsequently, we used a high-performance TSQ

C20:4-LysoPS, m/z 568.3 → 481.3 for C22:6-LysoPS, m/z
508.3 → 421.2 for C17:1-LysoPS, m/z 510.3 → 409.3 for

Quantiva mass spectrometer (Thermo Fischer Scientiﬁc) for

C16:0-LysoPT, m/z 538.3 → 437.3 for C18:0-LysoPT andm/z 536.3 → 435.3 for C18:1-LysoPT. The LC-MS/MS sys-

the detection of LysoPT. For the Quantiva analyses, we ﬁrst
performed product ion scanning using a chemically synthesized C18:1-LysoPT . The product ion scanning analyses
indicated that deprotonated precursor ions of C18:1-LysoPT
5)

tem was controlled by the Xcalibur software (Thermo
Fisher Scientiﬁc) and data were collected with the same
software.

(m/z 536.3) lost the corresponding polar head group (i.e.
threonine) upon collisional activation, yielding a character-

Method validation
A mixture of LysoPS species (C16:0, C18:0, and C18:1,

istic fragment ion of C18:1-LPA (m/z 435.3). The fragmentation was useful for SRM detection of LysoPT in the LC-MS/

each 1 μM) and LysoPT species (C16:0, C18:0, and C18:1,

MS analyses. We could also detect and analyze LysoPS spe-each 1 μM) was prepared as a CH3OH solution. It wascies using Quantiva by the same strategy.

ﬁnal concentrations of 100 nM, 50 nM, 10 nM, 5 nM, 1

Typical SRM chromatograms of LysoPT and LysoPS standards were obtained by LC-MS/MS analyses (Fig. 1A andnM, and 500 pM, respectively, each containing 100 nM

1C). This method was applied to mouse large-intestine

diluted with CH3OH to prepare six standard solutions with140

November 2022

Fig. 1.

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2(A, C) SRM chromatograms of LysoPT (A) standard samples (C16:0, C18:1 and C18:0, each 50 pmol), LysoPS (C)standard samples (C16:0, C18:1 and C18:0, each 50 pmol). (B, D) LysoPT species (C16:0, C18:2, C18:1, C18:0,
C20:4 and C22:6) (B) and LysoPS species (C16:0, C18:2, C18:1, C18:0, C20:4 and C22:6) (D) detected in the mouselarge intestine.

For the large-intestine sample, lipid extract corresponding to 10 mg of large-intestine tissue was injected. The retention times foreach LysoPT and LysoPS species were similar between the standard samples and large-intestine sample.

homogenates (Fig. 1B and 1D). Peaks with m/z values andretention time that correspond to C16:0-LysoPT, C18:0-Lys-

Method validation

oPT, and C18:1-LysoPT standards were detected in the mouselarge-intestine homogenates. Thus, we concluded that LysoPT

The linearity of the calibration curves was evaluated bydetermining the ion intensity of analytes in eight concentra-

Linearity

species with C16:0, C18:0, and C18:1 were present in mice.

tions (50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 50
141November 2022

Fig. 2.

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

Standard curves of LysoPT and LysoPS.
The curves were constructed by plotting the peak area ratio of the analytes at six concentrations (500 pM, 1 nM, 5 nM, 10
nM, 50 nM and 100 nM, n＝6, each) against the concentrations of the internal standard (C17:1-LysoPS). The precisions of
the method are shown in Table 1.

nM, and 100 nM). The calibration curves of C16:0-LysoPT,
C18:0-LysoPT, and C18:1-LysoPT were linear from 500

the internal standard (100 nM C17:1-LysoPS). Most intraand inter-day precision values were less than 10% (Table 1),

pM (5 fmol) to 100 nM (1000 fmol) (Fig. 2). Similarly,
C16:0-LysoPS, C18:0-LysoPS, and C18:1-LysoPS wereconﬁrming the accuracy and robustness of the present assay
method (Table 1).

detected with linearity from 500 pM (5 fmol) to 100 nM(1000 fmol) (Fig. 2). The present method enables us todetect both LysoPT and LysoPS at 20-fold lower concentra-

Recoverytions compared with the previous method. When we tested
50 pM (0.5 fmol), we could not detect C16:0-LysoPT,

ples with a known amount of LysoPT and determined theamount of recovered LysoPT by LC-MS/MS. The recover-C18:0-LysoPT, and C18:1-LysoPT (data not shown). At 100
pM (1 fmol), we could detect C16:0-LysoPT and C18:0-Ly-

ies were in the ranges 91.0‒119.7% for C16:0-LysoPT,
81.2‒122.8% for C18:0-LysoPT, and 91.7‒121.0% for

soPT with signal to noise ratio over 30 but could not detect
C18:1-LysoPT (data not shown). The linearity was

C18:1-LysoPT (Table 2).

observed from 500 pM and 100 nM for the three LysoPT

LysoPT and LysoPS distribution in mouse tissues

We examined the recovery of the assay by spiking sam-species (Fig. 2). Thus, the lower limit of quantiﬁcation

We then determined the distribution of LysoPT and Lys-

(LLOQ) and the lower limit of detection (LLOD) for
C16:0-LysoPT or C18:0-LysoPT is 100 pM and those foroPS in mice using the established method. Several LysoPT

C18:1-LysoPT is 500 pM.

and LysoPS species (C16:0, C18:0, C18:1, C18:2, C20:3,
C20:4, C20:5, and C22:6) species were detected in the sam-Assay precision

ples (brain, thymus, heart, lung, liver, spleen, kidney, stom-

tigated by determining the level of LysoPT and LysoPS inach, small intestine, large intestine, muscle, skin, plasma,and serum) (Fig. 3A and Table 3). Both saturated and unsat-

the standard solutions containing 500 pM, 1 nM, 5 nM, 10

urated LysoPT and LysoPS species were detected, indicat-

nM, 50 nM, and 100 nM LysoPT and LysoPS in addition to

ing that both phospholipase A1s and phospholipase A2s are

species (C16:0, C18:0, C18:1, C18:2, C20:4, and C22:6)

The intra- and inter-day precision of the assay was inves-

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Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

Table 1.

Precision of assays of each LysoPT and LysoPS species

Intra-day precision (%)
concentration (nM)fmol

LysoPT (16:0) LysoPT (18:0) LysoPT (18:1) LysoPS (16:0) LysoPS (18:0) LysoPS (18:1)

0.5

5

7.1

1.8

10.3

9.9

7.1

4.0

1

10

6.4

5.3

1.9

7.8

5.9

9.9

5

50

2.1

6.1

0.8

9.4

2.5

6.2

10

100

5.7

6.9

3.1

5.0

5.6

9.3

50

500

3.2

5.3

0.7

2.9

6.5

1.3

100

1000

4.1

3.8

2.4

0.7

2.6

0.6

Inter-day precision (%)
concentration (nM)fmol

LysoPT (16:0) LysoPT (18:0) LysoPT (18:1) LysoPS (16:0) LysoPS (18:0) LysoPS (18:1)

0.5

5

9.2

1.9

1.7

4.5

3.7

6.0

1

10

4.0

2.8

4.0

3.2

1.6

8.0

5

50

0.8

1.4

4.2

2.2

1.3

5.1

10

100

1.7

2.1

4.4

3.5

1.7

3.1

50

500

3.0

1.5

1.5

0.9

3.3

3.6

100

1000

5.0

0.9

1.9

2.3

2.5

2.1

Data are SD among intra-day or inter-day (n=6).

Table 2.

Extraction recovery of three LysoPT species from several tissues

Brain

Thymus

Heart

Lung

Liver

SpleenKidney Stomach Intestine Mascle

Skin

PlasmaLysoPT

50 nM

107.7

106.8

104.1

107.1

112.5

102.4

116.1

119.7

98.7

109.1

97.6

91.0

(16:0)

100 nM

101.0

98.9

107.7

109.8

110.9

104.8

113.6

106.4

98.4

111.5

105.0

103.2

LysoPT

50 nM

116.7

81.2

102.1

93.1

103.0

95.4

118.4

111.9

82.6

110.3

113.7

107.5

(18:0)

100 nM

113.6

86.1

95.5

84.2

102.8

92.9

109.4

115.2

107.1

109.0

122.8

94.0

LysoPT

50 nM

111.0

103.0

105.0

108.1

114.3

97.9

111.4

121.0

101.0

113.1

109.6

92.8

(18:1)

100 nM

106.6

101.0

103.9

107.5

91.7

104.1

112.9

111.4

114.2

120.2

112.0

108.9

Data are mean (n=3).

sues. For example, in the lung, spleen, kidney, small intestine, large intestine, and skin, the ratio of LysoPS to Lys-

involved in the production of LysoPT and LysoPS. LysoPT
and LysoPS levels were high in digestive tissues (stomach,large and small intestines) and kidneys. Patterns of LysoPT
species expression diﬀered between tissues (Fig. 3B). Lys-oPT was roughly 100:1, while in the heart, muscle, brain,oPT appeared to be most highly expressed in the stomach,followed by the large intestine. In these tissues, C18:0-Lys-

400:1, respectively.

oPT was the main LysoPT species (Fig. 3A and B).

(arachidonic (C20:4) and docosahexaenoic (C22:6) acids),

and liver/stomach, it was roughly 30:1, 60:1, 250:1 and
LysoPT species, especially with unsaturated fatty acids

Like LysoPT, LysoPS was detected in all tissues tested

were detected in the serum, while they were not detected in(Fig. 3B and C). Interestingly, the composition of LysoPS
and LysoPT species was similar in each organ. For exam-

the plasma (Fig. 3D), suggesting that they were produced
during the blood coagulation. LysoPS was detected in both
plasma and serum, with concentrations in serum approxi-

ple, C18:0-LysoPS and C18:0-LysoPT were the most abundant species in the stomach and kidney, followed by the
C16:0- and C18:1-species of LysoPS and LysoPT. In themately 50-fold higher than in plasma. The fact that LysoPTlarge intestine, C18:0-LysoPT and C18:0-LysoPS were thebelow the detection limit. Interestingly, the LysoPS species

most common molecular species. In contrast, the relative
concentrations of LysoPT and LysoPS varied among the tis-in serum and plasma diﬀered signiﬁcantly (Fig. 3D). In
plasma, C18:0-LysoPS were the main LysoPS species,

was not detected in plasma is likely because LysoPT was143

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Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

144

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Fig. 3.

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

Distribution of LysoPT and LysoPS in various mouse tissues.
(A) Concentrations of LysoPT (upper) and LysoPS (lower) species in tissues. (B and C) Concentrations of LysoPT (B)and LysoPS (C) species in A were shown for each tissue. (D) Concentrations of LysoPT and LysoPS species in plasmaand serum. The values are the means＋SD obtained from 3 mice. The values of the concentrations of each LysoPT and LysoPS species are shown in Table 3 (n＝3, values are means＋SD).

145

146

N.Q.

N.Q.

0.015±0.004

0.099±0.043

N.Q.

0.005±0.003

N.Q.

N.Q.

liver

spleenkidneystomachsmall-intestine

large-intestine

mascleskin

106.688±37.900

4.706±0.727

0.010±0.004

0.165±0.031

0.772±0.123

0.196±0.053

1.532±0.182

9.288±3.309

0.214±0.038

0.259±0.190

0.067±0.005

0.150±0.047

heart

lung

liver

spleenkidneystomachsmall-intestine

large-intestine

mascleskin

0.457±0.103

0.097±0.021

0.751±0.280

0.359±0.046

8.402±3.137

0.371±0.036

0.230±0.037

0.108±0.012

0.236±0.035

0.031±0.002

0.177±0.028

0.663±0.156

0.193±0.045

0.023±0.000

0.815±0.089

1.191±0.114

0.544±0.128

0.443±0.073

0.212±0.026

0.098±0.001

0.087±0.007

0.024±0.006

0.075±0.005

0.009±0.002

LysoPS (18:2)

N.Q.

N.D.

0.017±0.001

0.018±0.002

N.Q.

N.Q.

N.Q.

N.Q.

N.Q.

N.D.

N.Q.

N.D.

LysoPT (18:2)

0.042±0.016

0.006±0.001

0.240±0.071

0.152±0.016

0.046±0.004

0.113±0.018

0.061±0.008

0.048±0.008

0.066±0.001

0.008±0.002

0.068±0.008

0.025±0.011

LysoPS (20:3)

N.Q.

N.Q.

0.019±0.005

0.021±0.001

0.005±0.002

0.041±0.008

N.Q.

0.011±0.003

N.Q.

N.Q.

N.Q.

N.Q.

LysoPT (20:4)

0.139±0.026

0.019±0.002

0.354±0.095

0.361±0.028

0.281±0.015

2.485±0.442

0.342±0.012

1.032±0.232

0.198±0.015

0.033±0.010

0.117±0.004

0.130±0.026

LysoPS (20:4)

N.Q.

0.009±0.001

0.015±0.006

0.011±0.001

N.Q.

0.006±0.003

0.005±0.001

0.011±0.003

N.Q.

0.017±0.008

N.Q.

0.014±0.008

LysoPT (22:6)

Tissue concentrations of LysoPT and LysoPS species

LysoPS (18:1)

N.Q.

N.D.

0.012±0.005

N.D.

0.074±0.030

N.Q.

N.Q.

N.D.

N.Q.

N.D.

N.Q.

N.D.

LysoPT (18:1)

Data are mean±SD (n=3). NQ not quantiﬁed ND not detected.

2.161±0.454

0.614±0.178

10.557±9.512

5.740±2.019

1.545±0.344

2.548±0.402

1.029±0.264

0.377±0.099

1.088±0.193

0.108±0.020

1.133±0.287

0.089±0.012

thymus

LysoPS (18:0)

0.021±0.003

0.009±0.005

0.114±0.097

0.064±0.036

0.016±0.008

0.009±0.002

0.009±0.006

0.014±0.010

brain

LysoPS (16:0)

0.042±0.013

N.Q.

lung

Tissue0.406±0.196

N.D.

heart

0.006±0.002

N.D.

thymusN.Q.

LysoPT (18:0)

N.D.

LysoPT (16:0)

brain

TissueTable 3.

N.Q.

N.Q.

0.020±0.001

0.031±0.011

0.016±0.008

0.215±0.036

0.026±0.002

0.098±0.022

0.010±0.003

N.Q.

N.Q.

N.Q.

LysoPS (20:5)

0.064±0.007

0.020±0.002

0.098±0.060

0.077±0.008

0.442±0.188

0.079±0.011

0.102±0.016

0.054±0.006

0.091±0.012

0.018±0.004

0.064±0.005

0.021±0.010

LysoPS (22:5)

0.348±0.071

0.373±0.052

0.830±0.276

0.577±0.043

0.485±0.053

1.032±0.212

0.634±0.058

3.316±0.536

0.339±0.027

0.683±0.210

0.297±0.012

7.313±3.571

LysoPS (22:6)

November 2022
Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

November 2022

Medical Mass Spectrometry Lipidomics Vol. 6 No. 2

whereas in serum, C20:4- and C22:6-LysoPS were the main

vivo despite its much lower concentration. Recently, four

LysoPS species. This suggests that the synthetic pathways

GPCRs were identiﬁed for LysoPS, i.e., LPS1/GPR34,

of LysoPS (and possibly those of LysoPT) diﬀer between

LPS2/P2Y10,
15,16)LPS2 L/A630033H20Rik,

and

LPS3/

. However, LysoPT species (C16:0, C18:0 andserum and plasma.

GPR174

Discussion

C18:1) did not activate any of these receptors . Thus,
LysoPT is not a ligand for GPCR-type LysoPS receptors

6,16)

There has been no evidence for the in vivo presence of
LysoPT, since it was pharmacologically identiﬁed as a syn-and has a role in activating mast cell degranulation.
The present study also revealed that the molecular spe-

5,6)

. Our present results demonstratecies of LysoPT are similar to those of LysoPS. The onlythat LysoPT is present in several mouse tissues and thestructural diﬀerence between LysoPT and LysoPS is that
LysoPT has an additional methyl group attached to the ser-thetic LysoPS analogue

blood. We changed the MS instrument and optimized themethod for LysoPT analysis, which was also successfully
applied to the detection of LysoPS. In TSQ Quantiva sys-

ine residue of LysoPS. In addition, the tissue distributiontem (Thermo Fischer scientiﬁc), the lens instructed in thepatterns of LysoPT and LysoPS were quite similar (Fig. 3).
Thus, we speculate that they share similar synthetic path-ion source in front of Q1 improved the removal eﬃciencyof neutral molecules. As a result, the background was low-

ways. Interestingly, LysoPT species detected were found tohave a fatty acid at the sn-1 position as judged by the detec-

ered, and the signal-to-noise ratio was improved, enabling

13)
tion system for sn-1/sn-2 lysophospholipids . PT, the mostthe detection of LysoPT in biological samples. The LLOQ

likely precursor of LysoPT, is widely distributed in various
10‒12). These notions indicate that LysoPT is prospecies

was 100 pm for both LysoPT and LysoPS (except forC18:1-LysoPT LLOQ, which is 500 pm), which is 5‒20

duced from PT by phospholipase A2 like LysoPS from PS.

times more sensitive than the method of Koistinen KM et
14）
and 20‒100 times sensitive than our previousal.

In a biological membrane, PS is mainly located in the inner
leaﬂet of the plasma membrane. Thus, PT may show a simi-

13)

method . The previous methods were not sensitive enoughlar bilayer distribution pattern in biological membranes.

to detect LysoPS or LysoPT in biological ﬂuids such as

Further studies are required to understand the biologicalplasma and serum. The total LysoPS concentrations inplasma and serum were 10‒20 nM and 700‒800 nM,

signiﬁcance of LysoPTs. However, the analytical method

respectively. The recent identiﬁcation of four GPCRs spe15‒17)
has indicated that LysoPS is an emergciﬁc to LysoPSin vivo and in vitro.

described herein clearly shows that LysoPT is present both

ing lysophospholipid mediator with multiple biologicalroles like LPA and sphingosine 1-phosphate (S1P). Interest-

Acknowledgments

ingly, LPA also showed similar nM plasma concentration to(21gm0010004h9905) and KAKENHI (20K21379), both

This

18)

those of LysoPS , and the concentration is much lower

work

was

, providing further evidence that LysoPS

Conﬂict of Interest

behaves as a local lysophospholipid mediator. Higher levels

None to be declared.

16,17)

by

AMED-LEAP

for JA.

than the eﬀective concentration (EC50) values for LysoPS
receptors

supportedof LysoPT and LysoPS were detected in serum than inReferences

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