Shinshu Med J, 70⑵：91～100, 2022

Altered Serum Lysophosphatidylethanolamine Species Profile
in Patients with Autoimmune Pancreatitis
Makoto Yamaura and Hiroya Hidaka＊
Department of Biomedical Laboratory Science, School of Health Sciences, Shinshu University School of Medicine

Lysophosphatidylethanolamines (LPEs) are hydrolysis metabolites of polyunsaturated fatty acids and alkenylgroup-rich phosphatidylethanolamine (PE). It was reported that LPE functions as a lipid mediator in vivo and it
is involved in an inflammatory process of chronic autoimmune disease. LPE is present in not only cells but also
serum, but the pathophysiological role and metabolic details of serum LPE are not known. In this study, charac­terization of the serum LPE species in autoimmune pancreatitis (AIP) was performed by mass spectrometry.
Serum samples from patients with AIP (n＝7) and healthy subjects (n＝9) were examined for fatty acid composi­tions using gas chromatography mass spectrometry. LPE species were analyzed using matrix-assisted laser de­sorption/ionization-time of flight mass spectrometry. Blood lipid tests, cholesterol and triglyceride were almostin the criteria range in patients with AIP. Serum arachidonic acid (C20 : 4) levels were higher in patients with
AIP than in healthy subjects, but eicosapentaenoic acid (C20 : 5) and docosahexaenoic acid (C22 : 6) concentra­
tions were not significantly different. In patients with AIP, serum total LPE levels were lower than those inhealthy subjects, and LPE 20 : 4 levels were positively correlated with LPE 20 : 5 and LPE 22 : 6 levels. LPE
p16 : 0 (plasmalogen-type) level was negatively correlated with LPE 20 : 4, LPE 20 : 5 and LPE 22 : 6 levels. This
study showed that though serum lipids and the fatty acid compositions of patients with AIP resembled healthy
subjects, serum LPE level of patients were significantly low and some serum LPE species fluctuated in a cor­
related way. Shinshu Med J 70 : 91―100, 2022
(Received for publication September 10, 2021 ; accepted in revised form November 30, 2021)
Key words : autoimmune pancreatitis, lysophosphatidylethanolamine, plasmalogen, mass spectrometryⅠalkenylacyl-type (plasmalogen-type)8） (Fig. 1). Plas­

Introduction

malogen-type PE (PE-pl) bound an alkenyl group at

Phosphatidylethanolamine (PE) is found in a cellthe sn-1 position (a vinyl-ether bond) and includes

membrane and serum lipoprotein and occupies 17-

many polyunsaturated fatty acids (PUFAs) at the sn-2

1）

25 ％ of total phospholipid in mammalian cells . PE is

position9）. PE-pl is distributed through the whole ofassociated with the cell functions of various cellular

the body of the organism, particularly in cranial nerve

processes, namely the precursor of phosphatidylcho­

cellular tissue and inflammatory immunologic cells

line (PC) biosynthesis, autophagy and oxidative phos­(lymphocytes, macrophages and neutrophils)10）11）. PE

phorylation reaction2）-4）. It was reported that a disor­

has many kinds of molecular species by the combina­

der of PE metabolism is associated with chroniction of acyl group and alkenyl group. As an analysis

5）-7）

of molecular species, mass spectrometry is used, butdisease and an infection. PE can be a diacyl- or

the combinatorial molecular species of multiple sub­＊Corresponding author : Hiroya Hidaka
Department of Biomedical Laboratory Sciences, School of
Health Sciences, Shinshu University School of Medicine,
3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan
E-mail : hiroyan@shinshu-u.ac.jp

No. 2, 2022

stituents may be present in the same mass electriccharge ratio (m/z). Therefore, the analysis of PE spe­
cies is difficult to apply clinically due to its complexity.
Lysophosphatidylethanolamines (LPEs) are lyso91

Yamaura・Hidaka

LPE, the identification of molecular species by mass

PLA1

A

B

H2C-O-CO-R’
PLA2| Sn-1R”-OC-O-CH
O| Sn-2 ||H2C-O-P-O-CH2CH2NH2
Sn-3
|
OPLA1

PLA2 H2C-O-CH=CH-R’| Sn-1R”-OC-O-CH
O| Sn-2 ||H2C-O-P-O-CH
2CH2NH2
Sn-3|
O-

Fig. 1 Structures of diacyl (A) and alkenylacyl (B)phosphatidylethanoamine
R’ : saturated fatty acid (SFA), monounsaturated fatty
acid (MUFA) ; R” : poly unsaturated fatty acid (PUFA); sn-1, sn-2 and sn-3 glycerol position ; PLA1 : phospho­
lipase A1 ; PLA2 : phospholipase A2 ; dotted line : break­point of phospholipase A.

spectrometry is easier than the analysis of PE. It wasreported that LPE including the polyunsaturated
fatty acid provides anti-inflammatory effects17）. Re­
cently, it was reported that the decrease in serum
PE of NAFLD patients is associated with a decrease
in serum LPE18）. The serum LPE level may reflect
PE metabolism. However, a pathophysiological role ofserum LPE and the metabolic details are not known.
An analysis of the serum LPE species by liquid chro­
matography-tandem mass spectrometry (LC-MS/MS) was reported18）, but it is hard to handle opera­
tions. Matrix-assisted laser desorption/ionizationtime of flight mass spectrometry (MALDI-TOF MS)
is a simple analytical procedure for analysis of LPE
species.
Immunoglobulin4 related disease (IgG4-RD) is
characterized by high IgG4 serum levels as well as
growth, infiltration, and fibrosis of lymphocytes and
IgG4-positive plasma cells in organs throughout the
body. In organs such as the pancreas and bile ducts,
IgG4-RD manifests as chronic lymphoproliferative

type metabolites of PE by phospholipase A (PLA)

disease with fibrotic and mass lesions, hyperplastic

activity. LPE is an intercellular signaling molecule12）.

lesions, and cellular infiltration19）. Autoimmune pan­

It has been reported that LPEs are bioactive lipids increatitis (AIP) is an IgG4-RD, and its characteristics

an important process of life in cells such as an inflam­include obstructive jaundice, pancreatic calculi, duc­

matory cascade reaction and cell proliferation or celltus pancreatic stenosis, high serum IgG4 levels, and a13）

death . On the other hand, it was reported that pro­

distended spleen20）. The levels of the inflammatory

tracted and uncontrolled immune responses can leadmarker, such as serum C-reactive protein (CRP), areto chronic inflammation, irreparable tissue damages,

not always elevated in patients with AIP21）. The lyso­

and chronic disease14）. Uncontrolled immune responses

phospholipids are involved as bioactive lipids for an15）

also occur in many common autoimmune diseases .

inflammatory process of various chronic autoimmune

The endogenous bioactive lipids induce immunity indiseases, but the details are unknown. To the best of

an inflammatory process and adjust it and play an

our knowledge, no study has examined whether AIP

important role to set a limit.

is associated with a LPE metabolism disorder. In thisLPEs including acyl group (LPE-acyl) and alkenyl

study, we analyzed serum fatty acids using gas chro­groups (PE-plasmalogen ; LPE-pl) are generated by amatography mass spectrometry, and the serum LPE

response of the PLA to PE. LPEs including the PUFA

species from patients with AIP using MALDI-TOF

and alkenyl group are produced from PE primarilyMS.

by a response of phospholipase A1 (PLA1) and phos­Ⅱpholipase A2 (PLA2), respectively. Because the side
chain (acyl group or alkenyl group) of LPE is one, itＡMaterials and Methods

Chemical reagentsis a readily soluble molecule, and approximately sev­

Chloroform, methanol, dichloromethane, isopropanol,eral hundred ng/mL is present in human serum16）. In

and acetonitrile for HPLC were obtained from Wako

92

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Serum lysoPE species of AIP patients

Pure Chemical Industries, Ltd. (Osaka, Japan). Strata-

Lipid extract samples were applied to Strata-XL-

XL-AW tubes for solid-phase extraction (SPE) were

AW tubes equilibrated with DM/IP (9/1 v/v), andobtained from Phenomenex (Torrance, CA, USA).

the unbound fraction was eluted using 4 column vol­The matrix reagent, 9-aminoacridine (9-AA) andumes of DM/IP (6/1 v/v). Bound material was thenheptadecanoic acid standard (C17 : 0) were obtained

eluted using one column volume of methanol, and thefrom Sigma-Aldrich (St. Louis, MO, USA). The LPE-

samples were dried using a centrifugal concentrator.

C13 : 0 internal standard was obtained from Avanti

Ｆ

Solid-phase extraction (SPE) samples were dis­Polar Lipids, Inc. (Alabaster, AL, USA).
Ｂ

Study subjects

MALDI-TOF mass spectrometry

solved in 25 μL isopropanol/acetonitrile (IP/AN : 3/2

The study enrolled 9 healthy subjects (five malesv/v) ; then 25 μL 9-AA (10 mg/mL in IP/AN : 3/2 v/

and four females ; average age : 53.1±11.7 years old,v) was added. Following mixing, 0.25 μL was placed

mean±SD) and 7 patients with AIP (five males andin a sample well plate and allowed to dry naturally.

two females ; 69.1±10.6 years old). All patients with

Samples were then analyzed using a MALDI-TOF/

AIP had been on steroid drugs (prednisone : PSL at

TOF MS 5800 system (AB SCIEX, Framingham, MA,

1-5 mg/day in six patients, and Celestamine at 0.5

USA) in the negative ion mode. LPE-acyl and LPE-

mg/day in one patient) for treatment of AIP. Therepl peaks were detected in the region m/z 400-600.

were not dieting on using eicosapentaenoic acid (EPA)

The peak molecular species were determined using

drugs for patients with AIP. The blood glucose levelproduct ion analysis. MS data analysis was performedin three patients with AIP who had a past medical

by quantifying the peaks using image analysis soft­

history of diabetes mellitus was controlled to 121-131

ware (Data Explore, AB SCIEX).

mg/dL. The study protocol was approved by theＧ Gas chromatography mass spectrometry (GCEthical Review Board of Shinshu University Schoolof Medicine (approval number : No. 3954).
Ｃ

Preparation of serumMS)
Total FA in serum was analyzed using GC MS.
Serum (200 μL), the internal standard (C17 : 0 FA),

Participants fasted for 12 h, after which blood wasand a C/M mixed solution (2/1 v/v, 2 mL) were added

collected. For serum samples, blood was collected in

to a glass tube and mixed. Subsequently, 5 times thetubes containing a blood coagulation accelerant

quantity of water was added to the sample mixture.

(Venoject II, TERUMO, Tokyo, Japan). After 15 min

After centrifugal separation for 10 min at 2,500×g,

at room temperature, the samples were centrifuged

the lower layer liquid was dispensed, and the lipid

for 10 min at 2,500×g, and the supernatants (serum)

sample was dried. The lipid sample was subjected towere collected. Serum samples were stored at －80 ℃

trans-methyl esterification of all FAs using thefor a maximum of 1 week prior to analysis.

method described by Taketomi et al22）. The GC MS

Ｄ

device used was GC-MS-QP2010 (Shimadzu, Kyoto).

Lipid extraction
To a 200 μL sample, 10 μL LPE C13 : 0 internal stand­FA methyl esters were analysed using the tempera­ard (240 μmol/L) and 2 mL chloroform/methanol

ture rise method (70-280 ℃ ), with a cyanopropyl(C/M : 2/1 v/v) were added. The solution was mixed

polysilphenylene-siloxane GC capillary column

vigorously for 1 min, after which 400 μL distilled( B P X - 90 ; f i l m t h i c k n e s s : 0 . 25 μ m ; l e n g t h : 60

water was added. Following inversion mixing, them ; inside diameter : 0.25 mm ; SGE Analytical Sci­

sample was centrifuged for 10 min at 2,500×g. The

ence Pvt., Ltd., Melbourne, Australia).

bottom layer was separated and dried using a cen­Ｈ

Statistical analysis

trifugal concentrator. The resultant solid product was

Data are expressed as mean±standard deviation

dissolved in 600 μL dichloromethane/isopropanol (DM(SD). The Student’s t-test was used for a statistical/IP : 9/1 v/v) to prepare a lipid extract sample.

evaluation of significant difference between the twoＥgroups. Correlation was assessed by linear regression

Solid-phase extraction (SPE)No. 2, 2022

93

Yamaura・Hidaka
Table 1

Clinical characteristics of the patients with AIP and healthy subjects
Normal range Unit

Healthy subjects

years

53.1 ± 11.7

69.1 ± 10.6－
100.6 ± 27.8

23.7 ± 3.6
114.5 ± 10.6

Age
BMI

18.0 － 25.01）

GlucoseTC

73 － 109 (mg/dL)2）
142 － 248 (mg/dL)2）

AIP patietns

202.0 ± 26.9

184.1 ± 30.6

HDL-C

≧

40 (mg/dL)3）

66.6 ± 9.8

67.1 ± 18.2

LDL-C

≦ 140 (mg/dL)3）TG
CRP≦ 150 (mg/dL)3）≦ 0.14 (mg/dL)2）

115.4 ± 22.0
100.0 ± 36.8

90.6 ± 17.5
115.6 ± 40.3

＜ 0.100.090 ± 0.107

IgG4≦ 135 (mg/dL)2）

53.7 ± 24.6

493.9 ± 410.9

UA＜ 7.8 (mg/dL)2）

5.18 ± 1.28

6.01 ± 0.82

19.2 ± 4.5
15.7 ± 6.0

22.1 ±
18.6 ±

18.4 ± 5.1

19.1 ± 12.9

AST

13 －

30

(IU/L)2）

ALT
γ-GT

7－
9－

42
32

(IU/L)2）(IU/L)2）

6.5
8.1

1）

: Tanaka H, Kokubo Y : Epidemiology of Obesity in Japan. JMAJ 48(1) : 34-41, 2005. 2）: Japanese Committee
for Clinical Laboratory Standards (JCCLS) ; Common-Use Reference Intervals. 3）: Japan Atherosclerosis

Society (JAS) ; Guidelines for Prevention of Atherosclerotic Cardiovascular Diseases 2017. 4）: Japanese
association of IgG4-related disease ; Reference (2020). AIP : autoimmune pancreatitis, BMI : body-mass index,
TC : total cholesterol, HDL-C : HDL-cholesterol ; LDL-C : LDL-cholesterol, TG : triglyceride, CRP : C-reactive
protein ; IgG4 : immunoglobulin G4 isoform, UA : uric acid, AST : aspartate aminotransferase, ALT : alanine
aminotransferase, γ-GT : γ-glutamyl transpeptidase.

analysis. Statistical analysis was performed using StatＣ

We measured the LPE molecular species levels in

Flex Ver.6 software (Artech Co., Ltd, Osaka)
Ⅲ
Ａ

Results

Clinical laboratory data

Serum LPE species level

serum from patients with AIP and healthy subjects(Table 2). Patients with AIP showed lower serum
total LPE, LPE-pl, and LPE-acyl levels compared toTable 1 shows the clinical characteristics of thethose in healthy subjects. Furthermore, LPE species

seven patients with AIP and the nine healthy sub­analysis showed that patients with AIP had signifi­

jects. Except for one patient, the serum IgG4 levels

cantly lower levels of LPE p16 : 0, LPE p18 : 1, LPE

were 10 times higher (from 1.5) than the upper limit

p18 : 0, LPE 16 : 0, LPE 18 : 0, LPE 20 : 5, and LPEof the reference value. The lipid data, total cholesterol

20 : 4 than those of healthy subjects (Table 2).

(TC), high-density lipoprotein-cholesterol (HDL-C),

Ｄ

Correlation between LPE species

low-density lipoprotein-cholesterol (LDL-C), andWe examined the correlations between LPE species

triglyceride (TG) were within the normal range. Otherin serum from healthy subjects and patients with AIP.

clinical laboratory data were also within the normal

In healthy subjects, LPE 20 : 4 levels positively cor­

range.

related with LPE 20 : 5 levels (r＝0.67), but showed noＢcorrelation with LPE 22 : 6 levels (r＝0.12) (Fig. 3A).

Total FA compositions in serum
Serum total FA levels in patients with AIP and

In patients with AIP, there was a positive correlation

healthy subjects were measured using GCMS. AIP

between LPE 20 : 4 levels and LPE 20 : 5 and LPE

patient serum was found to have lower stearic acid

22 : 6 levels (r＝0.61 and 0.91, respectively) (Fig. 3B).

(SA ; C18 : 0) and higher arachidonic acid (ARA ; C20 :We also examined the correlations between the4) levels than those of healthy subjects (p＝0.048 andthree LPE-pls, LPE p16 : 0, LPE p18 : 1and LPEp≦ 0.001, respectively) (Fig. 2). Other serum fatty

p18 : 0, and LPE-PUFAs, LPE 20 : 4, LPE 20 : 5, andacids did not have a significant difference between

LPE 22 : 6, respectively. In healthy subjects, LPE

the two groups.

p16 : 0 levels positively correlated with LPE 22 : 6

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Serum lysoPE species of AIP patients

1,500

Concentration (mg/mL)

Healthy subjects
Patients with AIP

1,200
900
600
*

**

300
0

C16:0 C16:1 C18:0 C18:1 C18:2 C20:4 C20:5 C22:6
Fatty acid

Fig. 2 Concentration of total fatty acid in serum
Open column : healthy subjects ; closed column : patients with AIP. ＊p＜0.05 ; ＊＊p＜0.01.

Table 2Concentration of LPE species in serum

Healthy subjects

Patients with AIP

LPE

mean

SD

mean

SD

P-value

p16 : 0p18 : 1
p18 : 0
16 : 018 : 2
18 : 118 : 020 : 5
20 : 422 : 6

2.51
1.85
3.06
3.19
3.55
2.54
4.50
2.68
2.16
2.05

0.49
0.36
0.70
0.88
0.74
0.67
0.68
1.31
0.30
0.42

1.87
1.23
2.15
2.17
2.26
1.63
3.08
1.15
1.34
1.69

0.30
0.46
0.47
0.97
1.11
1.20
0.77
1.27
0.76
1.26

0.011
0.010
0.002
0.060
0.022
0.094
0.003
0.047
0.012
0.462

(μmol/ L)(μmol/ L)
LPE : lysophosphatidylethanolamine, plasmalogen-type : LPE p16 : 0, LPE p18 : 1 and LPE p18 : 0,acyl-type : LPE 16 : 0, LPE 18 : 2, LPE 18 : 1, LPE 18 : 0, LPE 20 : 5, LPE 20 : 4 and LPE 22 : 6.

levels (r＝0.60), but not with other LPE-PUFA levels

There was no correlation between LPE p18 : 0 and(Fig. 4A). LPE p18 : 1 and LPE p18 : 0 in healthy

other LPE-PUFA levels (Fig. 4F).

subjects did not have a correlation with other LPEPUFAs (Fig. 4B, C). In patients with AIP, LPE

Ⅳ

Discussion

p16 : 0 levels showed a strong negative correlation

This study showed that though serum lipids and thewith LPE 20 : 4, LPE 20 : 5, and LPE 22 : 6 levels (r＝fatty acid compositions of patients with AIP resem­

0.72, 0.63, and 0.68, respectively) (Fig. 4D). LPE p18 :bled healthy subjects, serum LPE levels were signifi­

1 level positively correlated with LPE 20 : 4 and LPE

cantly low and serum LPE species linked and fluctu­

22 : 6 levels (r＝0.59 and 0.55, respectively), but did

ated. LPE 20 : 4 was positively correlated with LPE

not correlate with LPE 20 : 5 levels (r＝0.26) (Fig. 4E).

20 : 5 and LPE 22 : 6, and LPE p16 : 0 (plasmalogen

No. 2, 2022

95

Yamaura・Hidaka

B 5.0○LPE 20:5 y= 2.9007x - 3.5930 r = 0.67
●LPE 22:6 y= 0.1683x +1.6887 r = 0.12

5.0

4.0

4.0

3.0

3.0

2.0

2.0

1.0

1.0
0.0

○ LPE 20:5 y= 1.0253x - 0.2166 r = 0.61
● LPE 22:6 y= 1.5214x - 0.3414 r = 0.91

LPE 20:5 and LPE 22:6 (μmol/L)

LPE 20:5 and LPE 22:6 (μmol/L)

A 6.0

1.5

2.0
2.5
3.0
LPE 20:4 (μmol/L)

3.5

0.0

0.0

1.0
2.0
LPE 20:4 (μmol/L)

3.0

Fig. 3 Correlation of LPE 20 : 4 versus LPE 20 : 5 and LPE 22 : 6 in serum of healthy subjects and patients with AIP
A : healthy subjects ; B : patients with AIP. Open circle (dotted line) : LPE 20 : 4 vs LPE 20 : 5 ; Closed circle (solidline) : LPE 20 : 4 vs LPE 22 : 6. LPE : lysophosphatidylethanolamine.

A

B

6.0

6.0

LPE-PUFA (μmol/L)

5.0△ LPE 20:4 y= 0.0813x + 1.9593 r = 0.13
○ LPE 20:5 y= - 0.0425x + 2.7900 r = 0.01
● LPE 22:6 y= 0.5252x + 0.7323 r = 0.60

5.0

C
6.0
△ LPE 20:4 y= 0.0436x + 2.0829 r = 0.05
○ LPE 20:5 y= 0.1150x + 2.4702 r = 0.03
● LPE 22:6 y= 0.5514x + 1.0309 r = 0.47

5.0

4.0

4.0

4.0

3.0

3.0

3.0

2.0

2.0

2.0

1.0

1.0

1.0

0.0

0.0

1.0

2.0
3.0
LPE p16:0 (μmol/L)

4.0

D

6.0

0.0

1.0
2.0
LPE p18:1 (μmol/L)

3.0

E

6.0

△ LPE 20:4 y= - 1.8074x + 4.7083 r = 0.72
○ LPE 20:5 y= - 2.6368x + 6.0722 r = 0.63
● LPE 22:6 y= - 2.8275x + 6.9667 r = 0.68

5.0

LPE-PUFA (μmol/L)

5.0

0.0

0.0

6.0
5.0

4.0

4.0

3.0

3.0

3.0

2.0

2.0

2.0

1.0

1.0

1.0

0.0

1.0
2.0
3.0
LPE p16:0 (μmol/L)

4.0

0.0

0.0

1.5

3.0
LPE p18:0 (μmol/L)

4.5

F△ LPE 20:4 y= 0.9780x + 0.1340 r = 0.59
○ LPE 20:5 y= 0.7294x + 0.2570 r = 0.26
● LPE 22:6 y= 1.4881x - 0.1377 r = 0.55

4.0

0.0

△ LPE 20:4 y= 0.0723x + 1.9452 r = 0.17
○ LPE 20:5 y= 0.1872x + 2.1109 r = 0.10
● LPE 22:6 y= 0.1948x + 1.4570 r = 0.32

0.0

1.0
2.0
LPE p18:1 (μmol/L)

3.0

0.0

△ LPE 20:4 y= - 0.1660x + 1.6942 r = 0.03
○ LPE 20:5 y= 0.2262x + 0.6684 r = 0.08
● LPE 22:6 y= - 0.5330x + 2.8390 r = 0.20

0.0

1.5

3.0
LPE p18:0 (μmol/L)

4.5

Fig. 4 Correlation between LPE-pl (plasmalogen-type) and LPE-PUFA species in serum of healthy
subjects and patients with AIP
A-C : healthy subjects ; D-F : patients with AIP. A, D : LPE p16 : 0 vs LPE-PUFA ; B, E : LPE p18 : 1 vs. LPEPUFA ; C, F : LPE p18 : 0 vs LPE-PUFA. Open triangle (dotted line) : LPE 20 : 4 ; open circle : LPE 20 : 5 (dashed dottedline) ; closed circle (solid line), LPE 22 : 6. LPE p16 : 0, LPE p16 : 1 and LPE p18 : 1. LPE-PUFA : LPE 20 : 4, LPE 20 : 5, andLPE 22 : 6.
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Serum lysoPE species of AIP patientstype) was negatively correlated with LPE-PUFAs. It

LPE decreased together in NASH patients18）. Serumwas reported that lysophospholipid functions as en­

LPE may provide an insight into PE metabolism.

dogenous bioactivity lipids for life activity in cells such

The lower LPE-pl (plasmalogen-type) levels in pa­

23）

as inflammatory cascade and cell death . The bioac­

tients with AIP may reflect lower PLA2 activity, andtive lipids are related to immune responses, and an

the lower LPE 20 : 4, LPE 20 : 5 and LPE 22 : 6 levels

association with common autoimmune disease is

may reflect lower PLA1 activity. Phospholipase iso­14）

found . It is reported that LPE-PUFA and LPE-plzyme, sPLA2-IID, is secreted by the dendritic cells(plasmalogen type) provide anti-inflammatory func­of lymph nodes, and shows high selectivity to PE25）.

tions17）. Water-soluble LPE is present in serum, but

It promotes selective hydrolysis of PUFA from PE ;pathophysiological roles and the metabolic details of

this has been reported to control inflammatory reac­

serum LPE are not known. The analysis of the serum

tions by suppressing the Th1 response of lymph

LPE species may contribute to explaining a mecha­

nodes26）. It is not clear whether PLA2 and PLA1 ac­nism of the lipid metabolism of patients with AIP.

tivities are mediated by the same or different enzyme

PEs are glycerophospholipids with two types,

proteins. PLA1, which can act upon PE, is intracellu­

diacyl-type and a plasmalogen-type. The former in­lar, and it has been found in cytosolic fractions andcludes two ester bonded fatty acid chains (acylmicrosomes27）. Pancreatic lipase (carboxylesterase) ofgroup) at sn-1 and sn-2 positions, and the latter in­

the pig pancreas and rat liver cytosol exhibit PLA1

cludes a vinyl-ether and an ester bond at the sn-1

activity, in addition to acylglycerol and carboxyles­

and sn-2 position, respectively. Particularly, PUFAs

terase activity28）. There are also phosphatidylserine

are included in sn-2 position of the PE24）. PUFAs and(PS)-specific PLA1 (PS-PLA1) activity and phos­

plasmalogens are known to play roles in an inflam­phatidic acid (PA)-selective PLA1α and PLA1β (PA-matory process. Because a difference of the side

PLA1α and PA-PLA1β) with only PLA1 activity29）.

chain influences the function of the PE, it is neces­

PS-PLA1 activity in stimulated rat platelets has beensary to identify molecular species. However, multiple

reported. Intracellular PLA1α and PLA1γ (iPLA1α

PE molecular species are present with the sameand iPLA1γ) develop by progress among intracellularmass electric charge ratio (m/z). It is difficult to

PLA1 (iPLA1) and exhibit PLA1 activity that uses

identify PE species using mass spectrometry. LPE is

phospholipids such as PE as substrates29）. However,a hydrolyzed metabolite by the phospholipase A

sufficient information on PLA1 for PE is lacking.

(PLA) of PE. LPE is isolated from a cell membrane

The LPE 20 : 4 concentrations of patients with AIP

easily and is present in serum ; the side chain of thecorrelated significantly with LPE 22 : 6 and LPE 20 : 5.

glycero-frame is one of the acyl group or alkenyl

However, LPE 22 : 6 did not correlate with the LPE

group. LPEs have an important function as bioactive

20 : 4 concentrations in healthy subjects. Furthermore,

lipids in an inflammatory process, but a pathophysio­

LPE 20 : 4, LPE 20 : 5 and LPE 22 : 6 of patients withlogical role of serum LPE and the metabolic details

AIP correlated negatively with LPE p16 : 0. These

are not known. Recently, the analysis of serum LPE

results suggested that the hydrolysis of LPE-PUFA

18）

but the clini­by PLA1 may be associated with the PLA2 which iscal application is difficult because the operation is

specific for LPE p16 : 0 generation in patients withcumbersome. MALDI-TOF MS is a simple and easy

AIP. It was suggested that the decrease in the plas­

analysis for LPE species. In this study, the character­malogen levels of the patients with AIP induces or

ization of the serum LPE species in autoimmune

worsens oxidative stress, because plasmalogen has

pancreatitis (AIP) was analyzed by MALDI-TOF

antioxidant properties30）. Kurano et al reported thatMS.

LPE-PUFA of patients with acute coronary syndrome

species by LC-MS/MS was reported

It was reported that the serum LPE level was as­

was increased31）. Arachidonyl-LPE (LPE 20 : 4) andsociated with a serum PE level, because the PE andLPE 22 : 6 by zymosan A-induced peritonitis model

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97

Yamaura・Hidaka

provide anti-inflammatory action17）, but the details

not the change of phospholipid molecular species isare not known. The change that LPE C20 : 4 and LPE

caused by a steroid, and it is a future problem.

C22 : 6 tuned to may be associated with anti-inflamma‑Ⅴ

tory action. Recently Yamamoto et al. reported thatConclusion

LPE molecular species decreased in NAFLD18）. It

This study showed that though serum lipids andwas reported that the inflammation of the liver inthe fatty acid compositions of patients with AIP re‑

NASH patients may increase lysophosphatidylcholine

sembled healthy subjects, serum LPE levels of pa‑

transferase three or four (respectively, LPCAT3, 4),tients were significantly lower and some serum LPE

expression of lysophosphatidylethanolamine trans­

species fluctuated in a correlated way. The analysis

ferase 1 (LPEAT1), and subsequently the serum LPEof the serum LPE species of MALDI-TOFMS maylevel was reduced. However, it is unknown about thecontribute to reveal the molecular mechanism andmetabolic mechanism of cells.

the pathophysiological role of the LPE metabolism in

A low dose of steroid (six patients : prednisone at 5

inflammatory disease.

mg/day, one patient ; Celestamine at 0.5 mg/day) was

Acknowledgements

given to patients with AIP. By the steroid therapy,side reaction of the disorder of lipid metabolism is

We would like to thank Emeritus Professor Ta‑

reported, but is primarily an increase of serum cho‑motsu Taketomi of Shinshu University for his valu‑

lesterol and the triglyceride. In the patients with AIP,

able advice and participation in the discussion.

the serum lipids were almost all within criteria rang‑

Conflict of interest

es. It was reported that the release of arachidonate isinhibited by steroid reagent32）. In this study, serum
ARA level of the patients with AIP was significantly

The authors declare that they have no conflicts ofinterest.

higher than in healthy subjects, and, as for serum

Funding

fatty acid composition of patients with AIP or other
PUFAs, there was no significant difference. The

This work was supported by JSPS KAKENHI Grant

effect of low dose steroid may be low in this study.

Number JP26460647 and 17K08980

However, we have not enough evidence whether orReferences
1） Vance JE : Phospholipid Synthesis and Transport in Mammalian Cells. Traffic 16 : 1-18, 2015
2） Menon AK, Stevens VL : Phosphatidylethanolamine is the donor of the ethanolamine residue linking a glycosylphos‑
phatidylinositol anchor to protein. J Biol Chem 267 : 15277-15280, 1992
3） Ichimura Y, Kirisako T, Takao T, et al : A ubiquitin-like system mediates protein lipidation. Nature 408 : 488-492, 2000
4） Böttinger L, Horvath SE, Kleinschroth T, et al : Phosphatidylethanolamine and cardiolipin differentially affect thestability of mitochondrial respiratory chain supercomplexes. J Mol Biol 423 : 677-686, 2012
5） Deleault NR, Piro JR, Walsh DJ, et al : Isolation of phosphatidylethanolamine as a solitary cofactor for prion formationin the absence of nucleic acids. Proc Natl Acad Sci USA 29 : 8546-8551, 20126） Nesic I, Guix FX, Vennekens K, et al : Alterations in phosphatidylethanolamine levels affect the generation of Aβ.
Aging Cell 11 : 63-72, 20127） Wang S, Zhang S, Liou LC, et al : Phosphatidylethanolamine deficiency disrupts alpha-synuclein homeostasis in yeast
and worm models of Parkinson disease. Proc Natl Acad Sci USA 111 : E3976-E3985, 20148） Wiesner P, Leidl K, Alfred Boettcher A, et al : Lipid profiling of FPLC-separated lipoprotein fractions by electrosprayionization tandem mass spectrometry. J Lipid Res 50 : 574-585, 20099） Braverman NE, Moser AB : Functions of plasmalogen lipids in health and disease. Biochimica et Biophysica Acta
98

Shinshu Med J

Vol. 70

Serum lysoPE species of AIP patients
1822 : 1442-1452, 2012
10） Dorninger F, Brodde A, Braverman NE, et al : Homeostasis of phospholipids - The level of phosphatidylethanolamine
tightly adapts to changes in ethanolamine plasmalogens. Biochim Biophys Acta 1851 : 117-128, 201511） Wallner S, Orso E, Grand M, et al : Phosphatidylcholine and phosphatidylethanolamine plasmalogens in lipid loaded
human macrophages. PLOS ONE 13(10) : doi.org/10.1371/journal.pone. 0205706, 201812） Lee LM, Park SJ, Im DS : Calcium signaling of lysophosphatidylethanolamine through LPA 1 in human SH-SY5Y
neuroblastoma cells. Biomol Ther 25 : 194-201, 201713） Caroline SB, Pamela JK, Michael HC, et al : Phospholipase A2-catalyzed hydrolysis of plasmalogen phospholipids inthrombin-stimulated human platelets. Thromb Res 120 : 259-268, 2007
14） Gilroy DW, Bishop-Bailey D : Lipid mediators in immune regulation and resolution. Br J Pharmacol 176 : 1009-1023,
201915） Chiurchiù V, Leuti A, Maccarrone M : Bioactive lipids and chronic inflammation : Managing the fire within. Front
Immunol 9 : doi : 10.3389/fimmu. 2018. 00038, 201816） Misra UK : Isolation of lysophosphatidylethanolamine from human serum. Biochimica et Biophysica Acta 106 : 371378, 196517） Hung ND, Kim MR, Sok DE : 2-Polyunsaturated acyl lysophosphatidylethanolamine attenuates inflammatory responsein zymosan A-induced peritonitis in mice. Lipids 46 : 893-906, 201118） Yamamoto Y, Sakurai1 T, Chen Z : Analysis of serum lysophosphatidylethanolamine levels in patients with nonalcoholic fatty liver disease by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 413 : 245-254,
201119） Della-Torre E, Lanzillotta M, Doglioni C : Immunology of IgG4-related disease. Clin Exp Immunol 181 : 191-206, 201520） Hamano H, Kawa S, Horiuchi A, et al : High serum IgG4 concentrations in patients with sclerosing pancreatitis. N
Engl J Med 344 : 732-738, 200121） Jungen T, Shaozhe C, Cong Y, Lingli D : Biomarkers in IgG4-related disease : A systematic review. Semin Arthritis
Rheum 50 : 354-359, 202022） Taketomi T, Hara A, Uemura K, et al : . A microwave-mediated saponification of galactosylceramide and galactosyl­
ceramide I3-sulfate and identification of their lyso-compounds by delayed extraction matrix-assisted laser desorption
ionization time-of-flight mass spectrometry. Biochem Biophys Res Commun 224 : 462-467, 199623） Sevastou I, Kaffe E, Mouratis, et al : Lysoglycerophospholipids in chronic inflammatory disorders : The PLA2/LPC
and ATX/LPA axes. Biochim Biophys Acta 1831 : 42-60, 201324） Brites P, Waterham HR, Wanders RJ : Functions and biosynthesis of plasmalogens in health and disease, Biochim
Biophys Acta 1636 : 219-231, 200425） Miki Y, Kidoguchi Y, Sato M, et al : Dual roles of group IID phospholipase A2 in inflammation and cancer. J Biol
Chem 291 : 15588-15601, 201626） Murakami M, Miki Y, Sato H, et al : Group IID, IIE, IIF and III secreted phospholipase A2s. Biochim Biophys Acta
Mol Cell Biol Lipids 1864 : 803-818, 201927） Arima N, Inoue A, Makide K, et al : Surface loops of extracellular phospholipase A1 determine both substratespecificity and preference for lysophospholipids. J Lipid Res 53 : 513-521, 201228） Xiao X, Lowe1 ME : The β5-loop and lid domain contribute to the substrate specificity of pancreatic lipase-related
protein 2 (PNLIPRP2). J Biol Chem 290 : 28847-28856, 201529） Morikawa RK, Aoki J, Kano F, et al : Intracellular phospholipase A1γ (iPLA1γ) is a novel factor involved in coatprotein complex I- and rab6-independent retrograde transport between the endoplasmic reticulum and the golgi
complex. J Biol Chem 284 : 26620-26630, 200930） Stadelmann-Ingrand S , Favreliere S, Fauconneau B, et al : Plasmalogen degradation by oxidative stress : production
No. 2, 2022

99

Yamaura・Hidaka
and disappearance of specific fatty aldehydes and fatty alpha-hydroxyaldehydes. Free Radic Biol Med 31 : 12631271, 200131） Kurano M, Suzuki A, Inoue A, et al : Possible Involvement of Minor Lysophospholipids in the increase in plasma
lysophosphatidic acid in acute coronary syndrome. Arter Thromb Vasc Biol 35 : 463-470, 201532） Lauritsen K, Laursen LS, Bukhave K et al : In vivo effects of orally administered prednisolone on prostaglandin andleucotriene production in ulcerative colitis. Gut 28 : 1095-1099, 1987

（2021. 9. 10 received ; 2021. 11. 30 accepted）

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