June 2020

Medical Mass Spectrometry Vol. 4 No. 1

Research Paper

Aging alters lipid mediator biosynthesis from polyunsaturated fatty acidsin the brain of senescence-accelerated micewith age-related cognitive dysfunction
1,3
1
1
1
Hideyuki Sasaki *, Yoshihisa Kaneda , Tomohiro Rogi , Hiroshi Shibata ,
2

3

3

2,3

Ryohei Aoyagi , Tsuyoshi Yamane , Kazutaka Ikeda , Makoto Arita
1

Institute for Health Care Science, Suntory Wellness Ltd., 8‒1‒1 Seikadai, Seika-cho, Soraku-gun, Kyoto 619‒0284, Japan
2
Division of Physiological Chemistry and Metabolism, Graduate School of Pharmaceutical Sciences, Keio University,1‒5‒30 Shibakoen, Minato-ku, Tokyo 105‒8512, Japan
3
Laboratory for Metabolomics, RIKEN Center for Integrative Medical Sciences,1‒7‒22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230‒0045, Japan

Abstract This study aimed to clarify whether long-chain polyunsaturated fatty acid (LCPUFA) metabolism in the brainthat causes an age-related decline of cognitive function changes with aging. The senescence-accelerated mice-prone 10(SAMP10) is an animal model used in studies related to aging. Young (two-month-old) mice and aged (nine-month-old)mice were fed control diet for 12 weeks. Age-related changes in lipid mediator proﬁles in the cortex and hippocampus wereassessed by comprehensive lipidomics, using liquid chromatography-tandem mass spectrometry after memory examination.
The expression of fatty acid metabolism-related mRNAs was evaluated in the hippocampus. Free arachidonic acid (ARA)
levels were extremely high among all detected free fatty acids, being approximately 8-fold higher than those of free DHA
in the cortex and hippocampus of both young and aged mice. Level of ARA-derived metabolites was extraordinarily highcompared to that of other PUFA-derived metabolites, whereas that of DHA-derived metabolites was very low in the cortex
and hippocampus of both mice. Aging decreased ARA-derived metabolites in the hippocampus, regardless of the biosynthetic pathway, such as cyclooxygenase, 5-lipoxygenase, 12/15-lipoxygenase, cytochrome P450, and non-enzymatic autoxidation. DHA-derived metabolites in the hippocampus were also decreased by age. There was no signiﬁcant diﬀerence inmRNA expression of the various enzymes related to metabolite biosynthesis between young and aged mice. The cognitive
function of SAMP10 was impaired due to aging. Based on analyzing a board range of LCPUFA-derived metabolites, we
propose that age-related decrease in LCPUFA metabolism in the hippocampus may be involved in the impaired cognitive
function in SAMP10 mice.
Key words: senescence-accelerated mice, polyunsaturated fatty acids, lipid mediator, brain

Introduction

Long-chain polyunsaturated fatty acids (LCPUFAs) are

the main constituents of biomembranes. They consist of
two series, namely n-6 and n-3. The major n-6 LCPUFA is* Corresponding author
Hideyuki Sasakiarachidonic acid (ARA), while the major n-3 LCPUFAs are

Institute for Health Care Science, Suntory Wellness Ltd., 8‒1‒
1 Seikadai, Seika-cho, Soraku-gun, Kyoto 619‒0284, Japan

docosahexaenoic acid (DHA) and eicosapentaenoic acid
(EPA). They have 20‒22 carbons with 4‒6 double bonds,

Tel: ＋81‒50‒3182‒0683, Fax: ＋81‒774‒98‒6262
E-mail: Hideyuki_Sasaki@suntory.co.jp

and play important roles in physiological homeostasis,

Received: February 27, 2020. Accepted: March 26, 2020.

including the modulation of membrane ﬂuidity, membrane

Epub May 21, 2020.

enzyme activities, and physiological responses

1,2)

.

Some of these responses are induced by the formation of

DOI: 10.24508/mms.2020.06.005
65

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Medical Mass Spectrometry Vol. 4 No. 1

lipid mediators from LCPUFAs themselves. These lipid

expression-associated metabolism of LCPUFA in the brain

mediators have been shown to exert a range of biologicaleﬀects. The 2-series prostaglandins and 4-series leukot-

of young and aged SAMP10 mice.

rienes, biosynthesized from ARA, are well-known lipid

Materials and Methods

mediators that are generally involved in the initiation ofAnimals and diets
All protocols for animal procedures were approved byacute inﬂammation. On the other hand, lipid mediators
derived from EPA and DHA, such as the 3-series prosta-

the Ethics Committee of Animal Experiment in accordance

glandins, 5-series leukotrienes, resolvins, protectin, andmaresin, exhibit anti-inﬂammatory eﬀects. The biosynthesis

with the Internal Regulations on Animal Experiments at
Suntory, which are based on the Law for the Humane Treat-of LCPUFA-derived lipid mediators is mainly evoked bycyclooxygenase (COX), lipoxygenase (LOX), and cyto-

ment and Management of Animals (Law No. 105, 1 Octo-

chrome P450 (CYP) enzymes. Lipid mediators are also partially produced through non-enzymatic autoxidation mecha-

were obtained from Japan SLC, Inc. (Shizuoka, Japan).

nisms

3,4)

ber 1973, as amended in 2 June 2017). Male SAMP10 mice
They were housed individually in polycarbonate cages withpaper bedding, which was changed once a week. The facil-. Recently, some studies have shown new

ity was maintained under speciﬁc pathogen-free conditions

physiological functions of several metabolites. For
instance, 18-hydroxyeicosapentaenoic acid derived fromat a temperature of 23±2°
C and humidity of 55±10%, witha 12 h light/dark cycle (light was switched on at 7:00).

EPA inhibits proinﬂammatory activation of cardiac ﬁbro5)
blasts , and DHA-derived protectin D1 (PD1) remarkably

Before the experiment, mice were acclimated to the facility

attenuates inﬂuenza virus replication and improves the sur-for more than a week. The mice had free access to waterand feed; we used the latter based on AIN-93M containing

6)

vival and pathology of severe inﬂuenza in mice .
LCPUFAs are the major components of brain phospho7)

4% fat with modiﬁed lipid compositions. The lipids con-8)

lipids and their levels in humans and animals have beenreported to decrease with aging. DHA/EPA or ARA supple-

sisted of palm oil, soybean oil, and linseed oil (SHOWAKOSAN CO., LTD., Minato-ku, Tokyo, Japan). The fatty

mentation has been shown to improve the cognitive func-acid compositions of the diet are shown in Table 1. All thediets were stored at 4°
C and changed twice a week to pre-9,10)tion in healthy elderly humans, and ameliorate the11,12).
Various mechanisms, such as synaptic plasticity, neurogen-vent oxidation. After more than 1 week of acclimation

esis, and membrane ﬂuidity have been shown to be associ-for 12 weeks, and we measured weight once a week andreduced cognitive function in aged animals as wellperiod, SAMP10 mice aged 2 or 9 months were fed the diet

2,11)

ated with this eﬀect in aged animals. A few metabolites,

observed general appearance twice a week.

such as PGE2 and PD1, have been shown to improve brain
13,14), though the relationship between lipid metabofunction

Gas–liquid chromatography (GC) for fatty acid composition

lites and brain function still remains unclear. Moreover,non-reported study has examined the changes in LCPUFA

Lipids in the diet were extracted and puriﬁed by themetabolite proﬁles in the brain with age-related impairmentof cognitive function.

Table 1.

The current study aimed to clarify changes in LCPUFA

Fatty acid composition of the diet

FA (AIN93M contained 4% fat)(%)

metabolism in the brain, which may be involved in an
age-related decline of cognitive function. SAMP10 strain

Palmitic acid (PA)was selected as a model of age-related neurodegeneration,
exhibiting age-related cerebral atrophy due to the loss andStearic acid (SA)

4.3

Oleic acid (OA)

31.2

perikaryal shrinkage of cortical neurons . Aged SAMP10

Linoleic acid (LA)α -Linolenic acid (ALA)

22.5

mice have a more deteriorated cognitive function thanDihomo- γ -linolenic acid (DGLA)

0.0

SAMR1 control mice. In the present study, we investigatedArachidonic acid (ARA)

0.0

137 molecular species of free fatty acids and metabolites bycomprehensive lipidomics systems using liquid chromatog-

Eicosapentaenoic acid (EPA)

0.0

Docosahexaenoic acid (DHA)

0.0

Others3.7

15)

raphy-tandem mass spectrometry (LC-MS/MS), and mRNA
66

26.9

11.3

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Medical Mass Spectrometry Vol. 4 No. 1

method of Folch et al . Phospholipids, which wereobtained using thin-layer chromatography (hexane : diethyl

d8, 15-HETE-d8, LTB4-d4, and PGE2-d4, respectively.

ether＝7 : 3), were methylesteriﬁed by incubating in methanolic HCl at 50°
C for 3 h, extracted with hexane, and sub-RNA sequencing

jected to capillary gas‒liquid chromatography (Agilent

ISOGEN (Nippon Gene, Tokyo, Japan) and puriﬁed using

7890B; Agilent Technologies, Santa Clara, CA) using an
SP-2330 column (30 m×0.32 mm×0.2μm; Supelco, Belle-

the RNeasy Mini kit (QIAGEN, Hilden, Germany) inaccordance with the manufacturer s instructions, as previ-

fonte, PA) with He (at 30 cm/s) as the carrier. The column

ously described . The integrity of isolated RNA was deter-

temperature was initially 180°
C for 2 min and thenincreased to 220°
C at a rate of 2°
C/min.

mined using a bioanalyzer (Agilent Technologies, Santa
Clara, CA, USA). RNA integrity numbers (RIN) of all sam-

LC-MS/MS analysis for lipidomics in brain samples

ples were identiﬁed higher than 8.6 and ﬁve samples wereselected in order of higher RIN value for library prepara-

16)

Total RNA was isolated from brain tissues using

19)

Brain tissues were homogenized on ice with methanol,tion and sequencing, for each young and aged mouse. The

and the extracted lipids were puriﬁed in solid-phase using
Sep-Pak C18 3 cc Vac cartridges (Waters) with deuteri-mRNA library preparation was based on SureSelectStrand-Speciﬁc RNA Library Prep for Illumina Multiplexedum-labeled internal standards (IS; AA-d8, 15-HETE-d8,
14,15-EET-d11, PGE2-d4, and LTB4-d4, each at a ﬁnal con-Sequencing (Agilent Technologies, version C.0, December2014). RNA-seq libraries were sequenced on a HiSeq 1500

pocampus of the aged mice could not be extracted lipids.

DNA Sequencer (Illumina, San Diego, CA, USA) in a
20)
50-base single-end read mode, as described previously .

centration of 10 pg/μL), excluding the sample that one hip-MS

The sequence reads were mapped to the mouse genome(NCBI version 37) using TopHat2 (version 2.0.8) and bot-

(QTRAP5500; SCIEX, Framingham, MA), as describedwie2 (version 2.1.0) with default parameters, and gene

Lipidomic analysis was performed using a UPLC system
coupledwithaquadrupole/linear

ion

trap

1,17)

previously . LC separation was performed using areversed-phase column [ACQUITY UPLC BEH C18 col-annotation was provided based on NCBI RefSeq. Transcript

umn (1.0×150 mm, 1.7μm particle size; Waters, Milford,

which was run with the same reference annotation asMA)]. Samples were eluted with a mobile phase composed

TopHat2 to generate FPKM (fragments per kilobase perof 0.1 vol% acetic acid in water and acetonitrile/methanol

million mapped reads) values for known gene models.

abundances were estimated using Cuﬄinks (version 2.1.1),(4 : 1, v/v) (73 : 27) for 5 min, and ramped to 30 : 70 after
15 min, to 20 : 80 after 25 min, and held there for 8 min,

Memory examination

ramping to 0 : 100 after 35 min, and held there for 10 min
with ﬂow rates of 70μL/min (0‒30 min), 80μL/min (30‒

We evaluated the cognitive function of mice from bothage groups using the Morris water maze test and the step-

33 min), and 100 μL/min (33‒45 min). MS/MS analyses

through test, which were modiﬁed from previously

were conducted in negative ion mode, and 137 molecular

described methods , after 12 weeks of feeding.

21)

species of free fatty acids and metabolites were identiﬁed

The Morris water maze pool consisted of a circular plas-(Supplement Table 1) and quantiﬁed by selected reaction

tic water tank, 90 cm in diameter and 60 cm in depth. The

monitoring (SRM). SRM transitions, declustering potential,

inside of the pool was painted black, and it was ﬁlled withentrance potential, collision energy, collision cell exit

water to a height of 40 cm at approximately 22°
C. The pool

potential, and retention time for all the analytes and internal

was divided into quadrants and a clear platform (8 cm in18)

standards are described previously . Compounds werediameter) was submerged 1 cm below the water level in one

quantiﬁed using stable internal standards. Extraction yieldand matrix recovery were determined using IS and quanti-of the pool quadrants; there were many cues in the internalpool, which could be used by the mouse for spatial localiza-

ﬁed with calibration curves. The lipid compounds weretion. These cues were held constant throughout the test.

grouped into 4 types, (group 1) free fatty acids, (group 2)

Mice were ﬁrst provided spatial learning trials for 4 days.
They were monitored via a video camera mounted over-monohydrides and epoxides, (group 3) diols, leukotrienes,
thromboxanes, lipoxines, and resolvins, and (group 4) pros-head, and the latency to escape onto the hidden platform

taglandins and others, and they were corrected with ARA-

was automatically recorded using a video tracking system
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Medical Mass Spectrometry Vol. 4 No. 1

in a trial. Each mouse was allowed to rest on the platform

Resultsfor 10 s after locating it. If it could not locate the platform

Free fatty acid levels

within 120 s, the trial was terminated, and researchers

We performed targeted lipidomic analysis on the cortex

placed it on the platform for 10 s. Mice received three trials

and hippocampus of young (5 months old) and aged

per day from diﬀerent start locations for a total of 4 days.

(12 months old) SAMP10 mice after intake of the control

After the ﬁnal training trial, mice were subjected to a single

diet for 12 weeks. There was no diﬀerence in body and120 s probe test on day 5. The platform was removed, andbrain weights between the young and aged mice (Table 2).
Levels of free PA, SA, and ARA were high in both the cor-time spent in target quadrant, the distance moved, andswimming speed were measured by a video tracking system. After a trial, the mouse was dried, and returned to its

tex and hippocampus of both the young and aged mice,compared with other FAs. Level of free ARA was approxi-

house cage.

mately 8-fold higher than that of free DHA. There was no

One day after Morris water maze test, the step-throughTable 2.

type passive avoidance test was carried out. The apparatus
consisted of two compartments separated by a sliding door.
One of the compartments was well-lit, and the other was

Body weight and brain weight of young and aged
micedarkened. The mouse was placed in the former compart-

Young (n＝8)
Mean±SE

Aged (n＝7)
Mean±SEment for 30 s. In an acquisition trial, the door to the dark

Body weight (g)29.7±0.927.3±1.2

compartment was opened, and the time before the mouse

Brain weight (mg)248±5246±10entered the dark compartment was recorded. Subsequently,

There was no signiﬁcant diﬀerence between the young and aged
mice for any of the variables (as per unpaired t-test).

the mouse was placed inside the dark compartment, thedoor was closed, and a slight foot-shock was delivered

Table 3.

(0.2 mA for 1 s). The mouse was then removed and returned
to its home cage. Next, in a retention trial, the mouse wasonce again placed in the well-lit compartment, and thepg/mgtissuelatency to re-enter the dark compartment was recorded up
to a maximum of 300 s. After each trial, the apparatus wascleaned.

Brain tissues

Free FA levels in the cortex and hippocampus
CortexHippocampus

Young (n＝8) Aged (n＝7)
Mean±SE
Mean±SE

Young (n＝8)
Mean±SE

Aged (n＝6)
Mean±SEPA

12164±952

11909±381

11501±712

11608±1417

POA

305±29264±18309±22

231±29SA

17235±822

17900±338

19998±678

OA

1347±871289±55

1816±125

19887±2326
1403±88*

LA

124±6

124±11173±10133±13*After we conducted two behavioral assays and the mice

ALA

ND

5±3

7±3

ND

were euthanized by exsanguination under anesthesia with

GLA

2±1

1±1

5±0

2±1

isoﬂurane, and brain tissues (cortex and hippocampus) wereimmediately harvested thereafter. Samples were immedi-

SDA

ND

ND

ND

ND

DGLA

66±7

52±4

106±14

89±14

MA

51±7

46±5

105±1295±8

ately stored at −80°
C until analysis.

Statistical analysis
All statistical analyses were performed using IBM SPSS
Statistics 25 (SPSS Inc., Chicago, IL, USA). Data areshown as the mean±standard error. All data were checked

ETA

ND

ND

ND

ND

ARA

10161±285

9561±487

14255±621

12988±611

EPA

40±4

43±8

64±6

48±5

AdA

115±8

122±11198±20154±17

DPA(n3)

33±3

33±4

52±5

40±4

DPA(n6)

15±2

15±1

22±3

22±4

1442±146

1380±156

1976±1621475±147

DHA

for normality of distribution and equality of variance using
the Shapiro‒Wilk test; homogeneity of variance was tested

Signiﬁcant diﬀerences were observed between the young and aged
mice (P＜0.05, *Mann‒Whitney test). PA, palmitic acid; POA,
palmitoleic acid; SA, stearic acid; OA, oleic acid; LA, linoleic
acid; ALA, α -linolenic acid; GLA, γ -linolenic acid; SDA, Stearidonic acid; DGLA, dihomo- γ -linolenic acid; MA, mead acid;
ETA, eicosatetraenoic acid; ARA, arachidonic acid; EPA, eicosapentaenoic acid; AdA, adrenic acid; DPA, docosapentaenoic acid;
DHA, docosahexaenoic acidusing Levene s test. Comparisons between the young andaged mice were performed using an unpaired Student s
t-test or a non-parametric Mann‒Whitney U test. For all
tests, P values less than 0.05 were considered statisticallysigniﬁcant.
68June 2020

Medical Mass Spectrometry Vol. 4 No. 1

diﬀerence in the levels of free fatty acids in the cortex

than in the young ones (Fig. 1B).

across the young and aged mice. Levels of free OA and LA
in the hippocampus were signiﬁcantly lower in the agedshows a comparison of the levels of ARA-derived metabolites across a variety of fatty-acid metabolizing

mice than in the young ones (Table 3).

enzymes or auto-oxidation. Levels of ARA-derived metabo-

Metabolite levels

lites in COX and CYP pathways in the cortex tended to belower in the aged mice (33.9±10.2 and 11.0±1.4 pg/mg tis-

A total of 40 metabolites, derived from LA, GLA,
DGLA, ARA, EPA, and DHA, were identiﬁed in the tar-sue, respectively) than in the young ones (43.5±4.4 pg/mg
tissue; Mann‒Whitney: P＝0.089 and 15.0±1.5 pg/mg tis-geted lipidomics. While the level of ARA-derived metabolites was high compared to that of other PUFA-derived

sue; unpaired t-test: P＝0.070, respectively). Moreover, lev-

Figure 2

els of these in the hippocampus were signiﬁcantly lower in
the aged mice (30.0±5.0 and 8.5±1.4 pg/mg tissue, respec-

metabolites, the level of n-3 PUFA-derived metabolites,such as DHA or EPA, was low in both the cortex and hippocampus. There was no diﬀerence in the levels of metabo-

tively) than in the young ones (52.1±8.5 pg/mg tissue;Mann‒Whitney: P＜0.05 and 17.4±1.9 pg/mg tissue;

lites in the cortex between the young and aged mice (Fig.
1A). Levels of ARA- and DHA-derived metabolites in theunpaired t-test: P＜0.05, respectively). Levels of ARA-derived metabolites in the 5-LOX and 12/15-LOX pathways

hippocampus were signiﬁcantly lower in the aged mice

were lower in the aged mice (21.3±1.5 and 92.1±8.5 pg/mg

Fig. 1.

Age-related changes in metabolite proﬁles in the cortex and hippocampus.
The total amounts of metabolites derived from ALA, EPA, DPA (n3), DHA, LA, GLA, DGLA, ARA, and DPA (n6) are presented.
Inset shows an enlargement of the small range. Data are presented as the mean±standard error (cortex (A); young mice (n＝8),
aged mice (n＝7), hippocampus (B); young mice (n＝8), aged mice (n＝6)). There was a signiﬁcant diﬀerence between the young#(Y) mice and aged (A) mice (P＜0.05, unpaired t-test; *Mann‒Whitney test). ALA, α -linolenic acid; EPA, eicosapentaenoic acid;
DPA, docosapentaenoic acid; DHA, docosahexaenoic acid; LA, linoleic acid; GLA, γ -linolenic acid; DGLA, dihomo- γ -linolenicacid; ARA, arachidonic acid69

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Medical Mass Spectrometry Vol. 4 No. 1

ARA-derived metabolites in non-enzymatic autoxidation
was lower in the aged mice (19.8±1.7 pg/mg tissue) than in
the young ones (31.1±2.3 pg/mg tissue; unpaired t-test: P＜0.05) in the hippocampus. Levels of that in the cortex were
not diﬀerent between the young (25.4±2.2 pg/mg tissue)
and the aged mice (22.9±3.1 pg/mg tissue).
A total of 19 metabolites derived from ARA were identiﬁed in both the cortex and hippocampus. Most of the
ARA-derived metabolites were signiﬁcantly lower in the
hippocampus of aged mice than in that of young ones, suchas thromboxane B2 (0.7±0.2 and 1.9±0.3 pg/mg tissue),
12-heptadecatrienoic acid (12.9±1.9 and 21.6±3.3 pg/mg
tissue), 5-hydroxyeicosatetraenoic acid (HETE; 11.7±0.8
and 15.9±1.0 pg/mg tissue), 5-oxo-eicosatetraenoic acid(oxo-ETE; 9.6±0.7 and 13.4±0.9 pg/mg tissue), 12-oxoETE (10.4±1.3 and 18.4±2.1 pg/mg tissue), 15-HETE (17.2±1.6 and 25.0±1.7 pg/mg tissue), 15-oxo-ETE (44.1±4.5
and 69.1±7.6 pg/mg tissue), Hepoxilin B3 (9.9±2.2 and17.1±1.9 pg/mg tissue), 8,9-epoxyeicosatrienoic acid (EET;

Fig. 2.

1.0±0.6 and 4.0±0.6 pg/mg tissue), 11,12-EET (3.1±0.3
and 5.2±0.5 pg/mg tissue), 14,15-EET (4.5±0.5 and 8.3±

Age-related changes in the levels of ARA-derived metabolites synthesized by enzymes and by
non-enzymatic autoxidation in the cortex and hippocampus.

0.9 pg/mg tissue), 11-HETE (7.3±0.7 and 11.6±1.0 pg/mg
tissue), and 16-HETE (1.0±0.2 and 1.8±0.1 pg/mg tissue).
There was no diﬀerence in ARA-derived metabolites in thecortex between the young and aged mice (Fig. 3, Supple-

The total amount of PGE2, PGD2, PGF2a, 15-keto-PGE2,
15-deoxy-PGJ2, 6-keto-PGF1a, TXB2, and 12-HHTrE
metabolized by COX (A). The total amount of 5,6-DHT,
8,9-DHT, 11,12-DHT, 14,15-DHT, 8,9-EET, 11,12-EET,
and 14,15-EET metabolized by CYP (B). The total amountof LTB4, LTB4-20OH, LTD4, 5-HETE, and 5-oxo-ETE
metabolized by 5LO (C). The total amount of LXA4,
LXB4, 5,15-diHETE, 8,15-diHETE, HxA3, HxB3, 12HETE, 15-HETE, 12-oxo-ETE, and 15-oxo-ETE metabolized by 12/15LO (D). The total amount of 8-HETE,
9-HETE, 11-HETE, 16-HETE, 17-HETE, 18-HETE, 19HETE, and 20-HETE synthesized by non-enzymatic
autooxidation (E). Data are presented as the mean±
standard error (cortex; young mice (n＝8), aged mice (n＝7), hippocampus; young mice (n＝8), aged mice (n＝6)).
There was a signiﬁcant diﬀerence between the young#
mice and aged mice (P＜0.05, unpaired t-test; *Mann‒
Whitney test). COX, cyclooxygenase; LO, lipoxygenase;
CYP, cytochrome P450

ment Table 2). The typical LC-MS/MS chromatograms of
ARA-derived metabolites, which changed in aging, areshown in Supplement Fig. 1.

mRNA expression of metabolism-related enzymes

The expression of mRNAs related to fatty acid-metabo-lizing enzymes is shown in Fig. 4. There was hardly any
diﬀerence in mRNA expression between the young andaged mice, excluding four mRNAs. Gpx4, Cyp2d22, andCyp2d26 mRNAs were signiﬁcantly higher in the aged
mice than in the young ones, whereas Agpat5 mRNA wassigniﬁcantly lower in the aged mice than in the young ones.
The rates of change were all small.

Evaluation of behavior
tissue, respectively) than in the young ones (29.4±1.9 and
143.2±11.1 pg/mg tissue; unpaired t-test: P＜0.05, respec-

escape latency to ﬁnd the submerged platform was signiﬁ-

We ﬁrst performed the Morris Water maze test. The

tively) in the hippocampus. Levels of these in the cortex

cantly higher in the aged mice than in the young ones

were not diﬀerent between the young (22.5±1.7 and 179.0

during the training days, excluding day 3 (Fig. 5A). In±9.6 pg/mg tissue, respectively) and the aged mice (21.3±

probe trial, the durations spent in the target quadrant were

1.8 and 165.4±17.1 pg/mg tissue, respectively). Levels of

37.9±9.8 and 20.7±10.3 s in the young and aged mice,70June 2020

Fig. 3.

Medical Mass Spectrometry Vol. 4 No. 1

Age-related changes in the levels of metabolites derived from ARA in the cortex and hippocampus.
Data are presented as the mean±standard error (cortex; young mice (n＝8), aged mice (n＝7), hippocampus; young mice (n＝8),
aged mice (n＝6)). There was a signiﬁcant diﬀerence between the young and aged mice (P＜0.05, *Mann‒Whitney test). COX, cyclooxygenase; LO, lipoxygenase; CYP, cytochrome P450

respectively, which did not diﬀer signiﬁcantly (Fig. 5B).

one-third and two-thirds of that in the young mice, respec-

The swimming speed, except in the target quadrant, did not
diﬀer across the young and aged mice (Fig. 5C). The dis-

tively. As far as we know, this is the ﬁrst study quantifying
the decrease in DHA- and ARA-derived metabolites in thetance moved was signiﬁcantly shorter in the aged mice thanin the young ones (Fig. 5D). We next performed the step-

hippocampus with age by a broad-targeted analysis ofthrough test. In acquisition trial, there was no diﬀerence inlatency time between the young mice (17±3 s) and aged

Metabolism of n-6 PUFA may occur selectively, since thelevels of n-6 free fatty acids, such as ARA, were higher

ones (14±3 s). In retention trial, however, the latency timethan those of n-3 free fatty acids in both the young mice

was lower in the aged mice (200±42 s) than in the young
ones (288±13 s, Mann‒Whitney, P＜0.05; Fig. 5E).

and the aged ones. The levels of free ARA were clearly

metabolites.

higher than those of other PUFAs (Table 3), and various
metabolites derived from ARA were produced in the brainof SAMP10 mice (Fig. 3). ARA-derived metabolites wereDiscussion

In the present study, the levels of metabolites biosynthesized from various fatty acids clearly indicated the diﬀer-

generated regardless of the type of enzymes, and metabo-ence in the metabolism of LCPUFAs, such as DHA anddetected in previous studies; PGE2 , LXA4, LTB4 , and23)
12-HETE were identiﬁed in the brain tissue. Moreover, inlites derived from nonenzymatic oxidation were also
14)

ARA, in the hippocampus of young and aged SAMP10
mice, but not in the cortex (Fig. 1). The results quantita-

22)

the cortex of SAMP8 mouse, COX metabolites, LOX

tively conﬁrmed the metabolic levels related to DHA andmetabolites, and products of nonenzymatic oxidation were

ARA in the hippocampus of aged mice to be approximately

detected in a previous study . Several metabolites play

24)

71

June 2020

Fig. 4.

Medical Mass Spectrometry Vol. 4 No. 1

Age-related changes in mRNA expression associated with fatty acid metabolizing enzymes in the hippocampus.

Data are presented as the mean of fold change (young mice (n＝5), aged mice (n＝5)). Signiﬁcant diﬀerences were observed between the young mice and aged mice (P＜0.05, *unpaired t-test).

pus or in the cortex (Fig. 1, Supplement Table 3). The cor-

important roles in neural function, such as spatial learning,
14)
synaptic plasticity (PGE2) , and resolution of inﬂamma-

tex and hippocampus are interconnected areas of the brain,

14)

tion (LXA4) , and act as second messengers in synaptic
25,26)
transmission and neuroprotection (12-HETE, 15-HETE).

supporting basic cognitive functions that are important for
the formation and retrieval of declarative memories. Metab-

However, whether other metabolites derived from ARA

olism of LCPUFA in the brain is probably aﬀected byplay roles in cognitive function remains unclear.
ARA-derived metabolites were synthesized by eachenzyme, such as COX, 5-LOX, 12/15-LOX, and CYP, asaging, especially in the hippocampus. A previous study
demonstrated no diﬀerence in 5-HETE, 8-HETE, 9-HETE,

well as by non-enzymatic autoxidation, in both the cortex
and hippocampus. Levels of these metabolites in the hippo-

and aged mice, and PGE2 and TXB2 in the cortex of aged

11-HETE, 15-HETE, PGF2a, and PGD2 between the young
SAMP8 mice were higher than those in young SAMP8
24)
mice . Overexpression of amyloid-beta (A β ) was

campus decreased with age, regardless of the biosynthetic
pathway, and the metabolites from COX or CYP in the cor-

27)

observed in the aged SAMP8 mice , and SAMP10 mouseserved as a unique model of age-dependence with inherited

tex also decreased with age (Fig. 2). Although the levels of
ARA-derived metabolites in the hippocampus of aged mice

cerebral atrophy and cognitive dysfunction . The mecha15)

were signiﬁcantly lower, they did not change with age inthe cortex (Fig. 3). The metabolism of LA, as an n-6 PUFA,

nism of aging might be diﬀerent between SAMP10 and

did not show a decrease with age, either in the hippocam-

increase in A β accumulation in the brain. Future studies are

SAMP8, for example, depending on whether there is an72June 2020

Fig. 5.

Medical Mass Spectrometry Vol. 4 No. 1

Age-related changes in Morris water maze test and passive avoidance response test in SAMP10.
Morris water maze test: escape latency to ﬁnd the hidden platform per group in three trials over 4 consecutive days (A). Time spent
in target quadrant (B), swimming speed (C), and distance moved (D) in the probe test. The step-through type passive avoidance
test: The latency time to enter the dark chamber in an acquisition trial or in a retention trial (E). Data are presented as the mean±
standard error (young mice (n＝8), aged mice (n＝7)). There was a signiﬁcant diﬀerence between the young and aged mice (P＜#0.05, unpaired t-test; *Mann‒Whitney test).

expected to reveal the eﬀects of aging on the metabolism of
LCPUFA in the brain of the wildtype mouse and/or othermembrane by maintaining its distribution in phospholipids.
13)
29)
DHA-derived metabolites, such as PD1 and Maresin ,species.

have also been reported to be useful in maintaining brain

The greater decrease in DHA-derived metabolite biosyn-

function; however, they were under the detection limit in
the resting state of the hippocampus and cortex. The pres-thesis in aged hippocampus might be inﬂuenced by the very

ence of a small amount of DHA-derived metabolite mightless free DHA available in the brain. The level of free DHA
was approximately one-eighth-fold lower than that of free
ARA, although both DHA and ARA are the main fatty acidhave an eﬀect on cognitive function and the decrease inbiosynthesis of these metabolites with age in the hippocam-

components in the brain. In the human orbitofrontal cortex,

pus might aﬀect brain function.

compositions of DHA and ARA in phospholipids wereSince LCPUFA metabolism became less active in theaged hippocampus, mRNA expression levels of LCPU-

7)

approximately 15% and 10%, respectively . Moreover,FA-metabolizing enzymes were evaluated by comprehensive transcriptome analysis. The results showed no signiﬁ-

compositions of DHA and ARA in the hippocampus of rat
8)

have been shown to be approximately 15% and 13.5% ,whereas those in the brain of SAMP8 mouse were 19.3%
21)
and 10.8%, respectively . In this study, fatty acid composi-cant diﬀerence in mRNA expression across various

tion in the hippocampus could not be tested owing to thelimited volume of tissue; however, in the previous study,

four mRNAs (Fig. 4). Decreased speciﬁc enzyme activity
or abnormal translation of enzyme genes in the hippocam-

we did conﬁrm their composition in the hippocampus ofpus may occur by aging. Thus, the molecular mechanisms

28)

SAMP10 mouse to be similar to that in SAMP8 mouse .

underlying downregulated LCPUFA metabolism in theThus, DHA is the most abundant LCPUFA in the brain,

aged hippocampus still remain to be elucidated.

enzymes related to metabolite biosynthesis, excluding only

probably implying its important role as a component of cell

Cognitive function in aged SAMP10 mice was impaired73June 2020

Medical Mass Spectrometry Vol. 4 No. 1

Conﬂict of Interest

compared to that in young mice, since the latency of aged
mice was lower than that of young mice in the step-throughAll other authors have no conﬂicts of interest.

test (Fig. 5). This result was in reasonable agreement with aprevious study . On the other hand, the result of Morris

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