June 2018

Medical Mass Spectrometry Vol. 2 No. 1

Research Paper

Development of targeted metabolomics for the determination ofornithine cycle compounds as possible biomarkers in cerebrospinal ﬂuid
regarding to Alzheimerʼs disease pathology using UHPLC-ESI-MS/MS
1,2

1

1,2

Haruhito Tsutsui , Yoshio Muguruma , Takumi Noda ,
3,4

5

1,

Hiroyasu Akatsu , Noriyuki Matsukawa , Koichi Inoue *1

Laboratory of Clinical & Analytical Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University,1‒1‒1 Nojihigashi, Kusatsu, Shiga 525‒8577, Japan
2
ONO Pharmaceutical Co., Ltd., 3‒1‒1 Sakurai, Shimamoto-cho, Mishima-gun, Osaka 618‒8585, Japan3
Department of Medicine for Aging Place, Community Health Care/Community-Based Medical Education,
Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467‒0001, Japan
4
Department of Neuropathology, Choju Medical Institute, Fukushimura Hospital, Toyohashi, Aichi 441‒8124, Japan
5
Department of Neurology, Nagoya City University, Graduate School of Medical Sciences, Nagoya, Aichi 467‒0001, Japan
Abstract

An early diagnosis of Alzheimer s disease can lead to better and more targeted treatment and/or prevention forpatients. Employing a pathological diagnosis by non-targeted metabolomics with ultra high performance liquid chromatography-electrospray tandem mass spectrometry, in our previous study, we found signiﬁcant changes in polyamine metabolites arising from the ornithine cycle in human brain samples of patients with Alzheimer s disease. In this study, we develop
a targeted metabolomics methodology for the separation and detection of compounds derived from the ornithine cycle inhuman cerebrospinal ﬂuid samples. For the targeted metabolomics, two derivatization reagents (9-ﬂuorenylmethyl chloroformate and 4-(N,N-dimethylaminosulfonyl)-7-ﬂuoro-2,1,3-benzoxadiazole) are utilized for sensitive and accurate determination of amine analytes. Validation tests conﬁrm a good linearity of r ＞0.99 or more in all calibration curves obtained us2

ing internal standards. The developed method is applied for the analysis of preliminary 15 metabolites including polyamine
metabolites in cerebrospinal ﬂuid samples classiﬁed according to brain autopsy Braak stages regarding to Alzheimer s disease and compared to a control samples. Utilizing the results obtained for the ornithine cycle, we expect to ﬁnd a metabolic
pathway that would be used as a potential biomarker in the future.
Key words: targeted metabolomics, liquid chromatography tandem mass spectrometry, Alzheimer s disease, ornithine cycle,
cerebrospinal ﬂuid

Introduction

ory, orientation and behavioral disturbances. The classicalAlzheimer s disease (AD) is a disorder that is seen frequently in the elderly and presents with symptoms of mem-

hallmarks of AD are increased presence of senile plaques,composed mainly of amyloid β (Aβ), and abnormal phos1)

phorylation of the tau protein in the brain tissue . The AD*Corresponding author

pathology has shown that accumulation of tau aggregates

Koichi Inoueand deposition of Aβ cause neuronal cell death, resulting inLaboratory of Clinical & Analytical Chemistry, College of
Pharmaceutical Sciences, Ritsumeikan University, 1‒1‒1 Noji-damage to higher brain functions/process referred to as the2)amyloid cascade hypothesis . In recent years, clinical tri-higashi, Kusatsu, Shiga 525‒8577, Japan
E-mail: kinoue@fc.ritsumei.ac.jp

Received June 29, 2017. Accepted November 29, 2017.

als targeting new drugs to address the amyloid cascade
hypothesis such as anti-Aβ antibodies and β-secretase

Epub March 7, 2018.

inhibitor have continued to fail

DOI: 10.24508/mms.2018.06.001

tance of clinical treatment facilitated by early diagnosis has
11

3‒7)

. Meanwhile, the impor-June 2018

Medical Mass Spectrometry Vol. 2 No. 1

been highlighted, with particular importance given to useful

Material and Methods

biomarkers that enable deﬁnitive prenatal diagnosis over

Materials. Interview scores, imaging, and biochemical

Overall, 15 analytes were used. Spermidine (SPD), sper-

methods have been reported as pathological indicators of

the world

8,9)

AD in clinical diagnosis . Cerebrospinal ﬂuid (CSF) ismine (SPM), putrescine (PUT), diaminopropane (DAP),
1
1
8
N -acetyl-spermidine (N -Ac-SPD), N -acetyl-spermidine

also employed as a diagnostic tool that reﬂects biochemical
11)changes in the nervous system . Already, many research-8
1 8
N N -diacetyl-spermidine(DAc-SPD),(N -Ac-SPD),1
1 12
N -acetyl-spermine (Ac-SPM), N N -diacetyl-spermine

ers have investigated CSF biomarkers for the diagnosis of(DAc-SPM), arginine (Arg), citrulline (Cit), and aspartate

10)

12)

the onset of AD . CSF metabolites in AD patients havebeen reported to change as early as several decades before(Asp) were purchased from Wako Pure Chemical Co.
1(Osaka, Japan); ornithine (ORN) and N -acetyl-putrescine

the appearance of dementia symptoms, and therefore, these(Ac-PUT) were purchased from Tokyo Kasei Co. (Tokyo,

markers have attracted considerable attention as potential
diagnostic markers for AD and mild cognitive impair-Japan); and cadaverine (CAD) was purchased from Kanto
Chemicals Co. (Tokyo, Japan). For internal standards (IS),13
13SPD-d8, SPM-d8, aspartate- C, arginine- C6, citrulline-d4

13,14)

ment.

and ornithine-d7 were obtained from Sigma-Aldrich Co. (St.
Louis, MO), and 1,6-diaminohexane (DAH) was purchased

Polyamines are physiologically active amines, present
widely in the world of biological organisms, from viruses

from Kanto Chemicals Co.. For derivatization, 9-ﬂuorenylmethyl chloroformate (F-MOC) was obtained from Wako

to humans. Polyamines promote primarily protein nucleic
acid synthesis by interacting with nucleic acids, especially15‒18)

RNA, and function as cell growth factors

Pure Chemical Co.. 4-(N, N-Dimethylaminosulfonyl)-7-ﬂuoro-2, 1, 3-benzoxadiazole (DBD-F) was obtained from. Polyamines

are also involved as modulators of biological function

Tokyo Kasei Co.. HPLC-grade water, acetonitrile and for-through interaction with glycosamine glycans such as
19)
N-methyl-D-aspartate receptor and heparin . In our previ-

mic acid (FA, for HPLC 99%) were obtained from Wako
Pure Chemical Co.. Sodium tetraborate (Borax) was pur-ous study, we detected that spermidine (SPD) and spermine(SPM) are over-expressed in brain tissue affected by ADchased from Kanto Chemicals Co.. Puriﬁed water wasusing a non-targeted metabolomics analysis of postmortem

obtained using a PURELAB ﬂex 5 system from ELGA Co(Woodridge, IL). Stock solutions were adjusted using aceto-brains of AD patients . We also found results that sug20)

gested activation of ornithine decarboxylase (ODC) by
highly sensitive determination of ornithine (ORN), putres-

nitrile/water (50 : 50, v/v).

cine, SPD, SPM, and their acetylated forms. In addition,

Human sample

Postmortem human tissues and CSF samples were col-immunostaining of brain tissue sections conﬁrmed thatODC was over-expressed in brains of AD patients whenlected from deceased bodies at the Choju Medical Institute
Fukushimura Hospital. We obtained written informed con-compared to non-AD patients . Therefore, the polyamines
regarding to ornithine cycle in central nervous system tis20)

sent for autopsy and the permission to use the obtainedsue such as CSF would be possible to use the preliminary

results for diagnosis, research, and genetic analysis fromthe patients guardians. The utilized tissues and CSF sam-biomarkers. On the other hands, there are few studies toinvestigate the relationship of polyamines and anticipatedAD pathology with CSF. In this study, we develop the tar-ples from the Fukushimura Brain Bank were used for accu-

geted metabolomic (auxiliary 15 analytes in ornithine
cycle) using derivatized ultra high performance liquid chro-

The volunteer s tissues and CSF samples were conﬁned tomatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) and measure these analytes inplaques/tangles, and the patients had been diagnosed with
gradual pre-mortem memory loss. The CSF samples wereconclusively diagnostic SCF based on AD pathology. On

dispensed into several tube, stored at −80°
C, and employed

the basis of this cause effect behavior of the ornithine cycle

for analysis after gaining approval from the authorities,

in CSF, our evidence will hopefully provide next trials.

including the Ritsumeikan University ethics panel. On the21)rate, reliable, and detailed pathological evaluation of AD .
speciﬁc brain region that is played visual pathology ofbasis of the pathological examinations, the CSF samples
were classiﬁed into AD and control groups. These data are12June 2018

Medical Mass Spectrometry Vol. 2 No. 1

Table 1.
GroupsAge

Sex

Control

71

Female

86

Male

76
96
A

B

C

Characterization of the AD and Control study groups
Pathological diagnosis

NFT braak stage

Grain stage

stroke, α -synuclein（̶）
MI, LB（̶）

I

（̶）

I

I

Male

MI, Bilateral Inferior olivary nuclear pseudohypertrophy,α-synuclein
（̶）

I

（̶）

Male

MI, arteriosclerosis, LB（̶）

II‒III

82

Male

Dropping of cerebellar Purkinje cells, LB（̶）

I

（̶）
（̶）

88

Female

I

77

Male

MI,α-synuclein（̶）
stroke,α-synuclein（̶），CAA (light)

85

Male

I

95

FemaleMI, Cerebellar infarction, CAA (heavy)
MI, CAA (light), α -synuclein（̶）

I

（̶）
（̶）

86

Male

MI, brain arteriosclerosis, LB（̶），CAA（̶）

I

（̶）

71

Male

I

78

Male

LB（̶）
，CAA, Cortical microinfarction
MI, Willis ring arteriosclerosis,α-synuclein（̶），TDP-43（̶）

（̶）
（̶）

84

Female90

FemaleWhite thin arteriosclerosis
LB（̶），Mild compression of the cervical spinal cord

III

n.d.
（̶）

89

Male

α-synuclein（̶），chronic subdural hygroma

II

n.d.

88

Male

IV

n.d.

88

FemaleAD, CAA (light),α-synuclein（＋）
AD, CAA (heavy),α-synuclein（̶）

VI

82

Male

75
92

I

I
III‒IV（̶）（̶）

V

FemaleAD, CAA,α-synuclein（̶）
AD, CAA,α-synuclein（̶）

（̶）
（̶）

VI

（̶）

Femalestroke, AD, CAA, LB（̶），α-synuclein（＋）

V

n.d.

Bottom note: MI: multiple infarct, CAA: cerebral amyloid angiopathy, LB: Lewy Body

shown in Table 1. In this study, we separated the four

800 L/h, respectively, and were obtained using a nitrogengroups based on the neuroﬁbrillary tangles (NFT) Braaksource (N2 Supplier Model 24S, Anest Iwata, Yokohama,stage. The Braak staging refers to classify the degree ofJapan).

pathology in AD. Thus, it is not direct diagnosis of clinical
AD disease.

Derivatization

UHPLC-ESI-MS/MS

Mixed standard (four analytes such as Asp, Arg, Cit, and
ORN) and IS solutions were diluted by the addition of acetonitrile/water (50 : 50, v/v) to give solutions at 10μM con-

The UHPLC system was a Waters Acquity H Class(Waters Co., Milford, MA). The reverse phase (RP) analysis was performed using an Acquity BEH C18 column

centrations. The solvent was removed from 30μL of eachof these two solutions, and the residues were re-dissolved in

5μL. The mobile phase consisting of solvent A (0.1% FA inwater) and solvent B (0.1% FA in acetonitrile), was deliv-

ture for 10 min. Subsequently, 50μL of 1% FA adjusted

ered at a ﬂow rate of 0.4 mL/min. The gradient elution wasusing acetonitrile/water (50 : 50, v/v) were added to theas follows: B%＝20, 60, 90, 98, 98, 20, and 20 (0, 8, 10, 11,

reaction mixtures to stop the derivatization reaction.

12, 13, and 20 min). A Waters Xevo TQD triple quadrupole
mass spectrometer was operated with an electrospray ion-

Finally, the derivatization solutions (5μL) were analyzed
by UHPLC-ESI-MS/MS.

ization (ESI) source in the positive mode. The ionization

Second mixed standard (remaining 11 analytes) and IS

source conditions were as follows: capillary voltage,
2.00 kV; cone voltage, 20‒70 V; collision energy, 10‒40 eV;

solutions were diluted by the addition of acetonitrile/water(1.7μm, 2.1 × 100 mm) at 40°
C. The injection volume was

50μL of 0.1 M Borax (pH 9.0) and reacted with an equalvolume of 40 mM F-MOC in acetonitrile at room tempera-

source temperature, 150°
C; and desolvation temperature,(50 : 50, v/v) to give solutions at 10μM concentrations. The
solvent was removed from 30μL of each of these two solu-

400°
C. The cone and desolvation gas ﬂows were 50 andtions, and the residues were dissolved in 150μL of 0.1 M
13June 2018

Medical Mass Spectrometry Vol. 2 No. 1

Borax (pH 9.0) and subsequently reacted with an equal volume of 40 mM DBD-F in acetonitrile at 60°
C for 30 min.

(metabolites and polyamines, acetylated polyamine in the

Next, the reaction mixtures were removed and re-dissolved

ornithine cycle; Fig. 1). For this experiment, the limited 15
analytes are selected for the wide-targeted metabolomics

in 100μL of mobile phase consisting of solvents A and B

regarding to ornithine cycle. If these analytes have the char-(each 50μL). Finally, the derivatization solutions (5μL)

acteristic values, we would expand the measurement ranges

were analyzed by UHPLC-ESI/MS/MS.

for various metabolic maps.

Sample preparation

Free amine analytes such as amino acids and polyamines
are difﬁcult to separate and detect using RP-LC-ESI-MS/

CSF samples were obtained from the Fukushimura Hos-

MS owing to their poor ionization efﬁciency, high polarity,

pital (as described above in Section Human sample) andand structural similarity. Derivatization of amine analytes iswere classiﬁed according to the level of seriousness interms of senile plaque levels (Non, n＝5; CERAD-A, n＝5;

one way of modifying their hydrophobicity and improving
24)
RP-LC separation and detection sensitivity in ESI-MS .

CERAD-B, n＝5; and CERAD-C, n＝5). The descriptive

F-MOC is a well-known derivatization reagent with thediagnosis of all study variables is summarized in Table 1. In
the AD group, the Braak phase was shown in the pathologi-

ability to improve LC analysis of amines and amino
25‒27). Moreover, in our study, the DBD-F-based deriacids

22)

cal diagnosis after death lasted from 6 to 20 years . In thepathological examination of the AD brain samples, we eval-vatization method was reported previously to yield highly
sensitive and simultaneous separation/detection of polyam-

uated the functional characteristics of visual pathology bydetermining the presence of plaque/entanglements in brain

ines in saliva . Based on our previous study, the
DBD-F-derivatized method was investigated by RP-UH-

tissues. After thawing, a CSF sample selected according to

PLC-MS/MS. In this result, the four analytes such as Asp,

the pathological investigation was added to IS and mixed

Arg, Cit and ORN showed the incompetent sensitive,

with an equal volume of acetonitrile, vortexed vigorously,

repeatability and detectable abilities to apply the pooled

and centrifuged at 15,000 rpm for 10 min. The supernatantsolution was subsequently removed, and the remaining resi-

CSF samples. However, at any experiment, these analytes

28)such as Asp, Arg, Cit and ORN should be monitored for the
possible biomarkers in CSF regarding to AD pathol-due was re-dissolved in 0.1 M Borax and derivatized using
the process described above for F-MOC and DBD-F. There-ogy

29,30). Thus, we tried to develop an analytical method

after, the sample was analyzed by UHPLC-ESI/MS/MS.

based on two different derivatization reagents such as
DBD-F for four analytes and F-MOC for others com-

Results and Discussion

31)
pounds . The derivatization reaction with F-MOC and
DBD-F generally proceeded efﬁciently under alkaline con-

Derivatization of analytes involved in ornithine cyclewith F-MOC and DBD-F

ditions. In this study, the alkaline conditions employed 0.1

Accumulation of ammonia produced by protein metabo-

M Borax (pH 9.0), and the reactions were carried out atroom temperature for 10 min for the F-MOC process and at23)

lism is associated with signiﬁcant neurotoxicity . Since
the ornithine cycle (also be known as the urea cycle) converts ammonia to non-toxic urea, disorders of this cycle can60°
C for 30 min for the DBD-F process.

lead to various diseases . Metabolites involved in orni-

UHPLC-ESI-MS/MS detection of derivatized analytes

23)

In order to perform simultaneous separation and analysis

thine cycle are contained in the body at high concentrations
and play important roles in anti-inﬂammatory and anti-fa-of the 15 derivatized amine analytes, we ﬁrst investigatedthe most optimal conditions for ESI-MS/MS. In particular,

23)

tigue activities . The term polyamine refers to a chemical
structure that possesses two or more amino groups. In gen-conditions such as product ions of F-MOC/DBD-F deriva-

eral, polyamines play important roles in cell proliferation

tives and collision energies were studied. In the fragment
pattern of each F-MOC derivative, a product ion at m/z

15,17)and differentiation. Additionally, in our previous study,

179.09 was detected arising from the C-O bond of F-MOC
cleavage in F-MOC. The ESI-MS/MS conditions for

we reported that the metabolism of polyamines, including
20)

ornithine, is involved in the pathology of AD . On the

DBD-F polyamine derivatives were utilized as reported pre28)
viously . Table 2 shows the ESI-MS/MS measurement

basis of these discussions, in this study, we tried to develop
a targeted metabolomics for preliminary 15 amine analytes
14June 2018

Fig. 1.

Medical Mass Spectrometry Vol. 2 No. 1

Structures and metabolic pathway related to the metabolites from the ornithine cycle and polyamines.

conditions tested for 15 amine derivatives and IS. On theseparated by UHPLC in the RP mode using a C18 column,

basis of the results presented in Table 2, precursor/productions were set, and selective and sensitive analysis was per-majority of them are eluted early in the polar mobile phaseas a result of their high polarity, thus affording poor resolu-

formed using UHPLC-ESI-MS/MS in the selected reaction
monitoring (SRM) mode. Next, in order to achieve simulta-

tion. However, by labeling the amines with two derivatizing reagents, their hydrophobicity was increased, and theretention time on the C18 column was prolonged. In addi-

neous separation of the 15 targeted analyte peaks in thetion, by optimizing the mobile phase, it was possible to per-chromatogram, the UHPLC conditions were examined.
Normally, when unmodiﬁed non-derivatized amines areform simultaneous separation and detection of 15 amines
15June 2018

Table 2.

Medical Mass Spectrometry Vol. 2 No. 1

Detective condition of metabolites on ornithine cycle and polyamines derivatized with F-MOC and DBD-F

Analytes

RT (min)

MRM transitions (m/z)

Cone voltage (V)Collision energy (eV)5.29

356 → 1792020

397 → 179

20

20

45

35

398 → 179

45

35

25

10

578 → 179

25

20

25

15

356 → 311

25

15

30

20

35

30

Asp
13
Asp- C

5.29

Arg

3.73

Arg- C6

3.73

Cit

4.75

13

Cit-d4

4.75

ORN

9.87

ORN-d7

9.87

Ac-PUT
DAc-SPD

2.99

DAP

6.73

PUT

7.31

3.17

CAD

7.95

1,6-Diaminohexane (I.S.)
1
N -Ac-SPD

8.56

N -Ac-SPD
DAc-SPM

6.38

SPD

9.02

SPD-d8Ac-SPM

9.02
8.16

SPM

9.82

SPM-d8

9.82

8

6.49
5.74

357 → 179

403 → 179402 → 179
584 → 179455 → 100525 → 437

Derivatization/
Internal standard
F-MOC/Asp- C
̶1313
F-MOC/Arg- C6
̶F-MOC/Cit-d4
̶
F-MOC/ORN-d7
̶
DBD-F/DAH
DBD-F/SPD-d8

DBD-F/DAH
DBD-F/DAH
DBD-F/DAH

539 → 451

45

25

45

25

567 → 479

45

20

45

20

̶

50

20

50

20

DBD-F/SPD-d8
DBD-F/SPD-d8

50

35

60

25

553 → 465

638 → 100
638 → 550

737 → 100822 → 733

829 → 741

920 → 835

1,104 → 1,015

1,112 → 1,024

DBD-F/SPM-d8
DBD-F/SPD-d8

50

30

̶

65

35

70

40

DBD-F/SPM-d8
DBD-F/SPM-d8

50

40

̶

within a measurement time of 15 min as shown in Fig.

assay method is sufﬁciently sensitive to enable simultane-2(A).

ous detection of polyamines present in CSF (Table 3).

Quantitative performance

Targeted metabolomics for the ornithine cycle in CSF

Firstly, a validation test was carried out. Calibration

The 15 selected amine analytes were measured in CSF

curves [X: concentration (nM), Y: derivative/IS peak area
ratio] of 15 amine analytes prepared using F-MOC andsamples using the UHPLC-ESI-MS/MS targeted metabolo-

DBD-F as derivatization reagents were obtained. We
obtained a calibration curve with high linearity and a cor-cycle. The obtained chromatograms are shown in Fig. 2(B).

relation coefﬁcient r of 0.996‒1.000 in the required con-shown in Fig. 2(A), the results, with clearly observed sharp

centration range for each analyte. Moreover, the limit ofpeaks, showed that simultaneous separation and detection isquantitation (LOQ) and the limit of detection (LOD) of
each metabolite were determined to be in the level of sev-

possible. The results from grouped CSF samples wereshown in Table 4. In our previous study, the polyamine lev-

eral nM. For the application of CSF for various types, it isels (SPD, SPM, PUT and Ac-SPD) in AD brain tissues were

infrequently observed to detect the protrude concentration

increased compared to control . Based on the evidence,from the calibration range. In this case, a diluted liquid isthe polyamine metabolite pathway in a speciﬁc brain region
is likely to be responsible for up-regulated polyamine

mics methodology developed speciﬁcally for the ornithine
As observed in the chromatogram of the reference standard

2

20)

prepared by adding not less than 10 parts of pure water to
one part of the CSF sample. On the basis of prior studies
for the direct/diluted methods, the recovery and repeatabil-

metabolism and elevated ornithine decarboxylase (ODC)

ity were considered acceptable and useful values (morethan 80% for recovery and less than 15% for repeatability,

result is not observed compared with previous study. In the8
kruskal-wallis test, the ORN, PUT, N -AC-SPD and

n＝6). These results suggest that the developed analytical

AC-SPM are less than 0.1 values. In addition, Arg is

activity. On the other hand, this study showed that clear

16June 2018

Fig. 2.

Medical Mass Spectrometry Vol. 2 No. 1(A) SRM chromatograms of amino acids and polyamines in standard solution. (B) SRM chromatograms of human
CSF samples.

17June 2018

Medical Mass Spectrometry Vol. 2 No. 1

Table 3.
Analytes

Calibration curves and linearity of metabolites on ornithine cycle and polyamines
Calibration range (nM)Linearity (R2)

LOQ (nM)

LOD (nM)

1.00‒100,000
10.00‒100,000

0.999

1

0.1

1.00

3

1

1.00‒100,000
10.00‒100,000

0.999

1

0.1

0.999

1

0.1

6.25‒2,000
6.25‒2,000

0.998

1

0.2

DAc-SPD

0.996

2.5

1

DAP

6.25‒2,000

0.997

1

0.1

PUT

6.25‒2,000
6.25‒2,000

0.996

0.5

0.1

0.997

0.5

0.1

50.0‒2,000

0.998

50

25

N8-Ac-SPD
DAc-SPM50.0‒2,00025.0‒2,000

0.998

50

25

0.997

25

20

SPD
Ac-SPM

25.0‒2,00050.0‒2,000

0.997

25

10

0.996

50

25

SPM

50.0‒2,000

0.998

50

25

Asp
Arg
Cit
ORN
Ac-PUT

CAD
N1-Ac-SPD

Table 4.
Analytes
Asp

Concentration levels (μM) of metabolites on ornithine cycle and polyamines in CSF samples
Group

P value(Kruskal‒Wallis test)

ControlA

B

C

11.1±10.2

6.6±6.2

12.3±9.8

2.4±0.627.2±2.8**

0.523

0.332

Arg

35.7±16.2

28.0±7.527.4±12.1**

Cit

3.8±3.0

3.6±2.0

3.6±2.9

1.9±0.5

0.452

ORN
Ac-PUT8.8±2.1

7.3±1.4

9.2±1.3

6.2±1.8

0.0871

0.3±0.9

0.3±0.2

0.4±0.2

0.2±0.1

0.354

DAc-SPD

0.2±0.3

0.3±0.1

0.2±0.1

0.3±0.1

0.617

DAP

0.4±0.2

0.2±0.2

0.5±0.2

0.257
0.0498PUT

1.6±1.0

0.7±0.4

1.5±0.7

0.3±0.1
0.6±0.2*

CAD
1
N -Ac-SPD

0.2±0.1

0.2±0.1

0.3±0.2

0.4±0.1*

0.147

0.3±0.3

0.3±0.2

0.3±0.2

0.1±0.03

0.224

N -Ac-SPD
DAc-SPM0.1±0.03

0.1±0.03

0.1±0.1

0.1±0.01

0.09680.9±0.3

1.3±1.0

0.7±0.3

1.2±0.5

0.501

8

SPD

12.3±11.1

4.8±5.7

20.9±19.4

2.3±0.6

0.161

Ac-SPM

5.9±2.99.0±13.2

7.5±1.1

9.5±3.90.0456SPM

16.8±16.4

3.6±3.513.4±8.3

13.5±16.1

0.267

Mean（μM）±S.D.
*: P＜0.05, **: P＜0.01 (vs. Control)
The variance in a group was evaluated using the F test.
The difference between two groups were evaluated using Student s t test or the Wilcoxon‒Mann‒Whitney test.

decreased between control and other groups. Trushina et al.

CSF based on AD pathology. Based on this result, PUT andreported that the altered canonical pathways in CSF
between the AD group vs. cognitively normal individuals

CAD in CSF showed a minimal change between controls
vs. group C. Recently, it is reported that hyperhomocystein-showed the cholesterol and sphingolipids transport, energy
metabolism, Krebs cycle, mitochondrial function, neu-

emia is a risk factor for development of dementia and AD
based on the effects of homocysteine, polyamines and oxi-

32)

33)

rotransmitter and amino acid metabolism . Thus, Arg is

dative metabolism including PUT, CAD and others .
Moreover, the modulation of learning and memory by poly-needed to investigate the exhaustive metabolic pathway in18June 2018

Medical Mass Spectrometry Vol. 2 No. 1

A, et al: Clinical and biomarker changes in dominantly in-amine metabolism was discussed and ﬁgured to ODC andthe sequential transfer of aminopropyl groups from S-ade-

herited Alzheimer s disease. N Engl J Med 367(9): 795‒

34)

nosylmethionine to PUT by SPD/SPM synthases .
Already, we reported that the ODC in polyamine metabo-

3) Puli L, Tanila H, Relkin N: Intravenous immunoglobulins

lism and ornithine cycle is an index enzyme-reaction for thefor Alzheimer s disease. Curr Alzheimer Res 11(7): 626‒

804, 2012.

20)

AD pathology monitoring using biological samples . In

636, 2014.

this study, it is unclear evidence to indicate the inevasible4) Jeffrey C, Paul SA, Bruno DB, Lutz F, Clifford RJ, et al:biomarker for AD severity. However, the possible pathwayof polyamine metabolism and ornithine cycle can be pre-Drug development in Alzheimer s disease: The path to2025. Alzheimers Res Ther 8: 39, 2016.

sented using the targeted metabolomics of CSF samples.
Future works would be needed to develop the wide-targeted5) David CH, Gad AM: Primary and secondary prevention
trials in Alzheimer disease: Looking back, moving for-metabolomics using UHPLC-MS/MS for various biologicalward. Curr Alzheimer Res 14(4): 426‒440, 2017.
6) Vassar R, Bennett BD, Babu-Khan S, Kahn S, Mendiaz

samples such as brain tissue, CSF, blood and others for AD

EA, et al: Beta-secretase cleavage of Alzheimer s amyloid

diagnosis.

precursor protein by the transmembrane aspartic protease

Conclusions

BACE. Science 286(5440): 735‒741, 1999.

In this study, we developed amine-targeted metabolomics
methodology employing UHPLC-ESI-MS/MS and two7) Graham WV, Bonito-Oliva A, Sakmar TP: Update on Alzheimer s disease therapy and prevention strategies. Annu

derivatization reagents (F-MOC and DBD-F). Furthermore,
we used this targeted metabolomics in a pathological inves-

Rev Med 68: 413‒430, 2017.
8) Kim D, Kim YS, Shin DW, Park CS, Kang JH: Harness-tigation to screen for CSF-based biomarkers suitable fordiagnosis of AD. The results identiﬁed polyamine metabo-

ing cerebrospinal ﬂuid biomarkers in clinical trials forlism and ornithine cycle as candidate markers in CSF thatand challenges. J Clin Neurol 12(4): 381‒392, 2016.

exhibit marked changes dependent on the AD pathology.

9) Parnetti L, Eusebi P, Lleó A: Cerebrospinal ﬂuid biomark-The methodology described here is demonstrated to be aneffective method for the identiﬁcation of possible AD mark-ers for target engagement and efﬁcacy in clinical trials for

ers in CSF.

rosci 39: 117‒123, 2016.

treating Alzheimer s and Parkinson s diseases: Potential

Alzheimer s and Parkinson s diseases. Front Neurol Neu10) Harrison JR, Owen MJ: Alzheimer s disease: The amyloid

Conﬂict of Interest

hypothesis on trial. Br J Psychiatry 208(1): 1‒3, 2016.

The authors have no conﬂicts of interest directly relevant11) Stephen PA, Richard BU, Laurel B, Tobias B, Kaj B, et
al: Cerebrospinal ﬂuid biomarkers for Alzheimer s dis-to the content of this article.

ease: A view of the regulatory science qualiﬁcation land-

Acknowledgements

scape from the coalition against major diseases CSF bio-

This study was supported by grants from the Japanesemarker team. J Alzheimers Dis 55(1): 19‒35, 2017.

Brain Bank Network for Neuroscience Research and theMinistry of Education, Culture, Sports, Science, and Tech-

12) Daniel F, Lilisbeth PP, Eric W, Lars-Olof W, Antonio S:

nology of Japan. We would like to thank Norihiro Ogawa,ease: The state-of-the-art after the new revised diagnostic

Takeshi Kanesaka and Chie Taniguchi for their technicalcriteria. Front Aging Neurosci 6: 47, 2014.

Meta-review of CSF core biomarkers in Alzheimer s dis-assistance in the clinical and pathological study, and all13) Igarashi K, Yoshida M, Waragai M, Kashiwagi K: Evalua-patients and relatives of the patients for the organ donation.

tion of dementia by acrolein, amyloid-β and creatinine.
Clin Chim Acta 450: 56‒63, 2015.

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