ISSN 2432-745X
2432-7441

Medical Mass Spectrometry
Vol. 8 No. 1, 2024
Guest Editor, Kaname Tsutsumiuchi
Preface
The 48th Annual Meeting of the Japanese Society for Biomedical Mass Spectrometry
Kaname Tsutsumiuchi, Makoto Arita, Toshimitsu Niwa
1
Research Papers
DESI-IMS coupled with triple quadrupole mass spectrometry demonstrated the capability of
NMN imaging in Shelfordella lateralis tissueZinat Tamannaa, Takumi Sakamoto, Md. Al Mamun, Soho Oyama, A.S.M. Waliullah,
Ariful Islam, Md. Mahamodun Nabi, Shuhei Aramaki, Eisuke Tasaki, Yuki Mitaka,
Mamoru Takata, Tomohito Sato, Tomoaki Kahyo, Kenji Matsuura, Mitsutoshi Setou 2
Sex-speciﬁc eﬀect of ethanol on colonic short-chain fatty acids and derivatives in the mouse modelusing targeted LC/MS
Siddabasave Gowda B. Gowda, Lipsa Rani Nath, Yonghan Li,
Jayashankar Jayaprakash, Divyavani Gowda, Shu-Ping Hui 11
Evaluation of four drug screening devices for detection of psychoactive drugs in pericardial ﬂuid
Masae Iwai, Tadashi Ogawa, Tomohito Matsuo,
Katsutoshi Kubo, Fumio Kondo, Hiroshi Seno 19
Plasma vitamin K dependent protein C as early diagnostic marker of feline chronic kidney disease
Hiroto Maeda, Yuiko Suzuki, Kokoro Masuda, Yoshio Kodera,
Toshifumi Watanabe, Kazuyuki Sogawa 27
Short Communication
Recovery of extracellular vesicles from liquid samples using polyamine solution
Arizumi Kikuchi, Azumi Naruse, Kenichi Nonaka, Motoki Mori, Miyuu Yamada,
Keiko Kano, Emi Mishiro-Sato, Kaname Tsutsumiuchi 35

Japanese Society for
Biomedical Mass Spectrometry

Medical Mass Spectrometry
Editorial Board
Editor-in-Chief
Makoto ARITA (Tokyo, Japan)
Associate Editors

Masayuki KUBOTA (Tokyo, Japan)

Yasuhiro MAEDA (Toyoake, Japan)

Fumio NOMURA (Chiba, Japan)

Tomoko OYA (Ichinomiya, Japan)

Daisuke SAIGUSA (Tokyo, Japan)

Emiko SATO (Sendai, Japan)

Masatoshi WAKUI (Tokyo, Japan)

Seiji YAMAGUCHI (Shimane, Japan)

Ikuko YAO (Sanda, Japan)

Editorial Board

Koutaro HASEGAWA (Hamamatsu, Japan)

Kazuo IGARASHI (Kobe, Japan)

Toyofumi NAKANISHI (Ichinomiya, Japan)

Toshimitsu NIWA (Ichinomiya, Japan)

Mamoru SATOH (Tokyo, Japan)

Hiroshi SENO (Nagakute, Japan)

Mitsutoshi SETO (Hamamatsu, Japan)

Yosuke SHIGEMATSU (Fukui, Japan)

Kaname TSUTSUMIUCHI (Kasugai, Japan)

Chunhua ZHANG (Yokohama, Japan)

International Advisory Board

Alma L. Burlingame (California, USA)

Neal Castagnoli (Virginia, USA)

Dominic M. Desiderio (Tennessee, USA)

Catherine Fenselau (Maryland, USA)

Published by Japanese Society for Biomedical Mass Spectrometry
Shubun University
6 Nikko-cho, Ichinomiya, Aichi 491‒0938, Japan
E-mail: jsbms.oﬃce@gmail.com
Homepage: http://www.jsbms.jp/english/

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Preface

The 48th Annual Meeting of the Japanese Society for
Biomedical Mass Spectrometry
1,

2,3,4

Kaname Tsutsumiuchi *, Makoto Arita

5, Toshimitsu Niwa

The 48th Annual Meeting of the Japanese Society for Biomedical Mass Spectrometry (JSBMS) was held on September 8th
and 9th, 2023, at the WINC Aichi, Nagoya, Japan. Fortunately, we had a fruitful time discussing our latest studies face-to-facein the meeting, which could not be held for three years because of COVID-19. The program comprised three award lectures,
three symposia with nine presentations, 46 short-oral and poster presentations, and two luncheon seminars.
This 8th issue of Medical Mass Spectrometry is based on the presentations at the 48th Annual Meeting of JSBMS (Nagoya).
The 49th Annual Meeting and the 3rd International Symposium of JSBMS and MSACL (Mass Spectrometry & Applications to the Clinical Lab) will be held on September 13th and 14th, 2024, organized by Prof. Ikuko Yao (Kwansei Gakuin
University). We look forward to seeing you in Kyoto and exchanging information on medical mass spectrometry.

1

College of Bioscience and Biotechnology, Chubu University* E-mail: tsutsu@fsc.chubu.ac.jp

2

Division of Physiological Chemistry and Metabolism, Keio University Faculty of Pharmacy

3

Laboratory for Metabolomics, RIKEN Center for Integrated Medical Sciences

4

Cellular and Molecular Epigenetics Laboratory, Graduate School of Medical Life Science, Yokohama City University

5

Shubun University

Epub May 17, 2024.
DOI: 10.24508/mms.2024.06.005
1

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Research Paper

DESI-IMS coupled with triple quadrupole mass spectrometry demonstrated
the capability of NMN imaging in Shelfordella lateralis tissue
1

1

1

1

1

1

Zinat Tamannaa , Takumi Sakamoto , Md. Al Mamun , Soho Oyama , A.S.M. Waliullah , Ariful Islam ,
1

1,2

4,5

4,6

4

Md. Mahamodun Nabi , Shuhei Aramaki , Eisuke Tasaki , Yuki Mitaka , Mamoru Takata ,
1,2

1,2

4

1,2,3

Tomohito Sato , Tomoaki Kahyo , Kenji Matsuura , Mitsutoshi Setou*

1

Department of Cellular & Molecular Anatomy, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-Ku, Hamamatsu, Shizuoka 431‒3192, Japan
2
International Mass Imaging Center, Hamamatsu University School of Medicine,1‒20‒1 Handayama, Higashi-Ku, Hmamatsu, Shizuoka 431‒3192, Japan3
Department of Systems Molecular Anatomy, Institute for Medical Photonics Research, Preeminent Medical Photonics Education &Research Center, 1‒20‒1 Handayama, Higashi-Ku, Hamamatsu, Shizuoka 431‒3192, Japan
4
Laboratory of Insect Ecology, Graduate School of Agriculture, Kyoto University,
Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto 606‒8502, Japan
5
Department of Life and Food Sciences, Graduate School of Science and Technology, Niigata University,
8050 Ikarashi 2-no-cho, Nishi-ku, Niigata 950‒2181, Japan
6
Department of Entomology, 2143 TAMU, Texas A & M University, College Station, Texas, 77843‒2143, USA
Abstract

Imaging mass spectrometry (IMS) has become an indispensable analytical tool for visualizing molecular

distributions in biological tissues. However, the detection of many important biomolecules, such as nicotinamide mononucleotide (NMN) in tissue by IMS is still challenging due to their lower abundance. Although the sensitivity of widelyused IMS instruments, desorption electrospray ionization (DESI) utilizing a time-of-ﬂight (TOF) mass analyzer, is not
suﬃcient to detect NMN in tissues, the recently developed DESI coupled with a triple-quadrupole (TQ) provides about ten
times higher sensitivity than the DESI-Q-TOF. In this study, we ﬁrst screened six insect species for NMN content using
ultra-performance liquid-chromatography-tandem mass spectrometry (UPLC-MS/MS). Interestingly, Shelfordella lateralis
showed signiﬁcantly higher NMN content (15.5 mg/100 g of dry weight) than ﬁve other insect species used in this study.
After screening, we tried to observe the distribution of NMN in S. lateralis using both DESI-Q-TOF and DESI-TQ, andonly the DESI-TQ successfully detected the NMN in S. lateralis. DESI-TQ revealed prominent distributions of NMN in theabdomen of S. lateralis in selective reaction monitoring mode. To the best of our knowledge, this is the ﬁrst report of NMN
imaging in animal tissue applying IMS. Our study will contribute to the extended application of IMS for NMN imaging in
mammalian tissues in the future. Furthermore, NMN imaging may help researchers to study nicotinamide adenine dinucle＋

otide (NAD ) metabolism in living organisms, which has signiﬁcant implications for medical research and diagnosis.
Key words: IMS, NMN, DESI-TQ, DESI-Q-TOF, UPLC-MS/MS, Shelfordella lateralis*Corresponding author
Mitsutoshi Setou

Introduction

Department of Cellular & Molecular Anatomy, Hamamatsu
University School of Medicine, 1‒20‒1 Handayama,

Imaging mass spectrometry (IMS) has been increasingly
used to visualize biomolecules due to its high sensitivity
and label-free nature. This technology has made it possible

Higashi-ku, Hamamatsu, Shizuoka 431‒3192, Japan
Tel: ＋81‒53‒435‒2086, Fax: ＋81‒53‒435‒2468

to reveal the identity and distribution of the speciﬁc mole1)
cule and the localization of the molecule in organs . Nowa-

E-mail: setou@hama-med.ac.jp

Received: August 16, 2023. Accepted: January 11, 2024.

days, imaging with desorption electrospray ionization(DESI), in combination with quadrupole-time-of-ﬂight

Epub April 19, 2024.
DOI: 10.24508/mms.2024.06.001
2

June 2024

Medical Mass Spectrometry Vol. 8 No. 1(Q-TOF) mass spectrometry, has been gaining much popu-

insects as a potential source of calories and protein . In

larity for visualizing molecular distribution on a sample
2)
surface under ambient conditions . DESI-Q-TOF has

traditional folk medicine and Chinese medicine, the whole22)

body extracts of several bees, wasps, ﬂies, butterﬂies,
moths, cockroaches, beetles, and other insects act as antivi-

shown the capability of visualizing spatial distributions of

21)
ral, antibacterial, and anti-cancer agents . While insects

molecules with diverse chemical natures, such as small
3)
4‒6)
7)
molecules , free fatty acids , phospholipids , lipid bio8)

9)

are consumed as food in various cultures around the world10)

marker , active compounds , and other metabolites . Oneof the signiﬁcant advantages of DESI-IMS is that it requires

and are known to be rich in protein and nutrients, there is
not adequate knowledge or research about their NMN con-a minimal sample preparation than traditional mass spectrometry, such as matrix-assisted laser desorption ionization

tent.

mass spectrometry (MALDI-MS) . Recently, DESI com-Cinnamomum verum J. Presl bark, a medicinal plant, using
23)
liquid chromatography mass spectrometry (LC-MS) .

Recently, our group has explored high NMN content in

11)

bined with triple quadrupoles (TQ) mass spectrometry has
emerged and oﬀers advantages over DESI-Q-TOF. The TQ

Applying the same method, we ﬁrst screened NMN content
in six insect species in this study, namely, the Turkestanmass analyzer consists of three quadrupole mass ﬁlters,
while DESI-Q-TOF consists of a single quadrupole mass ﬁl-cockroach Shelfordella lateralis, the ﬁeld cricket Gryllus
bimaculatus, the damp-wood termite Hodotermopsis sjost-ter. These three quadrupole mass ﬁlters make DESI-TQ
more speciﬁc for precursor-to-product ion transitions of theedti, the black soldier ﬂy Hermetia illucens, the Japanesetargeted compound analysis . This allows DESI-TQ to
exhibit higher speciﬁcity and excellent sensitivity in selec12)

subterranean termite Reticulitermes speratus, and the giant
mealworm Zophobas atratus. Finally, we employed both
DESI-Q-TOF and DESI-TQ to observe the distribution oftive reaction monitoring (SRM) mode than other high-reso13)

lution mass analyzers .

NMN in S. lateralis bodies, which showed the highest

Despite the signiﬁcant improvements in the sample

NMN content among the other species under this study.

preparation and instrumentations of IMS, detecting andimaging of many biologically important molecules in tissue
is still challenging. For example, nicotinamide mononucle-

Materials and Methods

otide (NMN), a well-known vital precursor of nicotinamide

We purchased the standard β-nicotinamide mononucleotide (β-NMN) from Tokyo Chemical Industry Co.

Chemicals and reagents＋

adenine dinucleotide (NAD ), has been found diﬃcult to be
detected by IMS. The analytical detection of NMN is chal-(Tokyo, Japan). LC/MS-grade formic acid (99%), acetonitrile (ACN), methanol, ethanol, and ultra-pure water were14)

lenging due to its physicochemical characteristics . NMN
is a nucleotide containing a pyridine base. It is an acid- andwater-soluble nucleotide found in all living cells as a coen-

obtained from Fujiﬁlm Wako Pure Chemical Corporation,
Ltd. (Osaka, Japan). Ammonium format solution was pur-zyme. Increasing NAD levels improves insulin sensitivity,
reverses mitochondrial dysfunction, and enhances lifes-

chased from Kanto Chemical Co., INC. (Tokyo, Japan).
Sodium formate was obtained from Sigma-Aldrich Co.

＋

15)

pan . When taken orally, NMN is quickly absorbed andLLC (St. Louis, MO, USA) and, leucine enkephalin from. NAD availability is essential for17)
slowing the aging process . Oral nicotinamide supplemen-

Waters Corporation (Milford, MA, USA).

tation within the daily acceptable upper threshold is an eﬃ-

Sample preparation for the quantiﬁcation of NMN

cient strategy to raise NAD levels and inﬂuence blood lipid

Two types of termite species (H. sjostedti, and R. speratus) were used in this study. H. sjostedti were collected ini-

＋16)

＋

converted into NAD＋

18)composition . Thus, NMN has important applications intially from logs from brown-rotted Luchu pine wood (Pinus

19)

medicine and healthcare , but the chemical synthesis of
20)

NMN is complex . Therefore, it is worth exploring a richluchuensis) in the forest of Amami Oshima Island in Japan.

source of natural NMN and its imaging in medical research.
Unlike other natural products, insects have received com-

And the colonies of R. speratus were collected from logs
from brown-rotted cedar wood (Cryptomeria japonica) inparatively little attention in imaging studies as sources ofmodern medicines . Insects can provide proteins forthe forest in Kyoto, Japan. The termite colonies were maintained at the Laboratory of Insect Ecology, Kyoto Univer-human use, and people in many parts of the world consume

sity, at about 25°
C under dark. The Individuals of H. sjost-21)

3June 2024

Medical Mass Spectrometry Vol. 8 No. 1

edti were randomly extracted from each of the six colonies

40°
C throughout the measurement. The electrospray ioniza-(colony code: KM022, KM024, KM025, KM103, KM109,
TMK091). And the individuals of R. speratus were ran-tion (ESI) source was used on the mass spectrometer, andthe analysis was performed in positive ion mode. The capil-

domly extracted from each of the three colonies (colony

lary voltage was set to 3.5 kV, the cone voltage to 22 V andthe source temperature was 150°
C. The desolvation tem-

code: MT616D, MT617E, and MT619G). We bought theperature was ﬁxed at 500°
C and the desolvation gas ﬂow

cockroach S. lateralis from Rakuten Group, Inc., Tokyo,
Japan, and the freeze-dried G. bimaculatus from Leaf Cor-rate was 600 L/h. The cone and nebulizer gas ﬂows were set
to 150 L/h and 7.0 Bar, respectively. Mobile phase A con-poration, Gunma, Japan. The two other insect species, dried

sisted of 0.3% formic acid in ACN, while mobile phase B

H. illucens, and dried Z. atratus, were purchased from GEX
Corporation, Osaka, Japan, and Sanko-Tokai Co., Ltd.,

was acetonitrile/100 mM ammonium formate (20:80, v/v).
The ﬂow rate used for elution was 0.6 mL/min. The follow-

Osaka, Japan, respectively.
All the samples were kept in a −80°
C freezer until
freeze dried treatment. The frozen insect samples were

ing setup was used to perform the gradient elution proce-freeze-dried (Freeze Dryer FD-1000, EYELA, Bunkyo,
Tokyo, Japan) and ground (Wonder Crusher WC-3 L, Osaka

2.5 to 4.5 min, 100% B; 4.5 to 4.6 min, 100% to 20% B; 4.6

Chemical Co. Ltd., Kita, Osaka, Japan) to form a powder.
The resulting insect powder (each about 5 g) was respec-

7 min. An aliquot of 5μL of the prepared sample wastively vacuum-sealed (Impulse Vacuum Sealer V301G-

constant temperature of 10°
C. The SRM transition from m/z

10WK, FUJIIMPULSE Co. Ltd., Toyonaka, Osaka, Japan)

335.1 to m/z 123.0 using a collision energy of 10.0 eV wasand kept in −20°
C freezer until use for analyses.

used for NMN quantiﬁcation.

Standard sample preparationacquire and analyze the UPLC-MS/MS data, and, TargetLynx software (Waters, version 4.1) was used for the quan-dure: 0 to 0.5 min, 20% B; 0.5 to 2.5 min, 20% to 100% B;
to 7.0 min, 20% B. The whole duration of the run time wasinjected into the system while the autosampler was kept at a

A MassLynx software (Waters, version 4.1) was used to
For standard sample preparation, an analytical balance(AUY220, SHIMADZU, Kyoto, Japan) was used to weight
the NMN standards. Then, 25% ethanol was used to dis-titative measurement. The statistical analysis was carriedout using GraphPad Prism (version 8.42).

solve the NMN standard. The prepared NMN standard was

Sample preparation for DESI-IMS analysis

then serially diluted to control solutions at concentrations

After collecting the samples, the whole body of the cockroach was embedded overnight by a super cryo-embeddingof 500, 200, 100, 50, and 20 ng/mL for calibration curve
preparation.

medium in the middle of a cryomold and frozen at −20°
C.

Insect extract preparation for UPLC-MS/MS

The frozen cockroach samples were then mounted on a

For each sample, approximately 50 mg of powder was

sample holder using the optimal cutting temperature (OCT)compound (Sakura Finetek Japan, Tokyo, Japan). The tis-weighed three times. Then 25% ethanol was added (40μL/

sues were sectioned sagittally at 20μm thickness at −20°C

mg of the sample) and extracted for one minute on ice witha mechanical homogenizer (HK-1, AS ONE, Osaka, Japan)

by a cryostat system (CM1950; Leica Biosystems, Wetzlar,

at 9000 rpm. The homogenized insect extracts were thenGermany). Then the sections were mounted on glass slides

centrifuged for 10 minutes at 3000 rpm in 4°
C. Then the(Matsunami, Osaka, Japan) and stored at −80°
C. Before
DESI-IMS analysis, the slices were air-dried.

supernatant was ﬁltered through a 0.42μm ﬁlter, and thecleared sample was transferred to LC-MS vials.

DESI-Q-TOF analysis

UPLC-MS/MS analysis

DESI-IMS analysis was performed in positive ion modeusing DESI source attached to a Q-TOF mass analyzer

ACQUITY (UPLC H-Class PLUS, Waters) coupled witha triple quadrupole mass spectrometer (Xevo TQ-XS,(Xevo G2-XS Q-TOF, Waters). Before the measurements,
sodium formate solution (500μM) in 2-propanol: water

Waters) was used to analyze the insect extracts. An Intrada
Amino Acid column (3μm, 3.0×100 mm, Imtakt) was car-(90:10, v/v) was used to calibrate the mass spectra externally, and leucine enkephalin solution (500μM) was used

ried out for chromatographic separation, which was kept at
4

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

to set up the detector. A solvent pump (ACQUITY UPLC

Results

Binary Solvent Manager, Waters) was used to deliver thespray solvent (98:2 methanol/water, v/v) at a ﬂow rate ofIn this study, we ﬁrst conducted a quantiﬁcation study of
NMN in six diﬀerent insect species S. lateralis, G. bimacu-

2μL/min. A 2D sampling stage was equipped with a DESI

latus H. sjostedti, H. illucens, R. speratus, and Z. atratus bysource to scan the designated area with a scan rate of 200μLC/MS (Fig. 1b). As seen in Fig. 1a, a linear calibration

m/sec, and the pixel size was 100μm × 100μm. The DESI

curve (Y＝9.3056X−154.914, R＝0.99) of NMN standards
was observed in the range of 20‒500 ng/mL. Chromato-source conditions were optimized using the following

grams showed the clear detection of NMN standard at eachpoint (Fig. S1a‒e).

parameters: a nebulizing nitrogen gas pressure of 0.4 MPa,a capillary voltage of 4.0 kV, a spray impact angle of 70°,a

A Chromatogram of NMN (SRM transition at m/z 335.1
to m/z 123.0) was observed with high intensities in S. later-

sampling cone of 50, and an inlet temperature of 120°C.
The setup for mass resolution, mass window, and collisionenergy were at 20000, 0.02 Da, and 4.00 V. The mass spec-

alis, G. bimaculatus (Fig. 1b, Fig. S2a‒b), H. sjostedti, H.
illucens (Fig. 1b, Fig. S3c‒d), and R. speratus (Fig. 1b, Fig.

tra were collected using a mass range of m/z 100 to 1000 of

S4e). On the other hand, NMN was not detected in the species of Z. atratus (Fig. 1b, Fig. S4f). The average concen-

the 1000 peaks with the highest intensities. The data werethen further processed at the range of m/z 300 to 400 to
detect the ion images of lower intensities.
DESI-Q-TOF data were acquired and processed using the

tration of NMN in S. lateralis, G. bimaculatus, H. sjostedti,

MassLynx software (Waters, version 4.1). And, HDI Imag-and 0.13 mg/100 g (dry weight) respectively. S. lateralis

ing software (Waters, version 1.4) was used to analyze theshowed signiﬁcantly higher NMN content than all otherion images.

species in this study (p＜0.0001) (Fig. 2). G. bimaculatus
showed the second highest content of NMN, which was sig-

DESI-TQ analysis

niﬁcantly higher than H. illucens, R. speratus, and Z. atra-

H. illucens, and R. speratus were 15.5, 6.93, 3.01, 0.143,

A Xevo TQ-XS equipped with a DESI source (Waters)tus (p＜0.005) (Fig. 2).
For the imaging study, we ﬁrst performed DESI-Q-TOF

has been employed for imaging in SRM mode. A syringe
pump delivered the DESI-IMS solvent (MeOH/H2O, 98/2,

analysis in the whole body of S. lateralis. But we could notv/v) at a ﬂow rate of 2μL /min. For NMN imaging, thedetect NMN in the entire body of S. lateralis (Fig. S5a), we＋
could only detect NMN at m/z 335.1 [M＋H] in NMN stan-

DESI source conditions were optimized using the following
parameters: N2 nebulizing gas pressure of 0.8 Mpa; capil-

dard (20μg/mL) (Fig. S5a). In this analysis, NMN was notlary voltage of 0.85 kV (Positive); MS source temperaturedetected with other adducts in both NMN standard and S.

of 150°C; sampling cone voltage of 20 V. The distances

lateralis (Fig. S5b).

between the emitter tip and the surface, the mass inlet andthe surface, and the mass inlet were roughly 2, 6, andNext, we performed DESI-TQ in the SRM mode to see
the distributions of NMN in the sagittal sections of whole-

0.5 mm, respectively. The identiﬁed areas on the glass slide

body S. lateralis. NMN (SRM transition of m/z 335.1 to m/

were scanned at a scan rate of 10 scan/sec. The pixel sizez 123.0) was successfully detected in almost entire regions

was set as 50μm × 50μm for tissue and 100μm × 100μm
for standard. A collision energy of 20 eV was used forof the S. lateralis section (Fig. 3b). To observe theregion-speciﬁc distribution of this molecule in S. lateralis,

NMN transition (m/z 335.1 to m/z 123.0). The SRM dwell

the histological images were overlaid on the ion image

time was set as 0.0016 sec/pixel. At least two serial sections(Fig. 3c). Interestingly, NMN was notably higher in someof the cockroach sample were analyzed under the same
experimental conditions. Following the measurement, tis-

speciﬁc parts of the S. lateralis abdomen, possibly in the
midgut. For further conﬁrmation of the NMN assignment,
pure NMN solution (1μg/mL) and extract from dry S. lat-sue sections were stained with hematoxylin and eosin(H&E) for histological analysis.
The ion image of DESI-TQ data was acquired and pro-eralis were spotted on the same slide and measured subse-

cessed using the updated version of MassLynx (Waters, ver-transition were clearly detected for pure NMN (Fig. 3d) andsion 4.2) software and HDI Imaging (Waters, version 1.6)

the S. lateralis extract (Fig. 3e).

quently. As expected, the ion images at the same NMN

software.
5June 2024

Fig. 1.

Medical Mass Spectrometry Vol. 8 No. 1

Calibration curve of NMN standards (a) and representative chromatogram of NMN obtained from six diﬀerent
species of insects (b).

Discussion

times higher sensitivity than the DESI-Q-TOF. In this study,
DESI-TQ successfully detected NMN in S. lateralis (Fig.

For the ﬁrst time, in this study, we demonstrated the
NMN imaging in biological tissue employing DESI-TQ in3), which could be attributed to its improved sensitivity. It
is to be noted that the sensitivity of DESI-TQ is inﬂuenced

SRM mode that showed higher sensitivity and excellent
selectivity than DESI-Q-TOF. Detection of NMN in tissue

by various factors, including dwell time. Dwell time refers

is challenging by IMS since this molecule is very labile andreadily undergoes in-source fragmentation under standard

to the duration the mass spectrometer spends analyzing aelectrospray ionization settings . DESI-Q-TOF is typically

Increasing dwell time leads to higher sensitivity as the
instrument has more time to accumulate ions from the sam-particular ion in a particular spot on the sample surface.

24)

used for untargeted analysis where the target ion is mixed
with myriads of other ions and could not be detected. In our
experience, DESI-TQ provides about ten to a thousandple. However, longer dwell times can compromise spatial
resolution. This is because the instrument spends more time6June 2024

Fig. 2.

Medical Mass Spectrometry Vol. 8 No. 1

Comparison of NMN content among diﬀerent
species of insects.
Diﬀerent letters indicate a signiﬁcant diﬀerence (p＜0.01) among diﬀerent species. One-way ANOVA was
performed followed by Tukeyʼs multiple comparison
tests among diﬀerent species of insects. All the data arerepresented as mean±SD. The same insect samples wereprepared in triplicate.

at each spot, resulting in a larger area being sampled, which
may reduce the ability to discriminate features at a ﬁner
spatial scale. Therefore, ﬁnding the optimal balance

Fig. 3.

between dwell time, sensitivity, and spatial resolution isrecommended.

Distribution of NMN in the whole body of S.
lateralis by DESI-TQ.
H&E images of S. lateralis section (a). Representative
DESI-IMS ion image for NMN (SRM transition at m/z
335.1 to m/z 123.0) in S. lateralis section (b), NMN
standard (d), and S. lateralis extract (e). Transparent
overlaid images between H&E and ion images for NMN
in S. lateralis (c).

The analysis of insects, such as S. lateralis, by IMS presents inherent challenges. Ideally, freezing the sample immediately after sacriﬁcing the animal or insect is crucial toprevent post-mortem degradation of certain analytes. However, the delicate anatomical structure of S. lateralis poses
diﬃculties in cryo-sectioning fresh-frozen samples without

dried the sample immediately after cryo-sectioning to minimize the NMN delocalization. Previously MALDI-IMS wasusing an embedding medium. To address this issue, we
employed a super cryo-embedding medium for the entire

used for peptide imaging in formalin-ﬁxed and paraﬃn-em-

body of S. lateralis. This approach facilitates cryo-section-25)

bedded (FFPE) S. lateralis . However, FFPE samples are
not suitable for NMN imaging since they require deparaf-

ing without compromising the mass spectral quality, thanks
to the mediumʼs monomeric property. It is important to notethat freezing samples using cryo-embedding medium

ﬁnization and extensive washing procedure.

requires a longer time compared to direct freezing, this pro-

(7 min run per injection) to screen and quantify NMN in
23)
plant extract using UPLC-MS/MS . SRM has been a gold

Our group recently developed a rapid SRM method

cedure may impact the imaging of certain biomolecules
susceptible to rapid post-mortem degradation. Fortunately,

standard for quantiﬁcation studies. We employed the same

there is no reported literature on the post-mortem degradation of NMN, supporting the compatibility of NMN imag-

method in our current study and found a signiﬁcantly
higher content of NMN in S. lateralis compared to otherspecies of insects. Cockroaches are one of the most suc-

ing in frozen and embedded samples.
One of the challenges in NMN imaging is the potential
delocalization during sample preparation. In our experi-

cessful and diverse insects in traditional and Chinese medi-

ence, NMN delocalization was observed when tissue sec-various natural raw foods, including edamame, broccoli,tion was stored in the freezer before analysis. This is

cucumber seed, cucumber peel, cabbage, avocado, tomato,

because NMN is highly soluble in water. In our study, wemushroom, beef, and shrimp. Among these, edamame

26)

cine . Previously Miles et al. investigated NMN content in7

June 2024

Medical Mass Spectrometry Vol. 8 No. 1(0.47‒1.88)
28)
mg/100 g . Considering the moisture content (60‒65%) ,showedthehighestcontentofimaging in tissue by DESI-TQ and revealed S. lateralis as a

NMN

27)

rich source of NMN, speciﬁcally in the abdomen. We

the NMN content in edamame should be approximately
1.18‒4.7 mg/100 g dry weight. In contrast, cockroaches

believe that, our method will help to develop other animal

have an NMN content of 15.5 mg/100 g dry weight (Fig. 2),

imaging in the ﬁeld of biomedical research. In the future,
NMN imaging could aid in understanding age-related

about 3 to 13 times higher than edamame. This is the ﬁrst

changes in NAD metabolism and its potential role instudy that revealed the surprisingly higher content of NMN

human health.

＋

in insects. Cockroach extracts have been used for centuries

Acknowledgment

to treat aches, pains, inﬂammation, and even chronic heart

This work was supported by the Cabinet Oﬃce, Government of Japan, Moonshot Research and Development Pro-29)

failure . The gut microbial metabolites of cockroach is avaluable source of new bioactive compounds with therapeu26)

tic promise for human health . Our study also indicatedthe promising use of this insect, particularly in pharmaceu-

gram for Agriculture, Forestry and Fisheries (funding
agency: Bio-oriented Technology Research Advancementticals. A recent study found that milk from cockroach species had a higher potential for nutrition than milk from typi-

Institution) Grant number JPJ009237 (to K.M.), JSPS
KAKENHI (Grant Number JP18H05268 to K.M.), and30)

cal mammals . As a result, it is thought of as a potential

JSPS KAKENHI (Grant Number JP23H69332 to K.M.).

new superfood that might be made accessible for ingestion
shortly. It is to be noted that cockroaches carry food-borne

Conﬂicts of Interest

pathogens that may transmit diseases to humans . There31)

The authors declare no conﬂict of interest.

fore, more research is required to focus on the sanitation
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10

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Medical Mass Spectrometry Vol. 8 No. 1

Research Paper

Sex-speciﬁc eﬀect of ethanol on colonic short-chain fatty acids andderivatives in the mouse model using targeted LC/MS
1,2#

Siddabasave Gowda B. Gowda*, Lipsa Rani Nath1#, Yonghan Li3,

Jayashankar Jayaprakash , Divyavani Gowda , Shu-Ping Hui *
1

1

2

2

Graduate School of Global Food Resources, Hokkaido University, Kita-9, Nishi-9, Kita-Ku, Sapporo 060‒0809, Japan
2
Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 060‒0812, Japan
3
Graduate School of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo 060‒0812, Japan

Abstract

Ethanol consumption aﬀects the human body, particularly the digestive system, leading to gut dysbiosis anddysregulation of metabolites. Although previous studies have focused on the eﬀects of ethanol on liver metabolism, colon-speciﬁc metabolites have not been well explored. In this study, we investigated the eﬀects of ethanol on colon-speciﬁcshort-chain fatty acids (SCFAs) and their derivatives in a mouse model using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Colonic ﬂush samples obtained from ethanol-fed (EF) and pair-fed (PF) mice of each sex were
subjected to targeted analysis of SCFAs and SCFA esters of hydroxy fatty acids (SFAHFA) using LC-MS/MS. The levelsof acetic acid (AA), propanoic acid (PA), and butyric acid (BA) were signiﬁcantly increased in the male mouse EF groupcompared to those in the PF group. In contrast, the levels of AA and PA were decreased in female mice in the EF groupcompared to those in the PF group. Furthermore, the levels of AA-derived SFAHFAs were decreased in the male EF groupbut increased in the female EF group compared to those in the control group. Compared to the control groups, hydroxy
fatty acid levels were signiﬁcantly decreased in the EF groups of both sexes. Overall, ethanol exerted diverse eﬀects oncolon-speciﬁc metabolites in both male and female mice. This study provides new insights into sex-dependent metabolicchanges associated with alcohol-induced gut microbiota dysfunction and their potential health impacts.
Key words: ethanol, short-chain fatty acids, SFAHFA, liquid chromatography, mass spectrometry, gut-microbial metabolites

1.

Introduction

health risks. Alcohol was ranked as the seventh leading

Alcohol is a globally prevalent psychoactive substancecause of death globally and contributed to 2.8 million

deeply rooted in various cultures that poses signiﬁcantdeaths in 2016 . The harmful eﬀects of alcohol on thedigestive system are closely related to the gut‒liver axis, in1)#

which the enterohepatic circulation is essential for trans-

Equally contributing authors*Corresponding author

12)

porting products from the gut to the liver . Alcohol disrupts
the gut‒liver axis, altering the microbial composition andSiddabasave Gowda B. Gowda

Faculty of Health Sciences, Hokkaido University, Kita-12,
Nishi-5, Kita-ku, Sapporo 060‒0812, Japan3)

gut barrier integrity , which leads to impaired nutrient
absorption and contributes to conditions such as alcoholicE-mail: gowda@gfr.hokudai.ac.jp
2
Shu-Ping Huihepatitis and cirrhosis. Dysbiosis disrupts tight junction
proteins, allowing the translocation of particles into portal

Faculty of Health Sciences, Hokkaido University, Kita-12,
Nishi-5, Kita-ku, Sapporo 060‒0812, Japan4)

circulation . Tight junction maintenance is facilitated byshort-chain fatty acids (SCFAs), which are byproducts ofTel: ＋81‒11‒706‒3693
E-mail: keino@hs.hokudai.ac.jp

anaerobic fermentation by the food ﬁbers . Fecal metabo5)lome analysis of patients with alcohol use disorders showeda decrease in SCFAs, which could be explained by dysbio-

Received: February 4, 2024. Accepted: February 28, 2024.
Epub April 16, 2024.

sis-impacting bacteria that produce SCFAs, including Fae-

DOI: 10.24508/mms.2024.06.002
11

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Medical Mass Spectrometry Vol. 8 No. 1

Chemical (Ann Arbor, MI, USA). Solid-phase extraction
cartridges (MonoSpin C18-AX) were provided by GL Sci-

6)

calibacterium .
Recent ﬁndings revealed a novel class of derivative lipids
of SCFAs, namely SCFA esters of hydroxy fatty acids
7)(SFAHFAs), which are abundant in the gut . Gut microbi-

ence (Tokyo, Japan). 2-Chloro-1-methylpyridinium iodide

ota is signiﬁcant in the formation of these lipids, which arevaleric acid (VA), and caproic acid (CA), were sourced(CMPI), along with propionic acid (PA), butyric acid (BA),8)

present in the colon and cecum . Understanding the eﬀectof alcohol on colonic-speciﬁc SCFAs and their derivatives

from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan).
The SFAHFA standards were synthesized in-house as per

was signiﬁcantly advanced when chemically generated

the protocols described in our previous study .

9)

SFAHFAs were measured in rat intestinal contents and
9)

2.2.

fecal samples .

Animal samples

Male and female C57BL6 mice (8‒10 weeks old) were

Although existing research has explored lipid metabolism in alcohol-induced gut dysbiosis, notable gaps exist in
our understanding of how ethanol speciﬁcally aﬀects

bred at the institutional core facility after obtaining
approval from The Institutional Animal Care and Use Com-

colonic SCFAs and SFAHFAs. A previous study used gas
chromatography‒mass spectrometry (MS) for analyzing

mittee at the University of Tennessee Health Science Center. Bothe male and female mice were fed a Lieber‒DiCarli

human stool samples and demonstrated that excessive alco-liquid diet (D710260, Dyets Inc., Bethlehem, PA, USA) for

hol consumption disrupts the intestinal microbiota andfour weeks with ethanol (0% for two days, 1% for twocauses dysbiosis, thus highlighting importance of fatty
acids (FAs) in intestinal health and linking microbiota dys-days, 2% for two days, 4% for one week, 5% for one week,
and 6% for one week) or pair-fed (PF) isocaloric maltodex-

10)function to alcohol-related pathologies . Furthermore,using liquid chromatography coupled with triple quadru-

trin (BioServ, Flemington, NJ, USA). At the end of the
four-week feeding period, both groups of mice were eutha-

pole and quadrupole time-of-ﬂight MS, previous studies

nized, and the colonic contents were ﬂushed with 3 mL of
0.9% saline. Flushed samples were frozen until lipid analy-

revealed that ethanol alters lipid metabolism by inﬂuencing
11,12)

the gut microbiota.

sis. All experiments were performed in compliance with the

However, the existing literature on lipid metabolism
in ethanol-induced gut dysbiosis is limited, and thesex-speciﬁc eﬀects of ethanol on gut lipid metabolism remain

relevant laws and/or institutional guidelines.

2.3.

unclear. Therefore, using liquid chromatography-tandem MS(LC-MS/MS), we investigated the sex-speciﬁc eﬀects ofExtraction and targeted analysis of SCFA by
LC-MS/MSThe extraction and analysis of SCFA were performed

ethanol on gut lipid metabolism in a mouse model. Unveil-using the method developed in our previous studies . Ini-

ing the intricate connections between alcohol consumption
and SCFA/SFAHFA variations, our ﬁndings contribute to atially, 80 μL of acetonitrile was added to 20 μL of colonic
ﬂush, followed by the addition of 100 μL of 20 μM AA-d4

nuanced understanding of the mechanisms, oﬀering thepotential for sex-speciﬁc therapeutic strategies against alco-

and 100 μL of 20 μM VA-d9 as internal standards. The mixture was then vortexed for 3 min at 3500 rpm and then cen-hol-related pathologies.

trifuged for 10 min at 15000 rpm and 4°
C. The supernatant
was carefully transferred into a new microtube, and derivat-

2.

Materials and Methods

2.1.

Materials

13)

ization was initiated by adding 20 μL of TEA and 10 μL of
CMPI of 2 mM each. The mixture was vortexed for 3 min at

LC/MS-grade solvents acetonitrile, methanol, and isopro-

3500 rpm, following which 20 μL of 2 mM DMED was

panol, 1 M aqueous ammonium acetate, and acetic acidadded to the microtube; the sample was further vortexed for(AA) were purchased from Wako Pure Chemical Industries,

30 min at 3500 rpm and centrifuged (10 min, 15000 rpm, 4°Ltd. (Osaka, Japan). The reagents triethylamine (TEA),
N,N-Dimethylethylenediamine (DMED), and acetic acid-d4

C). The centrifugate was transferred to an LC vial, and 5 μLof this solution was injected into the LC-MS system.

(AA-d4) and HPLC-grade chloroform were acquired from
Sigma-Aldrich (St. Louis, MO, USA). Pentanoic acid-d9

Targeted analysis was performed using a Prominence
ultrafast liquid chromatography (UFLC; Shimadzu, Kyoto,(PA-d9) and palmitic acid-d31 were obtained from Cayman

Japan) connected to a TSQ Quantum Mass Spectrometer
12June 2024

Medical Mass Spectrometry Vol. 8 No. 1

9)

System (Thermo Fisher Scientiﬁc Inc., San Jose, CA, USA)
operated in the positive-ion mode. The chromatographic

in our previous research . The elution solvents consisted of

separation was performed on a Hypersil GOLD C8 column(50 mm × 2.1 mm), which is a reversed-phase-type columnB), and methanol (pump C). An Atlantis T3 C18 column

with particle and pore sizes of 1.9 μm and 175 Å, respectively. The column oven was maintained at 40°
C through-for the chromatographic separation, which was maintained

10 mM ammonium acetate (pump A), isopropanol (pump(50 mm × 2.1 mm, 3 μm, Waters, Milford, USA) was used
at 40°
C with a ﬂow rate of 300 μL/min. The elution gradient
was set as follows: 40% B and 20% C (0‒7 min), 80% B

out the analysis. The mobile phase comprised a dual-solvent system: solvent A was Milli-Q water containing

and 10% C (7‒14 min), 30% B and 35% C (14‒18 min),

20 mM ammonium acetate, and solvent B was a mixture ofand returned to the initial conditions. For each injection, themethanol and acetonitrile (50:50, v/v). The ﬂow rate was

sample volume was set to 10 μL.

set to 0.3 mL/min. The gradient program was initiated with

2.5.

solvent B at 100% for the ﬁrst 1.5 min and held constant for

Statistical analysis

2 min. At 2.1 min, the proportion of solvent B was adjusted

Data were visualized using Microsoft Excel 2021 andto 50% and maintained for 7 min. Subsequently, solvent B

GraphPad Prism version 8.0.1. Statistical analysis involved
Studentʼs t-test with p＜0.05, which was considered signiﬁ-

was returned to 100% after 7.5 min and maintained for
10 min. The electrospray ionization (ESI) source parame-cant.

ters were as follows: the spray voltage was 3,500 V; the3.

sheath gas was 40 arbitrary units; the auxiliary gas was 25

Results
Fig. 1 shows the outcomes of SCFA analysis of the colon

arbitrary units; the capillary temperature was 250°
C; andthe HESI vaporizer temperature was 150°
C. The SCFA con-contents of EF and PF mice. The concentration of eachcentrations were calculated from the linearity curves constructed using Xcalibur 2.2 software (Thermo Fisher Scien-acquired SCFA level is shown in the bar graphs. Male PF

tiﬁc., San Jose, CA, USA).

mice. In particular, AA and PA levels signiﬁcantly

and EF mice exhibited lower SCFA levels than female
decreased in the EF group of females, whereas the trend

2.4.

Extraction and targeted analysis of SFAHFAs by
LC-MS/MS

was reversed in males. Conversely, BA and VA levels
increased in male and female mice in the EF group, respec-

The extraction and analysis of SFAHFAs were performed

tively. However, no signiﬁcant diﬀerences were observed in9)using a method developed in our previous studies .
Approximately 50 μL of colonic ﬂush from ethanol-fed (EF)CA levels between sexes.

mice samples were transferred to Eppendorf tubes. To this,samples are shown in Fig. 2 (A‒C). The overall SFAHFA

100 μL of an internal standard mixture in methanol wasconcentrations were higher in males than in females. In
particular, the levels of the AA ester of 2-hydroxy palmitic

The concentrations of SFAHFAs detected in colonic ﬂush

added, which comprised 5 μM deuterated SFAHFA standard
mixture (PAHPA-d30, BAHPA-d30, VAHPA-d30, andacid (2-AAHPA), AA ester of 2-hydroxy arachidic
acid (2-AAHAA), AA ester of 2-hydroxy behenic

HAHPA-d30). In addition, 50 μL of methanol and 400 μL of
Milli-Q water were added to the mixture. The resulting mix-

acid (2-AAHBA), AA ester of 2-hydroxy tricosylic
acid (2-AAHTA), AA ester of 2-hydroxy lignoceric acidture was vortexed (3500 rpm, 1 min) and centrifuged(15,000 rpm, 1 min, 4°C) to collect the supernatant for sol-

(2-AAHLA), and AA ester of 2-hydroxy pentacosylic acid
(2-AAHPSA) exhibited signiﬁcant increases in the EF

id-phase extraction using the MonoSpin C18-AX cartridges.
The detailed workﬂow for the solid-phase extraction forgroup of females compared to those in the PF group, whereas

9)

SFAHFAs is described in our previous study .

a reversed trend was observed in males. Furthermore, the AA
ester of 2-hydroxy heneicosylic acid (2-AAHHA), BA ester

Lipidomic analysis was conducted using a TSQ Quantum(Thermo Fisher Scientiﬁc, Waltham, MA, USA) coupledof 2-hydroxy behenic acid (2-BAHBA), PA ester of
2-hydroxy cerotic acid (2-PAHCA), BA ester of 2-hydroxywith the Prominence UFLC system (Shimadzu, Kyoto,
Japan) operating under ESI in negative mode. The LC-MS

cerotic acid (2-BAHCA), CA ester of 2-hydroxy cerotic
acid (2-CAHCA), BA ester of 2-hydroxy lignoceric acidconditions closely adhered to the methodology established(2-BAHLA), and PA ester of 2-hydroxy pentacosylic acid

Access MAX Triple Quadrupole Mass Spectrometer

13

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Medical Mass Spectrometry Vol. 8 No. 1

Fig. 1.

Amount of SCFAs in colon contents of PF and EF groups of male and female mice.

(M_PF: Male pair-fed, M_EF: Male ethanol-fed, F_PF: female pair-fed, F_EF: female ethanol-fed, n＝4 for each group). Studentʼs t-test: p＜
0.001 (a), p＜0.01 (b), p＜0.05 (c), and ns: non-signiﬁcant.

Fig. 2.

Amount of SFAHFAs detected in colon contents of the PF and EF groups of male and female mice.

The concentrations of SFAHFAs at the molecular level are shown in A, B, and C, and the total SFAHFAs of each SCFA-derived lipids areshown in D. (M_PF: Male pair-fed, M_EF: Male ethanol-fed, F_PF: female pair-fed, F_EF: female ethanol-fed, n＝4 for each group). Studentʼs t-test: p＜0.001 (a), p＜0.01 (b), p＜0.05 (c), and ns: non-signiﬁcant.

(2-PAHPSA) did not show any signiﬁcance between the EF

whereas no such diﬀerences were observed in male EF
mice. However, the levels of the VA ester of 2-hydroxy

and PF groups of sexes.
The levels of the AA ester of 2-hydroxy cerotic acid
(2 AAHCA) and PA ester 2-hydroxy lignoceric acid(2-PAHLA) were signiﬁcantly increased in female EF micecerotic acid (2-VAHCA) and VA ester of 2-hydroxy pentacosylic acid (2-VAHPSA) were signiﬁcantly decreased infemale EF mice, but no signiﬁcant diﬀerence was observed
14June 2024

Medical Mass Spectrometry Vol. 8 No. 1

in male mice. Fig. 2(D) shows the total amount of SFAH-

16)

lism . Therefore, metabolites (such as SCFAs) produced
by beneﬁcial bacteria in the gut aid absorption and utiliza-

FAs based on their SCFA acyl chains in the PF and EF miceof both sexes. In female mice, the levels of AA-derived

tion. These metabolites also support mucosal immunity,

SFAHFAs were signiﬁcantly increased in the EF group,

preserve intestinal homeostasis, and shield the intestinal

whereas the opposite trend was observed for the male mice.

mucosal barrier.

However, BA showed no signiﬁcant diﬀerences between
sexes. The levels of PA-derived SFAHFAs were signiﬁ-

As shown in Fig. 1, a signiﬁcant decrease in the SCFA
concentration was observed for the female EF mice com-

cantly increased in the female EF group, whereas those of
VA-derived SFAHFAs decreased. The concentrations of

pared to female PF mice. The relevance of these ﬁndings ishydroxy FAs (HFAs) in PF and EF mice of both sexes are

humans with alcohol use disorders. This independent study

shown in Fig. 3. Notably, higher concentrations of HFAs
were observed for females than males except for 2-hydroxy

revealed a reduction in SCFA levels, which is consistentpalmitic acid (2-HPA) in both the PF and EF groups. A sig-

attributable, in part, to dysbiosis, which negatively aﬀects
6,10)
SCFA-producing bacteria, namely Faecalibacterium .

further supported by a study on the fecal metabolome inwith our results. The observed decrease in SCFAs is likely

niﬁcant decrease in HFA concentration was observed in

The observed increase in SCFA levels in male EF mice may
be attributed to inherent sex-speciﬁc hormonal and physio-

female mice with EF. However, no signiﬁcant variations
were observed for HFA concentrations in male EF mice,
except for 2-HPA and 2-hydroxy behenic acid (2-HBA),

logical diﬀerences. This is in agreement with a recent study
on Chinese bamboo rats, which showed signiﬁcant diﬀer-

which were signiﬁcantly decreased in the EF group. The
concentrations of all the SCFA, SFAHFA, and HFA ana-

ences in microbial composition and genetic/metabolic path-

provided in Table 1. The data shows a large deviation in theways between female and male rats despite shared captive
17)
and feeding conditions . These sex-speciﬁc variations may

concentration levels among each animal, suggesting that

contribute to unique responses to alcohol exposure, inﬂu-

additional experiments with a large animal number would

encing SCFA levels. The congruence between our mousebe essential to draw any further conclusions.

model results and the insights from the bamboo rat study
highlights a robust association between ethanol consump-lyzed in the colonic ﬂush samples of PF and EF mice were

4.

Discussions

tion, gut dysbiosis, and alterations in SCFA levels in a
sex-speciﬁc manner. Furthermore, parallel ﬁndings across

Alcohol-induced dysbiosis has a detrimental eﬀect on gut

integrity, leading to the development of acute (alcoholic
hepatitis) and chronic (alcohol-related cirrhosis) liver disor-

diﬀerent species enhance our understanding of the intricate

ders. Many metabolites such as SCFAs are produced by theand downstream physiological eﬀects.

relationships between alcohol consumption, gut microbiota,

anaerobic gut microbiota as byproducts of the fermentation

The modulation of SFAHFAs in the PF and EF groups of

14,15)and have been demonstrated to havemultiple favorable eﬀects on mammalian energy metaboof dietary ﬁbers

Fig. 3.

both sexes is detailed in Fig. 2. Previous investigations into
SFAHFAs have indicated a decline in their levels in theAmount of hydroxy fatty acids detected in the PF and EF groups of male and female mice.

(M_PF: Male pair-fed, M_EF: Male ethanol-fed, F_PF: female pair-fed, F_EF: female ethanol-fed). Studentʼs t-test: p＜0.001 (a) and p＜0.01 (b).

15

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Table 1. The concentrations of short-chain fatty acids, SFAHFAs, and hydroxy fatty acids analyzed in the colonic ﬂush samples
Male mice
PF1

PF2

PF3

PF4

EF1

Female mice
EF2

EF3

EF4

PF1

PF2

PF3

PF4

EF1

EF2

EF3

EF4

SCFA
Acetic acid
Propanoic acid
Butyric acid
Valeric acid
Caproic acid170.27
12.49
4.52
12.41
0.32

179.21
13.89
4.27
15.56
0.83

141.17
9.47
4.21
9.97
0.59

72.10
7.98
1.15
3.67
0.78

347.47
22.05
28.96
12.65
1.24

359.45
23.56
35.08
14.94
0.28

Concentration (μmol/L)
303.28 361.51 1938.33 1781.13 2181.98 2822.14 1205.66 1526.45 1266.54 1112.41
21.44
18.05 113.18 118.68 123.34 180.34
72.94
69.97
63.74
65.13
26.96
32.18 381.42 375.36 428.11 440.38 388.65 410.53 384.06 309.70
10.60
13.81
16.82
18.53
21.19
17.47
22.38
23.92
26.68
17.78
2.33
0.33
0.51
1.48
0.95
1.73
1.87
1.75
2.05
0.68

SFAHFA
2-AAHPA
2-AAHAA
2-AAHHA
2-AAHBA
2-BAHBA
2-AAHTA
2-AAHCA
2-PAHCA
2-BAHCA
2-VAHCA
2-CAHCA
2-AAHLA
2-PAHLA
2-BAHLA
2-AAHPSA
2-PAHPSA
2-VAHPSA
AA_SFAHFA
PA_SFAHFA
BA_SFAHFAVA_SFAHFA

13.14
9.86
4.69
82.97
14.76
53.74
24.07
4.93
59.86
11.10
200.01
86.57
9.73
8.80
4.69
7.44
10.54
279.72
22.10
83.43
21.64

16.03
13.74
3.27
125.93
19.52
40.22
24.41
15.74
40.31
15.50
190.97
58.90
4.99
1.34
10.92
0.54
13.67
293.42
21.26
61.17
29.17

18.49
33.51
14.75
206.27
8.33
56.92
27.96
8.32
94.74
20.02
207.92
75.62
2.63
5.80
6.39
9.40
11.44
439.9120.35
108.87
31.46

24.00
56.24
14.28
210.27
8.45
71.75
26.50
7.66
108.01
20.39
165.63
95.98
6.17
11.45
15.34
4.28
10.61
514.3518.11
127.91
31.00

0.78
4.31
5.58
18.07
0.00
8.25
23.91
4.33
77.16
20.21
200.45
20.05
13.49
0.00
2.21
2.17
13.28
83.16
20.00
77.16
33.49

4.00
0.59
2.59
5.62
0.00
3.45
21.59
22.30
56.25
20.60
208.75
13.44
13.10
0.88
5.40
2.28
12.37
56.67
37.68
57.13
32.97

Concentration (pmol/L)
3.62
1.99
0.87
0.00
4.91
2.68
1.90
1.20
0.62
4.80
2.48
3.41
12.66
14.81
4.11
8.23
6.20
17.58
23.43
7.32
11.69
20.53
5.33
6.45
25.06
27.32
17.04
17.26
8.36
6.18
1.74
1.76
83.23
67.73
67.42
78.72
23.68
13.51
17.03
16.63
191.88 158.62 135.42 112.72
22.17
13.00
11.16
13.41
3.13
5.24
0.50
5.43
17.23
0.00
1.37
0.00
4.91
2.64
3.23
2.64
7.06
1.90
12.05
6.17
17.55
11.49
7.23
11.35
85.63
87.77
46.12
52.59
18.55
13.33
14.29
13.35
106.66
85.31
92.22
86.05
41.23
25.00
24.25
27.98

0.48
0.07
0.74
0.43
0.96
0.08
0.11

0.40
0.04
0.58
0.30
0.66
0.06
0.09

0.48
0.04
0.50
0.23
0.54
0.05
0.08

0.98
0.08
1.27
0.69
1.60
0.12
0.11

0.23
0.07
0.60
0.28
0.68
0.08
0.10

0.15
0.04
0.30
0.13
0.28
0.05
0.07

HFA
2-HPA
2-HAA
2-HHA
2-HBA
2-HTA
2-HLA
2-HPSA

Concentration (nmol/L)
0.20
0.28
0.29
0.05
0.06
0.20
0.36
0.42
1.90
0.19
0.20
1.04
0.44
0.49
2.33
0.05
0.07
0.21
0.09
0.10
0.33

0.49
0.41
6.79
2.13
4.87
0.45
0.66

0.00
0.87
0.00
6.56
0.00
1.70
22.09
4.32
47.28
18.31
109.77
11.19
2.12
3.27
2.22
4.45
10.07
44.63
10.89
50.55
28.38

0.56
2.33
4.09
8.57
2.62
3.43
23.29
1.80
104.3520.12
128.03
7.28
0.94
0.69
4.01
2.55
11.41
53.55
5.29
107.66
31.53

3.94
3.69
0.93
31.37
2.39
19.71
26.82
3.10
76.47
18.54
109.25
36.32
31.76
0.00
7.38
3.13
5.81
130.16
37.99
78.86
24.35

5.77
4.58
2.16
18.15
13.73
7.34
38.15
0.00
78.12
14.51
150.49
30.99
7.71
0.00
6.74
4.61
7.49
113.89
12.32
91.85
22.00

7.77
10.88
1.08
70.79
0.00
29.85
27.33
4.94
50.83
13.28
68.24
115.21
66.58
4.29
15.12
5.24
7.00
278.04
76.76
55.13
20.28

4.59
14.77
1.81
74.44
1.29
28.01
21.54
2.89
60.93
11.31
63.15
119.97
28.26
1.95
14.14
11.52
7.06
279.26
42.67
64.17
18.37

0.52
0.44
5.78
2.19
5.11
0.47
0.71

0.38
0.31
2.95
1.50
3.41
0.32
0.53

0.21
0.06
0.66
0.31
0.80
0.09
0.14

0.18
0.04
0.59
0.21
0.58
0.07
0.10

0.41
0.10
1.39
0.47
1.20
0.11
0.14

0.39
0.12
1.38
0.51
1.29
0.11
0.12

colons of rats subjected to a high-fat diet, in contrast to an

observed. As the generation of HFAs is linked with theincrease observed in mice infected with the inﬂuenza
7,18)
virus . The observed downregulation of speciﬁc SFAH-

abundance of free FAs (FFAs), these HFA results are inagreement with previous research that highlighted the piv-FAs derived from PA and BA in antibiotic-treated mouseotal role of FFA metabolism in the progression of alcoholic
22)
liver disease . The implications of FFAs in alcohol-in-

fecal samples underscores their speciﬁcity for the gut
12)
microbiota . Previous studies have consistently demon-

duced gut dysbiosis, characterized by microbiota imbalances and potential contribution to gastrointestinal disor-

strated that supplementation with SCFAs, achieved throughinterventions such as a high-ﬁber diet or probiotics,

23)

ders, have been accentuated in numerous studies . An
animal study has suggested that maintaining optimal intesti-enhanced gut epithelial integrity and mitigated liver injury
19,20,21). Considering the apparent associin alcoholic models

nal FFA levels, particularly saturated fatty acids, canenhance and preserve the integrity of the intestinal gut bar-

ation between SFAHFAs and SCFAs, these two biomole-rier . The observed decline in HFA concentration after eth24)

cules are reasonably correlated. This consistency strongly
supports the continued exploration of interventions involv-

anol consumption in our study adds to this understandingby emphasizing the intricate interplay between alcohol con-ing SCFAs and SFAHFAs as promising strategies foraddressing pathologies associated with alcohol consump-

sumption, FA metabolism, and gut health. These ﬁndings
reinforce the importance of targeted interventions to modu-

tion.

late HFAs, particularly in maintaining intestinal barrier
integrity, as a potential strategy for addressing alcohol-re-

A notable decrease in the HFA concentration in female
EF mice is shown in Fig. 3. In male EF mice, except for
2-HPA and 2-HBA, no signiﬁcant alterations werelated pathologies. The ﬁndings of the study are summarized16June 2024

Fig. 4.

Medical Mass Spectrometry Vol. 8 No. 1

Summary of changes in the precursor (FA 22:0(2OH), AA) and product (2-AAHBA) SFAHFA metabolism.

English editing expenses.

in Fig. 4 using a representative FA and its metabolites. The
concentration of precursor FA 22:0(2OH) was decreased in

Conﬂict of interest

the EF group of both sexes, and that of AA was increased in
the male EF mice but decreased in female EF mice. How-

The authors declare no competing ﬁnancial interest.

ever, an opposite trend was observed for 2-AAHBA in the
EF groups of both sexes. This study has some limitations,

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18

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Research Paper

Evaluation of four drug screening devices for
detection of psychoactive drugs in pericardial ﬂuid
1,2
1,2
1,2
Masae Iwai *, Tadashi Ogawa , Tomohito Matsuo ,
1,3

1,4

1,2

Katsutoshi Kubo , Fumio Kondo , Hiroshi Seno
1

Department of Legal Medicine, Aichi Medical University School of Medicine,1‒1 Yazakokarimata, Nagakute, Aichi 480‒1195, Japan
2
Poison Analysis Center, Aichi Medical University School of Medicine,1‒1 Yazakokarimata, Nagakute, Aichi 480‒1195, Japan3
Department of Oral Pathology/Forensic Odontology, Aichi Gakuin University School of Dentistry,
1‒100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464‒8650, Japan
4
Department of Biomedical Sciences, Chubu University College of Life and Health Sciences,
1200 Matsumoto-cho, Kasugai, Aichi 487‒8501, Japan
Abstract
SIGNIFY

We evaluated the applicability of four immunoassay-based drug screening devices, Triage TOX Drug Screen,®

TM

ER, IVeX-screen M-1, and DRIVEN-FLOW M8-Z, in the detection of psychoactive drugs in pericardial ﬂuid

as an alternative to urine. A total of 38 pericardial ﬂuid samples from forensic autopsies were analyzed with the four drug
screening devices. To conﬁrm the results, the concentrations of psychoactive drugs in pericardial ﬂuid samples were measured together with those in urine and blood samples by liquid chromatography-tandem mass spectrometry. Only IVeXscreen M-1 precisely detected psychoactive drugs without false positive results, whereas Triage TOX Drug Screen and®

SIGNIFYTM

ER showed several false positive results, and DRIVEN-FLOW M8-Z led to many false positive results. These

results suggest that IVeX-screen M-1 is more useful than other screening devices for psychoactive drugs in pericardial ﬂuid
and that pericardial ﬂuid is a valid alternative material when urine is not available.
Key words: drug screening devices, psychoactive drugs, pericardial ﬂuid, liquid chromatography-tandem mass spectrometry

3‒5)

Introduction

detection of amphetamines. These false positive results
are particularly important in the context of forensic autop-

The detection of possible drug consumption is a central

sies, as they can be exacerbated by the production of putrefactive amines, such as 2-phenethylamine, by saprogenic

1)

aspect of forensic toxicological examinations . Devices used
for urine drug screening can provide a rapid indication of the2)

presence of analytes of interest during autopsy , but thesebacteria in moderately or heavily decomposed bodies.

devices are associated with several drawbacks. For example,

In our previous report, we evaluated the performance of®

®

Triage DOA frequently returns false positive results in theﬁve drug screening devices, Triage TOX Drug Screen,TM
ER, IVeX-screen M-1, Status DS10, andSIGNIFY* Corresponding author

DRIVEN-FLOW M8-Z, in the detection of amphetamines

Masae Iwai

and methamphetamines in urine containing putrefactive
amines. The results suggested that DRIVEN-FLOW M8-Z

Department of Legal Medicine, Aichi Medical University
School of Medicine, 1‒1 Yazakokarimata, Nagakute, Aichi

was more useful than other screening devices for screening

480‒1195, Japan
E-mail: masaoshi@aichi-med-u.ac.jp

Received: November 30, 2023. Accepted: March 31, 2024.

of methamphetamines in the presence or absence of
2-phenethylamine, while none of the tested devices

Epub May 20, 2024.

detected amphetamines precisely .

6)Another issue with the forensic detection of drug use is

DOI: 10.24508/mms.2024.06.003
19

June 2024

Medical Mass Spectrometry Vol. 8 No. 1that collection of urine is not always feasible. In these

zepam and ﬂunitrazepam (Eisai, Tokyo, Japan); brotizolam

cases, blood, pericardial ﬂuid, cerebrospinal ﬂuid, vitreous
humor, or other alternative ﬂuids must be analyzed. In par-(Sumitomo Pharma, Tokyo, Japan); estazolam (Takeda
Pharmaceuticals, Tokyo, Japan); phenobarbital, amitripty-

ticular, pericardial ﬂuid often can be readily obtained fromline, nortriptyline, diazepam and triazolam (Fujiﬁlm Wako
Pure Chemical, Osaka, Japan); temazepam, zolpidem, diaz-the pericardial cavity. Typical volumes of pericardial ﬂuid
amount is suﬃcient for forensic drug testing. Because of

epam-d5, and phenobarbital-d5 (Sigma-Aldrich, St. Louis,
MO, USA). High-performance liquid chromatography

the potential utility of this material in forensic analyses, in(HPLC)-grade methanol was obtained from Fujiﬁlm Wako

the present study we evaluated the applicability of four®
drug screening devices, Triage TOX Drug Screen, SIGNI-

Pure Chemical (Osaka, Japan). Other common chemicals

7)

taken at the time of autopsy range from 5 to 35 mL ; thisFY

TMused were of the highest purity commercially available.
Laboratory distilled water was puriﬁed using a Direct-Q

ER, IVeX-screen M-1, and DRIVEN-FLOW M8-Z, tothe detection of psychoactive drugs in pericardial ﬂuid from
autopsy samples. In this analysis, liquid chromatogra-

UV 3 system (Millipore, Molsheim, France).

phy-tandem

Biological samples

mass

spectrometry(LC-MS/MS)

was

Pericardial ﬂuid, urine and blood samples were obtained

employed as a sensitive and quantitative tool to compare
the performance of these immunoassay-based devices.

from autopsy cadavers at Aichi Medical University from2021 to 2023. Samples were collected in 5- or 15-mL tubes

Materials and Methods

and stored at −80°
C until analysis. Additionally, pericardial

Chemicals and materials

ﬂuid samples were assessed without any pretreatment.

Amphetamine was kindly provided by Dr. Kenji Hara
(Fukuoka University). The following standard drugs were

Devices and their principles of detection

used: methamphetamine (Dainihonseiyaku, Osaka, Japan);amobarbital (NIPPON SHINYAKU, Kyoto, Japan); broma-

Triage TOX Drug Screen (Alere San Diego, CA, USA),TM
SIGNIFY ER (Innovacon, CA, USA), IVeX-screen M-1

Table 1.

®

Cutoﬀ values of psychoactive drugs for four drug screening devices
Cutoﬀ values (ng/mL)

Drug classiﬁcation

Barbiturates
Sedative-hypnotics

Tricyclic antidepressantsStimulants

Drug name

Abbreviation*

1®

Triage TOX
Drug Screen

SIGNIFYER

TM

IVeX-screen
M-1

DRIVENFLOW M8-Z

Phenobarbital

BAR

230

100

150

2200

Amobarbital

BAR

250

300

200

2000

Bromazepam

BZO/BZDBZO/BZD

750
̶*2

1562
̶*2

600

Brotizolam＞10000Estazolam

BZO/BZD

400

2500

400

500
̶*2
̶*2

Flunitrazepam

BZO/BZD

200

390

400

̶*2

Lorazepam

BZO/BZD

200

1562

2000

Midazolam

BZO/BZD

200

12500

Temazepam

BZO/BZD

200

98

10000
̶*2

550
̶*2
̶*2

Triazolam

BZO/BZD

300

ZOL

2500
̶*2

600

Zolpidem

100
̶*2

50

50

Amitriptyline

TCA

600

1500

300

1000

Nortriptyline

TCA

900

1000

1000

1000

Amphetamine

AMP

500

10000085000

MethamphetaminemAMP/METH/MET

500

1000
̶*2

500

500

*1BAR; barbiturates, BZO/BZD; benzodiazepines, ZOL; zolpidem, TCA; tricyclic antidepressants, AMP; amphetamines, mAMP/METH/
MET; methamphetamines.
*2Not available.

20June 2024

Medical Mass Spectrometry Vol. 8 No. 1(Biodesign, Tokyo, Japan) and DRIVEN-FLOW M8-Z

m/z 225 → 42 for amobarbital, m/z 136 → 91 for amphet-(Alfa Scientiﬁc Designs, CA, USA) were assessed in thisstudy. All four devices are based on a competitive immuno-

for amitriptyline, m/z 264 → 233 for nortriptyline, m/zamine, m/z 150 → 91 for methamphetamine, m/z 278 → 91

316 → 209 for bromazepam, m/z 393 → 314 for brotizolam,assay and give qualitative responses to the presence orm/z 285 → 193 for diazepam, m/z 295 → 267 for estazolam,m/z 314 → 268 for ﬂunitrazepam, m/z 301 → 255 for temaze-absence of drugs, and they are recommended to be used forurine only by their manufacturers. Cutoﬀ values that arepam, m/z 343 → 308 for triazolam, m/z 308 → 235 for zolpidem, m/z 290 → 154 for diazepam-d5 and m/z 236 → 42 forused by the devices to determine positive results are shownin Table 1.

phenobarbital-d5.

LC-MS/MS analysis

Ethics approval

Samples of human pericardial ﬂuid, urine, or blood(100 μL) were mixed with 100 μL methanol and 200 μL ace-

All experiments were approved by the Ethics Committeeof Aichi Medical University (approval no. 2020-172).

tonitrile after addition of 40 μL internal standard solution(0.5 μg/mL diazepam-d5 and 25 μg/mL phenobarbital-d5).

Results

The mixture was vortexed for 60 s and centrifuged at

A total of 38 pericardial ﬂuid samples with matched

15,000 g for 10 min, and the supernatant was transferred toanother tube, followed by addition of 100 μL of 0.1% triﬂu-

urine and blood samples were obtained during forensic

oroacetic acid in acetonitrile. The solvent was removed
with a centrifugal evaporator (CVE-200D; Tokyo Rikaki-

autopsies. The presence of psychoactive drugs was ﬁrst®
evaluated in urine by Triage TOX Drug Screen or SIGNI-

kai, Tokyo, Japan). The residue was reconstituted in 100 μL
methanol and centrifuged at 15,000 g for 1 min. A 5 μL ali-

FY

quot of supernatant was subjected to analysis by LC-MS/
MS. Samples containing high concentrations of target com-

The concentrations of the drugs in urine and blood samples
were also determined by LC-MS/MS. The concentrations ofpounds were analyzed after dilution as needed.
LC-MS/MS analyses were performed using a Nexera X2

drugs that were present in these samples were determined
to be within the ranges of 2.0 to 20,300 ng/mL in urine sam-liquid chromatograph coupled to an LCMS-8040 massples (Table 2) and 0.50 to 3230 ng/mL in blood samples

spectrometer (Shimadzu, Kyoto, Japan). For separation, a(Table 3). Because of low sample volumes, four urine andthree blood samples were not analyzed by LC-MS/MS.

TM

ER. Nine urine samples (cases 1‒9) tested positive

for the presence of at least one psychoactive drug (Table 2).

Kinetex column (2.1 mm I.D. × 100 mm, particle size
2.6 μm; Phenomenex, Cheshire, UK) was used. The column

Pericardial ﬂuid samples were then subjected to analysis

temperature was maintained at 40°
C. The gradient systemwith four drug screening devices: Triage TOX Drug
TM
ER, IVeX-screen M-1 and DRIVScreen, SIGNIFY®

used for separation included mobile phase A (a solution ofand mobile phase B (a solution of 0.1% formic acid in

EN-FLOW M8-Z; these samples were also analyzed by
LC-MS/MS. LC-MS/MS analysis revealed that 9 samples

10 mM ammonium formate in methanol). The ﬂow rate was(cases 1, 2, 3, 6, 7, 8, 9, 11, 14 and 16) contained at least

0.5 mL/min. The elution gradient involved a linear increase

one type of psychoactive drug at a concentration between
9.8 and 3270 ng/mL (Table 4). A representative LC-MS/MS

0.1% formic acid in 10 mM ammonium formate in water)from 5% B to 95% B over 3.0 min, followed by constant
5% B over 0.01 min and maintained at 5% B for 3.0 min to

analysis of a pericardial ﬂuid sample (case 7) shows peaks
with the same retention times as those of standard amitrip-

equilibrate the column for the next sample. The desolvation

tyline (6.25 min) and nortriptyline (6.30 min) (Fig. 1a).

line temperature and heat block temperature were 250°
C

Diagnostic fragment ions and their ion abundance ratios
were also fully consistent with those of standard amitripty-

95% B for 1.5 min. The mobile phase was then returned toand 400°
C, respectively.

line and nortriptyline, thus conﬁrming the presence of these

Electrospray ionization was applied in the negative mode

compounds in this pericardial ﬂuid sample (Fig. 1b).

for phenobarbital and amobarbital and positive mode forother compounds. Quantiﬁcation was performed by

The results from the application of the four drug screen-

selected reaction monitoring (SRM) using the peak area.

ing devices to pericardial ﬂuid samples are summarized in

The SRM transitions were m/z 231 → 42 for phenobarbital,®

Table 4. The Triage TOX Drug Screen device returned
21June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Table 2.
Case

Summary of results for drug screening devices and LC-MS/MS in urine samples

drug screening device®

mAMP, BZO, BAR

Phenobarbital (4630), Methamphetamine (1140),
Amphetamine (247), Estazolam (37.3)®

AMP, mAMP, BZO

Methamphetamine (1520), Amphetamine (317),
Bromazepam (150),

1

Triage TOX Drug Screen Drug Screen

2

Triage TOX Drug Screen

3

SIGNIFYTM

BAR

Phenobarbital (6250)®

ER

mAMP, BZO

Methamphetamine (13260), Amphetamine (970),
Temazepam (55.3), Diazepam (7.0)®

BZO

Lorazepam (4.0)®

BAR

Phenobarbital (1030)®

BZO, TCA

Amitriptyline (513), Nortriptyline (200),
Triazolam (1.2), Flunitrazepam (0.89)®

BAR

Amobarbital (380)®

AMP, mAMP, BZO

Methamphetamine (20300), Amphetamine (2680),
Brotizolam (7.4), Flunitrazepam (5.2)
̶
NT*

4

Triage TOX Drug Screen

5

Triage TOX Drug Screen

6

Triage TOX Drug Screen

7

Triage TOX Drug Screen

8

Triage TOX Drug Screen

9

Triage TOX Drug Screen

10

Triage TOX Drug Screen

11

SIGNIFY

TM

ER

̶

12

SIGNIFY

TM

̶

®

13
14

ER

̶

®

Triage TOX Drug Screen

̶

®

̶

®

̶

NT*
̶

®

̶

Zolpidem (0.20)
̶

®

̶

̶

Triage TOX Drug Screen

15

Triage TOX Drug Screen

16
17‒28

Triage TOX Drug Screen

29‒38

LC-MS/MS (concentration, ng/mL)

Positive test result

NT*
NT*

®

Triage TOX Drug Screen or
IVeX-screen M-1
Triage TOX Drug Screen or
SIGNIFYTM ER*Not analyzed due to insuﬃcient sample quantity.

Table 3.

Quantiﬁcation of psychoactive drugs in blood samples by LC-MS/MS

Case

Psychoactive drug (concentration, ng/mL)

1

Phenobarbital (3230), Estazolam (117), Methamphetamine (28.0), Amphetamine (5.1), Flunitrazepam (13.3)

2

Bromazepam (280), Methamphetamine (40.4), Nitrazepam (23.5), Amphetamine (17.5)

3

Phenobarbital (2610)

4

Methamphetamine (23.0), Temazepam (3.1), Amphetamine (3.0)

5Lorazepam (3.7)

6

Phenobarbital (1750)

7

Amitriptyline (986), Nortriptyline (221), Flunitrazepam (6.9), Clonazepam (4.8), Triazolam (2.9)

8Amobarbital (670)9Methamphetamine (190), Amphetamine (38.3), Flunitrazepam (19.1)
̶

10
11
12

Midazolam (3.4)
NT*

14

NT*
NT*

15

̶

16

Zolpidem (0.50)
̶

13

17‒28
29‒38

̶

*Not analyzed due to insuﬃcient sample quantity.

22

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Summary of results for four drug screening devices and LC-MS/MS in pericardial ﬂuid samples

Table 4.
®

Triage TOX
Drug Screen

SIGNIFY

1

BAR

BAR

2

̶

3

BAR

4

AMP*
̶

Case

5
6

TM

ER

Phenobarbital (744), Methamphetamine (7.0),
Amphetamine (0.95) Estazolam (35.8)

BZO* , TCA*̶

TCA* , ZOL*

2

Bromazepam (24.1), Methamphetamine (8.9),
Amphetamine (2.5)

BAR
̶

BAR
̶

2
BAR, ZOL*
2ZOL*

Phenobarbital (3270)

̶

̶

ZOL*
2
ZOL*

2

BAR
3
TCA*

BAR
3
TCA*

BAR

8

3
BAR*

̶

9

BZO*

3
BAR*
3
METH*

3

11

̶

12

̶

13
14

BZO*
3
BZO*

TCA*
̶
̶

̶

15

*1

̶

̶

16
17‒28

̶

̶

̶

29‒38

̶

1

2

̶
̶

2

̶

̶

Methamphetamine (6.3), Amphetamine (0.60)
̶
Phenobarbital (505)

3

̶

3
BZO*

2

2

TCA

AMP*
2
TCA*

10

LC-MS/MS (concentration, ng/mL)

2
2
TCA* , ZOL*

7

1

DRIVENFLOW M8-Z

BAR
3

1

IVeX-screen
M-1

TCA*
2
ZOL*

Amitriptyline (488), Nortriptyline (33.1)

MET
2
2
TCA* , ZOL*

Methamphetamine (73.2), Amphetamine (9.8)
̶

2
ZOL*
2
2
TCA* , ZOL*

Midazolam (1.6)
̶

2
ZOL*
2
2
TCA* , ZOL*̶

Amobarbital (116)

2
ZOL*
3
ZOL*

̶

2
ZOL*
2
2
TCA* , ZOL*

Midazolam (38.7)
̶Zolpidem (0.928)
̶̶

2* Undeterminable. * False positive.
*3Positive even though the sample drug concentrations determined by LC-MS/MS analyses were under the cutoﬀ values.

tive result using DRIVEN-FLOW M8-Z. Other samples

three ambiguous results (cases 4, 9 and 15) for unknown
reasons, and it returned one false positive result for benzodiazepines (case 13). The SIGNIFY

TM

returned negative results for the spiked psychoactive drugs.

ER device produced

three false positive results for tricyclic antidepressants. The
IVeX-screen M-1 device returned no false positive results.

Discussion

The DRIVEN-FLOW M8-Z device returned false positive

devices with respect to their performance in detecting psy-

results for zolpidem or tricyclic antidepressants in 35 of thechoactive drugs in forensic pericardial ﬂuid samples. Oneof these devices, IVeX-screen M-1, was found to be moreIn the present study, we investigated four drug testing

38 cases.

useful than the other three screening devices. This device
accurately detected phenobarbital (cases 1, 3 and 6), ami-

Eleven samples (cases 2, 7, 8, 9, 11, 14 and 16) tested
positive for psychoactive drugs according to drug screening
devices, even though the sample drug concentrations deter-

triptyline (case 7), amobarbital (case 8) and methamphet-mined by LC-MS/MS were under the immunoassay cutoﬀ
values that are considered to indicate positive results (Tableamine (case 9), and returned no false positive results. On®
the contrary, the Triage TOX Drug Screen and SIGNI-

4). Therefore, to investigate this potential discrepancy,

FY

blank pericardial ﬂuid samples spiked with psychoactive
drugs at the concentrations found by LC-MS/MS analyses

positive results. To our knowledge, the only informationTM

ER devices returned several false positive results,
and the DRIVEN-FLOW M8-Z device returned many false

were subjected to analyses with the drug screening devices.

concerning the applicability of drug testing device to theUsing a blank sample spiked with the concentration of TCA

detection of psychoactive drugs in pericardial ﬂuid has®

been reported by Tominaga et al . This report showed thatTOX Drug Screen; the concentration of BZO from case 14

Triage TOX Drug Screen returned a few false positive

found in case 7 returned a positive result using Triage

5)

®

®

returned a positive result using Triage TOX Drug Screen;
and the concentration of ZOL from case 16 returned a posi-

results as ours and recommended the combined usage ofanother device to minimize misinterpretation prior to instru23

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Fig. 1. Representative LC-MS/MS analysis of a pericardial ﬂuid sample (case 7).
(a) Selective ion monitoring liquid chromatograms show the same retention times (6.25 min and 6.30 min) as those of standard
amitriptyline and nortriptyline. (b) Diagnostic fragment ions with ion abundance ratios of the noted fractions from the pericardial
ﬂuid sample were fully consistent with those of standard amitriptyline and nortriptyline, thus conﬁrming the presence of these compounds in this pericardial ﬂuid sample.

the pericardial ﬂuid is found in a closed compartment, con-mental analysis.
The principal advantage of the use of pericardial ﬂuid istamination by microorganisms is less likely as compared tothat pericardial ﬂuid is easily obtained from the pericardial

blood and cerebrospinal ﬂuid, and pericardial ﬂuid is thus

cavity, and the usual volume of pericardial ﬂuid taken at therelatively stable in the postmortem period . In the present

8)

7)time of the autopsy is enough for forensic drug testing . As

study, however, we found that drug concentrations were24

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

lower in the pericardial ﬂuid than in the urine. Speciﬁcally,

the immunoassay-based devices for the detection of psycho-

the concentrations of methamphetamine and amphetamine

active drugs. Thus, we recommend that positive resultsin pericardial ﬂuid were considerably lower than those in

obtained using the screening devices should be conﬁrmed
by LC-MS/MS.

urine samples. We conclude, then, that pericardial ﬂuid is
an important alternative material when urine is not avail-It should be noted that the concentrations of the drugs inand amphetamine because of low concentrations compared

the blood samples seem to be higher than those in the pericardial ﬂuid, except for a couple of drugs such as phenobar-

to urine.

bital in case 3. Because drug concentrations in pericardialable, but it is unsuitable for detecting methamphetamine

ﬂuid can be thought to be inﬂuenced from, or almost thesame with those of blood from anatomical perspective, peri-

One of the disadvantage of immunoassays, including
those investigated in the present study, is that antibodies
tend to be variable in terms of their potentials for cross-re-cardial ﬂuid thus has the potential to be very useful alterna-

activity . This cross-reactivity can lead to inconsistent
4)

tive when blood is not available.

results or false positive signals. In our previous study, we
found that the DRIVEN-FLOW M8-Z device was particu-

In conclusion, pericardial ﬂuid is an important alternative
material for forensic analyses when urine is not available.
Among the four drug screening devices tested for this appli-

larly useful for the detection of methamphetamines inforensic urine samples . One advantage of this device

cation, we found IVeX-screen M-1 to be the most useful, as

when screening urine samples was the lack of false positive

it accurately detected the highest number of psychoactive

signals even in the presence of the putrefactive amine
2-phenethylamine. However, in the present study, thisdrugs without returning false positive results. Despite the6)accuracy of this device, it is strongly recommended
LC-MS/MS methods should be used to conﬁrm any posi-device led to many false positive results for zolpidem andtricyclic antidepressants. False positive results are rela-tive results achieved with immunoassay-based screening

tively common with immunoassays, as antibodies tend tobe variable in terms of their potentials for cross-reactivity.

devices.

Conﬂict of Interest

In this case, we hypothesize that antibodies employed by
the DRIVEN-FLOW M8-Z device may cross-react with

The authors declare no conﬂicts of interest.

substances commonly found in the pericardial ﬂuid, thus
References

limiting its usefulness in this context.
We also investigated the potential discrepancy that

1) Harper L, Powell J, Pijl EM: An overview of forensic

eleven samples returned positive results for TCA, BZO and
ZOL, even though the sample drug concentrations deter-

drug testing methods and their suitability for harm reduc-mined by LC-MS/MS were under the immunoassay cutoﬀ

2) Towler S, Concheiro M, Pearring S, Rodda LN: Evalua-values. As a result of test spiked with psychoactive drugs at
the concentrations found by LC-MS/MS analyses, threetion and applicability of Alere iCup DX 14 for rapid post-

samples returned positive results and other samples

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tion point-of-care services. Harm Reduct J 14: 52, 2017.

mortem urine drug screening at autopsy. J Forensic Sci

returned negative results. These discordant results remainunresolved, and further studies incorporating LC-MS/MS

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analyses will be performed to clarify the reasons for theseanalysis of morphine (heroin) and cocaine in forensic tox-

discrepancies and to accurately evaluate the performance of

icology. Forensic Sci Int 164: 168‒171, 2006.

Ventura R, et al: Application of pericardial ﬂuid to thethe drug screening devices.
We used LC-MS/MS analyses to evaluate the results

4) Maharjan AS, Johnson-Davis KL: Issues of interferencesfrom the drug screening devices. LC-MS/MS has been
widely used in recent bioanalytical work, since it is a pow-

in urine, in Dasgupta A (ed): Critical issues in alcohol

erful analytical technique that combines the resolving
power of liquid chromatography with the detection speciﬁc-2019.

with immunoassays used for screening of drugs of abuseand drugs of abuse testing. p. 129, Elsevier, Amsterdam,5) Tominaga M, Michiue T, Maeda H: Evaluation of the onsite immunoassay drug-screening device Triage-TOX in

9)

ity of mass spectrometry . In the present study, we foundthat LC-MS/MS was more accurate and sensitive than wereroutine forensic autopsy. Legal Med 17: 499‒502, 2015.
25

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Medical Mass Spectrometry Vol. 8 No. 1

6) Iwai M, Ogawa T, Matsuo T, Kondo F, Kubo K, et al:

Valverde JL, et al: Validation of a procedure for the gas

Evaluation of ﬁve drug screening devices for testing ofchromatography-mass spectrometry analysis of cocaine

amphetamines and methamphetamines. Med Mass Spec-and metabolites in pericardial ﬂuids. J Anal Toxicol 31:

tom 6: 64‒69, 2022.

75‒80, 2007.

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9) DʼOvidio C, Locatelli M, Perrucci M, Ciriolo L, Furton

novel bioanalytical method for the determination of opi-

KG, et al: LC-MS/MS application in pharmacotoxicologi-

oids in blood and pericardial ﬂuid. J Anal Toxicol 44:

cal ﬁeld: current state and new applications. Molecules754‒768, 2020.

28: 2127, 2023.

8) Contreras MT, González M, González S, Ventura R,

26

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Research Paper

Plasma vitamin K-dependent protein C as early diagnostic marker
of feline chronic kidney disease
1

2

2

3

4

2

Hiroto Maeda , Yuiko Suzuki , Kokoro Masuda , Yoshio Kodera , Toshifumi Watanabe , Kazuyuki Sogawa
Maeda Veterinary Hospital, 1‒4‒8 Yamatemachi, Tomakomai, Hokkaido 053‒0851, Japan
Department of Biochemistry, School of Life and Environmental Science, Azabu University,
1‒17‒71 Fuchinobe, chuo-ku, Sagamihara, Kanagawa 252‒5201, Japan
3
Center for Disease Proteomics, Kitasato University School of Science,
1‒15‒1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252‒0373, Japan
4
DVMs Animal Medical Center Yokohama, 2‒2 Sawatari, Kanagawa-ku, Yokohama, Kanagawa 221‒0844, Japan
1

2

Abstract

Chronic kidney disease (CKD) is a common disorder and cause of death in cats. In the classiﬁcation proposedby the International Renal Interest Society (IRIS), stage 1 and 2 CKD are diﬃcult to diagnose accurately using markers,
in comparison with normal control cats. We recently described a simple and highly reproducible tandem mass tag (TMT)
labelling method for identifying potential disease-marker candidates among low-abundance plasma proteins. In the current
study, plasma samples were obtained from 90 normal control cats and 50 cats with CKD (stage 1). To identify new plasma
biomarkers for CKD, six plasma samples (three from normal control cats and three from CKD stage 1 cats) were extractedwith SSA lectin magnetic beads, diﬀerentially labelled with TMTs, digested with trypsin and subjected to analysis using
LC-MS/MS. Sialylated Vitamin K-dependent protein C was identiﬁed as a protein with lower levels in CKD stage 1 catscompared to normal control cats. An ELISA of plasma sialylated vitamin K-dependent protein C showed within-run (4.3‒
4.9%) and between-day (4.3‒5.2%) reproducibility. Plasma sialylated vitamin K-dependent protein C levels measured withthis assay were signiﬁcantly greater in normal control cats than in CKD stage 1 cats (1.40±0.13 vs. 1.07±0.11 AU/mL, p＜
0.001). These results indicate that sialylated vitamin K-dependent protein C may be useful as a complementary marker withplasma creatinine, BUN and SDMA for detection of CKD stage 1 in cats.
Key words: chronic kidney disease, feline, plasma, vitamin K-dependent protein C

Introduction

2)tion of survival and improvement of QOL . However, there

Feline renal diseases are increasingly common in veteri-are only a few reports on this area in clinical veterinary

1)3)nary practice . It is important to diagnose and identify the

medicine .

pathological basis of renal dysfunction accurately at an

In daily clinical practice, plasma creatinine (p-Cre) andearly stage, since early diagnosis and treatment can delay
progression of renal dysfunction, which leads to prolonga-

serum symmetric dimethylarginine (SDMA) are used as
markers based on the stage classiﬁcation criteria of the
4)International Renal Interest Society (IRIS) . However,many of these markers have low speciﬁcity for renal func-* Corresponding author
Kazuyuki Sogawa

tion and there is a blind area without abnormal values,

Department of Biochemistry, School of Life and Environmental Science, Azabu University, 1‒17‒71 Fuchinobe, Chuo-ku,

despite development of mild renal dysfunction. Therefore,

Sagamihara, Kanagawa 252‒5201, Japan
Tel: ＋81‒42‒769‒1924, Fax: ＋81‒42‒769‒1924

it is especially important to evaluate the transitional period

Received: February 6, 2024. Accepted: April 3, 2024.

to identify a mild decrease of renal function in the early

Epub May 10, 2024.

phase using the available markers in clinical practice. In

DOI: 10.24508/mms.2024.06.004

stage 1 there may not be any reduction in GFR or surrogate

from a normal status to Stage 1 in the IRIS classiﬁcationfor early diagnosis of renal function. However, it is diﬃcult

E-mail: sogawa@azabu-u.ac.jp

27June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Magnetic bead sample preparation using the Glycoprotein Isolation Kit

markers of GFR, despite ongoing active disease in the kidney.
We recently described a simple and highly reproducible
tandem mass tag (TMT) labelling method for identifying
potential disease-marker candidates among low-abundanceParamagnetic non-porous particles coupled with a sambucus sieboldiana lectin ligand (MB-SSA; Bruker Dalton-

8)
plasma proteins . In this study, we used the lectin and tandem mass tag (TMT) method to generate comparative pro-ics, Bremen, Germany) were used to process the plasma
samples. Binding, washing and elution solutions were pre-tein proﬁles of plasma samples from normal control catspared according to the manufacturerʼs instructions. MB-SSA

and CKD stage 1 cats. Plasma levels of candidate proteinsbinding solution (10 μL) and plasma (5 μL) were transferred
to a 0.2‒ mL thin-walled tube. A homogenous magnetic-par-were compared to evaluate their potential to discriminate
between the normal control cats and CKD cats. Novelticle solution (10 μL) was added, mixed, and incubated for
5 min. The tubes were placed in an 8×12-well magnet-

plasma biomarkers were identiﬁed that may be superior to

ic-bead separator (MBS; Bruker Daltonics) for 30 sec formagnetic ﬁxation of the MB-SSA particles. The supernatantclassical markers for early and speciﬁc detection of CKD
stage 1 in cats.

was aspirated and the tubes were removed from the MBS

Materials and Methods

device. Washing solution (100 μL) was added and carefully

Animals

mixed with the magnetic beads. The tube was placed back

Plasma samples from 50 cats with stage 1 CKD and 10

in the MBS device and moved back and forth sequentiallycats with stage 2 CKD brought to Maeda Veterinary Hospital between January 6, 2017 and February 20, 2018 (Ameri-

between adjacent wells on each side of the magnetic bar of

can Shorthair: 5, Chinchilla: 2, Mix: 53; 29 males and 31
females; age 2‒13 years) were used in the study. Plasma

supernatant was aspirated. This washing procedure was

samples from 90 cats aged 2‒13 years (Scottish Fold: 4,

bound molecules were eluted by incubation with 10 μL
MB-SSA elution solution for 1 min, before the eluate was

the device. After ﬁxation of the magnetic beads for 30 s, therepeated three times. After the ﬁnal washing step, theAmerican Shorthair: 2, Mix: 84; 43 males and 47 females)
were used as normal control cats. Cats clinically diagnosed
as normal based on serum biochemistry and ultrasonogra-

collected using the MBS device.

phy tests were deﬁned as normal cats, and cats showing

Tandem mass tag (TMT) labelling

clinical signs of CKD in blood chemistry were deﬁned as

Reduction and alkylation were performed as describedthose with stage 1 CKD. Criteria for normal urinalysis wereurine speciﬁc gravity (USG) ＞1.030, urinary protein to cre-previously . A TMT sixplex Isobaric Label Reagent Set(Thermo Scientiﬁc, Rockford, IL, USA) was used accord-

atinine ratio (UPC) ≤ 0.2, and a negative bacteriologic urine

ing to the manufacturer's instructions. Tubes containing the7)

5)culture . Diagnosis of CKD was made prior to inclusion in

diﬀerent isobaric chemical tags (0.8 mg each) were added

the study, based on clinical and laboratory ﬁndings. After5)
stabilization, cats were classiﬁed into 2 groups with p-Cre

to 41 μL of anhydrous acetonitrile and dissolved for 5 minwith occasional vortexing at room temperature. TMT solu-of ＜1.6 and 1.6‒2.8 mg/dL in stages 1 and 2, based on thetion (20 μL) was added to each tube and allowed to react at

IRIS guidelines. Diagnosis of stage 1 CKD was made if
p-Cre was ＜1.6 mg/dL, urine concentrating ability had

room temperature for 60 min. A volume of 4 μL of 5%

been lost, other disorders related to decreased urine concen-

was incubated for 15 min to quench the reaction . Samples
were ﬁnally pooled and lyophilized. Analyses were per-hydroxylamine was added to each sample and the mixture
7)

trating ability were excluded, and ultrasonographic changes
or absent renal corticomedullary diﬀerentiation) . Samples

formed for samples of 20 μL of glycoprotein-isolated
plasma from normal control cats (n＝3) (TMT6-126, 127

were centrifuged at 1,710×g for 10 min (Model 6200;

and 128) and stage 1 CKD cats (n＝3) (TMT6-129, 130 and

Kubota, Tokyo, Japan) and then stored at −80°
C for further

131). Plasma samples were randomly drawn.

were consistent with CKD (e.g., small kidney size and poor
6)use. The ethics committee for Maeda Veterinary Hospital
approved the protocol (2016-02). All owners gave signed

Protein identiﬁcation and quantiﬁcation by LC-MS/
MS analysis

informed consent to participation of their animal in the

Trypsin-digested peptides were injected into a trap col-study.
28

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

umn (C18, 5 μm, 0.3×5 mm) (IonPac; Thermo Scientiﬁc,

IL, USA) at 0.5 mg/well and incubated for 1 day at 4°C.

Rockford, IL, USA) and an analytical column (C18, 3 μm,

The plate was washed three times with PBS containing
0.05% Tween-20 (PBST). The microtiter plate was coated0.075×120 mm) (C18 packed emitter column；Nikkyo
Technos, Tokyo, Japan) attached to an HPLC system (Ulti-

with 20% NOF102 containing 10% sucrose for 1 day at 4°C.

mate 3000; Thermo Scientiﬁc). The ﬂow rate of the mobile

ELISA conditions

phase was 300 nL/min. The solvent composition of themobile phase was programmed to change in 120 min
cycles, with varying mixing ratios of solvent A (2% aceto-

After washing the microtiter plate with PBST, 100-μL aliquots of 10-times diluted plasma samples were added innitrile and 0.1% formic acid) to solvent B (90% acetonitrile

duplicate to wells. The plates were incubated at room temperature for 1 h and then washed three times. HRP-conju-

7)

and 0.1% formic acid), as described previously . Puriﬁed
peptides from HPLC were moved to a hybrid ion-trap Fou-gated SSA lectin (Vector Laboratories, Burlingame, CA,rier transform mass spectrometer (LTQ-Orbitrap XL;
Thermo Scientiﬁc) and analyzed using previously estab-USA) in PBST (100 μL) was added to each well and thelished parameters . A database search engine (Proteome

plate was incubated at room temperature for 30 min. The
plate was washed three times and then 100 μL of TMB solu-

Discoverer; Thermo Scientiﬁc) was used to identify andtion (FUJIFILM, Osaka, Japan) was added. After incuba-quantify proteins from the mass, tandem mass and reportertion at room temperature for 10 min, 100 μL of stop solution

ion spectra of peptides. Peptide mass data were matched by

was added and the absorbance at 450 nm was measured.

searching the UniProt database (https://www.uniprot.org/
proteomes/?query＝*) using the search parameters: peptide

Other procedures

8)

Creatinine was measured enzymatically with creatinine
deiminase using a Fuji DRI-CHEM Slide CRE-PIII kit

mass tolerance＝2 ppm and fragment tolerance＝0.6 Da, withthe enzyme parameter set to trypsin, allowing up to one
missed cleavage variable modiﬁcation, methionine oxidation, and cysteine alkylation. The minimum criteria for pro-

(FUJIFILM, Osaka, Japan). Blood urea nitrogen (BUN)
was measured using a N-Assay BUN-L Nittobo D-Type kit

tein identiﬁcation were ﬁltered with Xcorr vs. charge state(Nittobo Medical, Tokyo, Japan). Plasma SDMA was mea-

and a false discovery rate (FDR) ＜1%.

sured using a Cat SDMA ELISA Kit (MyBioSource, San
Diego, CA, USA). Serum levels of vitamin K-dependent

ELISA
Immunization and establishment of hybridoma celllines

protein C was determined by enzyme-linked immunosorbent assays according to the manufacturerʼs instructions(ELISA Kits for vitamin K-dependent protein C, LifeSpan

Synthetic peptides of 20 amino acids (vitamin K-depen-

Biosciences. Seattle, WA, USA).

dent protein C N peptide; FUJIFILM, Osaka, Japan) wereused for immunization of BALB/c mice. Hybridoma cell
9)line vitamin K-dependent protein C N-01 was established

Statistical analysis

and antibody isotypes were determined using a Mousetistics 19 (SPSS, Chicago, IL, USA). Numerical data arepresented as the mean±standard deviation (SD). Diﬀer-

Statistical analysis was performed using IBM SPSS Sta-

Monoclonal Antibody Isotyping Test Kit (AbD Serotec,

ences in non-parametric data between two groups were analyzed by Mann-Whitney U-test, three groups were analyzed

Kidlington, United Kingdom). To obtain pure monoclonal
antibodies on a large scale, BALB/c mice were initiallystimulated with 1.0 mL pristine (Sigma-Aldrich, St. Louis,

by Steel-Dwas test, with P＜0.05 considered signiﬁcant.
Receiver operating characteristic (ROC) curves were con-

MO, USA) and then inoculated 2 weeks later. Monoclonal
9)

antibodies were puriﬁed as described elsewhere .

structed to assess the sensitivity, speciﬁcity, and areas under
the ROC curves (AUCs).

Immobilization of antibodies on a polystyrene microtiter
plate

Results

The anti-vitamin K-dependent protein C N peptide antibody dissolved in PBS buﬀer was dispensed into a 96-well

Identiﬁcation of candidate proteins as markers of
CKD stage 1
Clinical characteristics and biochemical variables of thepolystyrene microtiter plate (Thermo Scientiﬁc, Rockford,29June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Establishment of an ELISA for sialylated vitamin
K-dependent protein C
Range, dilution analysis and detection limit

normal control cats and CKD groups are shown in Table 1.
To identify new plasma biomarkers for CKD, six plasma
samples (three normal control cat and three CKD stage 1)

A standard curve was drawn based on the colorimetricintensity of diluted recombinant cat vitamin K-dependent

were alkylated with iodoacetamide, digested with trypsin,
diﬀerentially labelled with TMTs, pooled and subjected toanalysis using LC-MS/MS (Fig. 1). The three normal con-protein C (Cusabio Technology) to establish the relationship of intensity with the sialylated vitamin K-dependent

trol cat samples were labelled with TMTs with reporter ionsat m/z＝126, 127 and 128, and the three CKD stage 1 samples were labelled with TMTs with reporter-ions at m/z＝protein C concentration (Fig. 2). The assay had a working
range of 0‒10 AU/mL and gave linear results from 0 to

129, 130 and 131. After comparing the proﬁles of proteins
by LC-MS/MS using an LTQ Orbitrap XL mass spectrome-

10 AU/mL (y＝0.021×−0.0018, r ＝0.9993, p＜0.0001). The
detection limit was estimated by assaying the zero concen-ter, 18 sialylated glycoproteins with unique peptide

tration eight times, and was deﬁned as the sialylated vitamin K-dependent protein C “zero” concentration＋3SD. The

2

sequences were found with higher levels in normal control

limit was found to be 0.18 AU/mL.

cats compared to CKD stage 1 cats (Table 2). Among these
proteins, hemoglobin subunit alpha and vitamin K-depen-

Within-run and between-run reproducibility

dent protein C had plasma levels elevated more than two-

The precision of the assay was determined using two

fold in all three normal control cats compared to the CKD
stage 1 cats. Vitamin K-dependent protein C was selectedsialylated vitamin K-dependent protein C concentrations of
0.5 and 2.0 AU/mL. Within-assay CVs were determined

for further analysis.

with eight replicates of each sample. Between-assay CVs
Table 1.

Clinical characteristics and biochemical variables of normal control cats and CKD cats
Variables

Normal control cats

CKD groupStage 1

Stage 2

p-ValueAge (Mean±s.d.)6.8±3.6

6.7±3.3

6.8±3.6

0.825

Sex (male, female)

90 (43, 47)

50 (24, 26)

10 (5, 5)

0.793

Blood urea nitrogen (mg/dL) (Mean±s.d.)p-Creatinine (mg/dL) (Mean±s.d.)24.1±5.7

25.6±6.8

26.8±5.90.714

0.96±0.23

1.20±0.23

1.99±0.24

0.015

Symmetric dimethylarginine (mg/dL) (Mean±s.d.)5.9±0.8

6.4±1.0

10.9±1.6

0.009

Urine speciﬁc gravity (Mean±s.d.)1.053±0.006

1.051±0.005

1.045±0.016

0.618

Urinary protein to creatinine ratio (Mean±s.d.)0.13±0.04

0.15±0.05

0.17±0.06

0.024

Fig. 1.

Identiﬁcation of candidate proteins using TMT labelling and LC-MS/MS analyses.
30June 2024

Table 2.

Medical Mass Spectrometry Vol. 8 No. 1

Plasma sialylated glycoproteins detected at higher levels in normal control cats compared to CKD stage 1 cats inproteome analysis

Database
Accession
No.

ID

Coverage(%)

TMT

Number ofmatching
peptidesNormal control cats

Stage 1 CKD cats

126

127

128

Average

129

130

131

Average

0.268

0.233

0.226

0.242

0.206

0.134

0.152

0.164
0.161

M3WPG6Alpha-2-macroglobulin
Apolipoprotein A-I

12.41

4

0.298

0.240

0.167

0.235

0.236

0.108

0.139

M3WG78ATP synthase subunit beta

8.00

3

0.212

0.158

0.133

0.168

0.277

0.106

0.113

0.166

4.69

6

0.260

0.134

0.307

0.234

0.180

0.164

0.133

0.159

2.86

2

0.357

0.164

0.154

0.225

0.282

0.122

0.121

0.175

M3W3E7M3W7M4 Attractin
M3WB05

Bifunctional purine biosynthesis protein ATIC

3.46

6

M3VZY7

Ceruloplasmin

5.63

4

0.230

0.142

0.268

0.213

0.230

0.136

0.142

0.169

M3WKP2Clusterin

16.63

13

0.391

0.132

0.263

0.262

0.140

0.157

0.140

0.146

M3VU42Complement factor I

2.31

2

0.394

0.162

0.227

0.261

0.106

0.263

0.219

0.196

P14450Fibrinogen alpha chain(Fragment)

100

1

0.191

0.191

0.245

0.209

0.198

0.160

0.175

0.178

P14469Fibrinogen beta chain

5.43

3

0.217

0.237

0.251

0.235

0.270

0.214

0.156

0.213

M3WB06

Fibronectin

4.24

8

0.194

0.155

0.270

0.207

0.297

0.181

0.131

0.203

P07405

Hemoglobin subunit alphaPhosphatidylinositol-glycanspeciﬁc phospholipase D

18.44

2

0.480

0.336

0.198

0.338

0.167

0.130

0.133

0.143

3.93

3

0.487

0.114

0.220

0.274

0.122

0.130

0.224

0.159

P49064

Serum albumin

31.58

19

0.251

0.177

0.130

0.186

0.278

0.131

0.128

0.179

M3WEV9Transthyretin
UDP-glucose 4-epimerase

8.84

2

0.169

0.121

0.188

0.159

0.118

0.189

0.116

0.141

2.66

2

0.152

0.208

0.217

0.192

0.251

0.174

0.099

0.174

Q28412

Vitamin K-dependent protein
C

14.21

3

0.280

0.291

0.289

0.287

0.132

0.140

0.137

0.136

M3W9I5

Vitronectin

5.47

2

0.337

0.343

0.388

0.356

0.236

0.197

0.186

0.206

M3WEG4

M3WFT7

Interference
Interference was assessed in samples containing 1.0 AU/
mL sialylated vitamin K-dependent protein C. Potential interference materials were added to urine at various concentrations. There was no substantial interference from hemoglobin(up to 5000 mg/L), free bilirubin (up to 207 mg/L), ditaurobilirubin (up to 204 mg/L), chyle (up to 1400 formazine turbidity units, equal to 1176 mg/L triglyceride), ascorbic acid (upto 500 mg/L), and rheumatoid factor (up to 500 U/L).

Fig. 2.

Recovery test

Standard curves for plasma sialylated vitamin
K-dependent protein C in the ELISA.

To evaluate recovery in the ELISA, 0.5 and 2.0 AU/mLof recombinant Cat vitamin K-dependent protein C were

Colorimetric intensity and plasma vitamin K-dependent
protein C concentration were related in the range of0‒10 AU/mL. Four concentrations of plasma sialylated
vitamin K-dependent protein C were determined by
ELISA.

added to pooled plasma (1.6 AU/mL). The percentage
recovery ranged from 94.3 to 102.7%.

Sialylated vitamin K-dependent protein C levels are
decreased in plasma of cats with CKD

were determined based on assays performed on 5 diﬀerentdays (two replicates of each sample per day). The within-run

Plasma vitamin K-dependent protein C levels andsialylated vitamin K-dependent protein C levels were mea-

CV was 4.3‒4.9% and the between-run CV was 4.3‒5.2%.
31

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

sured in stage 1 CKD cats (n＝50), stage 2 CKD cats (n＝mL and an AUC of 0.989, with sensitivity of 97.5% and10) and normal control cats (n＝90). Plasma vitamin K-de-

speciﬁcity of 97.5%.

pendent protein C values for normal control cats, stage 1

Discussion

CKD cats and stage 2 CKD cats were 3.36±0.32 μg/mL,

In this study, the lectin and TMT method was used to

3.46±0.31 μg/mL and 3.53±0.30 μg/mL, respectively (Fig.
3). Plasma sialylated vitamin K-dependent protein C was

detect 18 proteins in plasma from CKD stage 1 cats andnormal control cats. Sialylated vitamin K-dependent protein

signiﬁcantly higher in normal control cats than in stage 1

C was identiﬁed as the protein with the largest diﬀerence inlevels between the two groups. The vitamin K-dependent

CKD cats (1.40±0.13 vs. 1.07±0.11 AU/mL, p＜0.001),
and in stage 1 CKD cats compared to stage 2 CKD catsprotein C is synthesized in the liver in a single-chain form,(1.07±0.11 vs. 0.82±0.14 AU/mL, p＜0.001) (Fig. 4). ROC
curves were constructed to evaluate the cut-oﬀs for plasma
sialylated vitamin K-dependent protein C, plasma creati-

and through several proteolytic steps is secreted into blood
primarily as a two-chain form, containing a light chain andnine, BUN, and SDMA for distinguishing stage 1 CKD catsa heavy chain linked via a single disulﬁde bond . Sialic

from normal control cats. The AUCs were 0.989 for plasma
sialylated vitamin K-dependent protein C, 0.698 for plasma

acids are derivatives of the negatively charged acidic sugar

creatinine, 0.554 for BUN, and 0.568 for SDMA. These
results suggest that plasma sialylated vitamin K-dependent

the sialic acid family have been discovered to date ;N-acetylneuraminic acid, N-glycolylneuraminic acid, andprotein C may be a better diagnostic stage 1 CKD bio-

deaminoneuraminic acid are representative members of thismarker than plasma creatinine, BUN and SDMA (Fig. 5).
The ROC curve for plasma sialylated vitamin K-dependent

family. Sialic acids usually form the terminal ends of the
carbohydrate groups of glycoconjugates. All sialyltransfer-protein C showed an optimal diagnostic cut-oﬀ of 1.22 AU/

ases characterized in animals to date, ranging from insects

10)

neuraminic acid. Over 40 naturally occurring members of
11)

Fig. 4.
Fig. 3.

Plasma vitamin K-dependent protein C levels innormal control cats and stage 1 and stage 2 CKD
cats.

Plasma sialylated vitamin K-dependent protein C
levels in normal control cats and stage 1 and stage
2 CKD cats.

Plasma sialylated vitamin K-dependent protein C wassigniﬁcantly higher in normal control cats than in stage 1
CKD cats (1.40±0.13 vs. 1.07±0.11 AU/mL, p＜0.001;
Mann‒Whitney U-test) and in stage 1 CKD cats compared
to stage 2 CKD cats (1.07±0.11 vs. 0.82±0.14 AU/mL,
p＜0.001).

Plasma vitamin K-dependent protein C values for normal
control cats, stage 1 CKD cats and stage 2 CKD catswere 3.36±0.32 μg/mL, 3.46±0.31 μg/mL and 3.53±
0.30 μg/mL, respectively.

32

June 2024

Fig. 5.

Medical Mass Spectrometry Vol. 8 No. 1

Plasma sialylated vitamin K-dependent protein C is more eﬀective than plasma creatinine, BUN and SDMA for early
detection of stage 1 CKD.
ROC analyses were performed for serum levels of plasma sialylated vitamin K-dependent protein C, plasma creatinine, BUN and
SDMA in stage 1 CKD and normal cats. The AUCs were 0.989 for plasma sialylated vitamin K-dependent protein C, 0.698 forplasma creatinine, 0.554 for BUN, and 0.568 for SDMA.

with CKD have signiﬁcantly higher FVII, FVIII, ﬁbrinogen18)
and D-dimer levels and a signiﬁcantly lower level of pro-to mammals, feature a type II transmembrane topology andusually localize to the Golgi apparatus. The enzyme isgrouped into 4 families according to the type of carbohy-tein C. However, it has not been reported in the sialylated
vitamin K-dependent protein C. Similar results were obtained

drate linkage they synthesize: β -galactoside α 2,3-sialyltransferases (ST3Gal-I-VI), β -galactoside α 2,6-sialyltrans-in this study, with plasma sialylated vitamin K-dependent

ferases (ST6Gal-I and -II), GalNAc α 2,6-sialyltransferases(ST6GalNAc-I‒VI), and α 2,8-sialyltransferases (ST8Sia-I‒

protein C found to be signiﬁcantly higher in normal control
cats (1.40±0.13 AU/mL) than in stage 1 (1.07±0.11 AU/mL)

12)VI). Sialic acid was added by these enzymes . During
coagulation, plasma vitamin K-dependent protein C is acti-

and stage 2 (0.82±0.14 AU/mL) CKD cats.

presence of protein S, activated protein C (APC) functions

In this study, we showed that plasma sialylated vitamin
K dependent protein C is a potential biomarker for CKD. Various attempts have been made to identify new plasma biomark-as an anticoagulant by proteolytic degradation of factors Va

ers for CKD, and some molecular targets have been suggested

11)

vated by a thrombin/thrombomodulin complex , and in the

12)

5)

and VIIIa , the only protein substrates hitherto identiﬁed
for vitamin K-dependent protein C. Several animal models,

as diagnostic or prognostic markers . The AUC for plasma
sialylated vitamin K-dependent protein C was 0.989, with sen-including canine, bovine and feline vitamin K-dependent

sitivity of 97.5% and speciﬁcity of 97.5%. These results suggest that plasma sialylated vitamin K-dependent protein C may

protein C, have been used to examine the physiologicalmechanisms of vitamin K-dependent protein C, and spe-be complementary to plasma creatinine, BUN and SDMA as acies-speciﬁc eﬀects have been found for the anticoagulant

marker for detection of stage 1 CKD in cats. We note that the

13,14). Thus, in squirrel

study is limited by the number of samples, and evaluation ofmonkeys, human APC has an anticoagulant eﬀect, but nomore samples from multiple facilities is required. Longitudinalfollow-up of cats in stage 1 CKD is also important to identify

and ﬁbrinolytic functions of APC
15)

proﬁbrinolytic activity , whereas human APC shows boththose with true CKD, which is progressive, and those recover-16)

anticoagulant and ﬁbrinolytic responses in cats .
A decrease in vitamin K-dependent protein C has been

ing from AKI when samples were taken.

reported in renal disease in humans, but not in cats. Patients

Acknowledgmentswith CKD commonly have blood coagulation disorders, andseveral tests can be used to measure procoagulant biomark-This work was supported by a Grant-in-Aid for Scientiﬁc

17)

ers in CKD . Compared to normal control cats, patients

Research (KAKENHI C: 22K07406).
33

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Medical Mass Spectrometry Vol. 8 No. 1

Conﬂict of Interest

et al: Development of a novel fragments absorbed immunocapture enzyme assay system for tartrate-resistant acid

The authors declared no potential conﬂicts of interest

phosphatase 5b. Clin Chim Acta 376: 202‒212, 2007.

with respect to the research, authorship, and/or publicationof this article.

11) Varki A: Diversity in the sialic acids. Glycobiology 2: 25‒

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nolytic eﬀects of activated protein C. Thromb Res 63:

matol 86: 590‒600, 1994.

123‒131, 1991.

10) Ohashi T, Igarashi Y, Mochizuki Y, Miura T, Inaba N,

34

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Short Communication

Recovery of extracellular vesicles from liquid samples
using polyamine solution
1,2
1
1,3
2
2
Arizumi Kikuchi *, Azumi Naruse , Kenichi Nonaka , Motoki Mori , Miyuu Yamada ,Keiko Kano , Emi Mishiro-Sato , Kaname Tsutsumiuchi
442

Daiyukai Research Institute for Medical Science, 25 Azaicho, Ichinomiya, Aichi 491‒0113, Japan
College of Bioscience and Biotechnology, Chubu University, 1200 Matsumoto-cho, Kasugai-shi, Aichi 487‒8501, Japan3
Department of Surgery, Daiyukai General Hospital, 1‒9‒9 Sakura, Ichinomiya, Aichi 491‒8551, Japan
4
Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464‒8601, Japan
1

2

Abstract

Liquid biopsies mainly analyze nucleic acids and proteins in the free-state or extracellular vesicles (EVs) in

non-solid biological samples, primarily blood. Collecting and processing liquid biopsy samples is challenging due to thelarge volume of samples and reagents and the need for special equipment. In a previous study, we reported a method forenriching free nucleic acids using a polyamine solution that is eﬀective for liquid biopsy. We also investigated the reactivity of this method for EV recovery in cell culture supernatants using mass spectrometry. Samples were prepared from thecell line NCI-N87 supernatants after 48 h of culture in a serum-free medium. For comparison, samples were treated using asolution containing polyamines (PA method) or ultracentrifugation (UC method). The liquid chromatography-tandem mass
spectrometry (LC-MS/MS) using the single-pot solid-phase-enhanced sample-preparation (SP3) method revealed diﬀerences between the two methods in the total ion chromatogram of the sample. However, the results of the Gene Ontology (GO)analysis showed that both methods achieved the best enrichment in GO terms related to EV. In addition, the volcano plot
analysis revealed that proteins suggested to exist in EVs were distributed in areas consistent with both methods. These results indicated that the PA method can recover EV proteins in liquid samples, and their comprehensive analysis is possibleusing the SP3 method.
Key words: extracellular vesicles, liquid biopsy, LC-MS/MS, polyamine method, single-pot solid-phase-enhanced samplepreparation (SP3) method

Introduction

state nucleic acids and proteins in extracellular vesicles

Blood samples from patients with cancer contain circulating tumor DNA (ctDNA), cell-free DNA (cfDNA), circulat-(EVs), including exosomes . Liquid biopsy can provide
information on the nature of cancer without requiring inva-

ing tumor cells, and microRNAs (miRNAs) derived fromsive procedures associated with conventional tissue collec-

2)

1)

3)

cancer cells . Liquid biopsy is a method for the genetic and1)cytological analysis of these samples . It analyzed free-tion .
EVs, including exosomes, contain proteins, mRNA,
miRNA, and DNA on their surface or inside; they are stable*Corresponding author

in body ﬂuids such as blood, urine, saliva, spinal ﬂuid, andArizumi Kikuchi

4)

breast milk after being secreted from cells . EVs have

Daiyukai Research Institute for Medical Science, 25 Azaicho,
Ichinomiya, Aichi 491‒0113, Japan

attracted attention as critical biological factors that convey
messages locally and remotely and are deeply involved invarious diseases and biological phenomena, including can-Tel: ＋81‒586‒533661, Fax: ＋81‒586‒533771
E-mail: akikuchi@daiyukai.or.jp

5)

cer and immune and neurological diseases . They can berecovered using centrifugation, immunoprecipitation, parti-

Received: February 13, 2024. Accepted: May 9, 2024.
Epub June 5, 2024.

cle size classiﬁcation, column adsorption, or ultracentrifu-

DOI: 10.24508/mms.2024.06.006
35

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

6)gation . Each method has diﬀerent recovery rates, speciﬁci-

Polyamine method

ties, and the time and cost required for operation.

To begin with, 9.0 mL of the treated cell culture superna-

Therefore, the method selected depends on its application.
Although ultracentrifugation is considered the gold stan-tant was transferred into a 15 mL conical centrifuge tube
(Nunc, ThermoFischer Scientiﬁc, USA). Then, 1/10

7)

dard , it has limitations, such as poor recovery rates, andvolume of 5 M NaCl solution and 1/10 volume containing
0.01% Spermidine (191-13831, FUJIFILM Wako

8)

requires time and specialized equipment . Moreover, weconsidered the possibility that the PA method coprecipitates
EVs along with nucleic acids. In a previous study, we have

Chemicals, Osaka, Japan) were added to the tube. After the
mixture was allowed to stand for 10 min at room tempera-

devised a method for enriching free nucleic acids using
9)
polyamine-containing solutions .

ture, it was centrifuged at 27,210×g (13,000 rpm) for
60 min using a micro-cooled centrifuge (Model 3700,

Here, we investigated, using liquid chromatography-mass
spectrometry (LC-MS/MS), whether this method can be

KUBOTA CORPORATION, Tokyo, Japan). Then, the cellused to recover EV proteins in cell culture supernatant.

before analysis.

Materials and Methods

Mass spectrometry

culture supernatant was removed and dissolved in PBS

Cell culture

Sample preparation was performed using the single-pot
solid-phase-enhanced sample-preparation (SP3) technology.

NCI-N87 (ATCC, Manassas, VA, USA).
The cells were cultured in RPMI-1640 with L-Glutamine

The protein in the sample was adjusted to 1‒200 μg/mL,

and Phenol Red (Fuji ﬁlm Wako, Tokyo, Japan) with 10%

25 mM. After 30 min at room temperature, 1/10 volume of
500 mM 3-methyl-1-butanol was added and allowed to

For our LC-MS/MS analysis, we used a cell line, that isand dithiothreitol was added to a ﬁnal concentration of2

fetal bovine serum at 37°
C in a 5% CO incubator. When
Advanced RPMI 1640 Medium (Thermo Fisher Scientiﬁc,stand for 30 min at room temperature. Particles and SeraTM
SpeedBead Carboxylate-Modiﬁed [E3] Magnetic
Mag

Waltham, MA, USA).

Particles (Global Life Sciences Technologies Japan K.K.,

the cells reached conﬂuency, they were treated with

After 48 h of culture, the collected media were ﬁrst

Tokyo, Japan). The bead mixture (10 μL) was added to thecentrifuged at 1,000×g for 10 min at room temperature(20‒25°C) to pellet and remove cells. All the centrifugation

sample for 10 μg of protein. Then, three times the volume

steps were performed at 4°
C. Next, the supernatant wasfor 10 min at RT, and 500 μL of 80% ethanol was added,

centrifuged at 2,000×g for 20 min. The media supernatant
was ﬁltered through a 0.22-μm pore ﬁlter (HAWACH

followed by washing three times using a magnetic stand.

Scientiﬁc Co. LTD, Xian City, China). Each method waswas added. After sonication, the mixture was incubated at

performed using a ﬁltered culture medium.

37°
C for 3 h. Trypsin solution was added, and the sampleof ethanol was added, the sample was mixed at 1,000 rpm

Then, 100 μL of lysyl endopeptidase plus 50 mM Tris buﬀer

was mixed at 1,500 rpm at 37°
C. for 20 h. After mixing,

Ultracentrifugation
Treated cell culture supernatant (50 mL) was used for

20% triﬂuoroacetic acid was added to the volume 1/20, thebeads were attached to a magnetic stand, and the superna-

ultracentrifugation to collect EVs. The ultracentrifugation
was performed using an Optima L-90K ultracentrifuge

tant was collected. Then, the supernatant was desaltedusing GL-Tip SDB (GL Sciences Inc., Tokyo, Japan). The(Beckman Colter, Fullerton, CA, USA) at 150,000×g forresulting solution was vacuum dried, and resuspended in

100 min in a SW28.1 Roter Swinging Bucket rotor(Beckman Colter). After centrifugation, phosphate-buﬀered

0.1% TFA in 2% acetonitrile to extract the peptides.
The LC-MS/MS analysis was performed using a PAL

saline (PBS) was added to the top of the tube, and ultracen-

Systemtrifugation was performed again under the same conditions.

Switzerland) and Q Exactive Hybrid Quadrupole-Orbitrap

Finally, the supernatant was removed, and the collected

Mass Spectrometer (Thermo Fisher Scientiﬁc). The samples
were concentrated on a C18 trap column (5-μm particle size,

EVs were suspended in PBS.

autosampler(CTC Analytics AG,

Zwingen,

300 μm inner diameter, 5 mm length; Chemical Evaluation
and Research Institute, Tokyo, Japan) and separated on a36

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

C18 column (3-μm particle size, 100-μm inner diameter,

10)

accession ID, PXD035832) .

125 mm length; Nikkyo Technos, Tokyo, Japan) at a ﬂow

Furthermore, we used the proteins described in
6)

MISEV2018

and examined the validity of the proteinsrate of 0.5 μL/min with an injection volume of 7.5 μL. The
mobile phases were solvent A (0.5% acetic acid) and sol-detected. Among the proteins listed, we identiﬁed peptide

vent B (0.5% acetic acid in 80% acetonitrile). The elution

spectrum matches (PSM) for signiﬁcant proteins. We also

gradient for solvent B was as follows: 5% to 40% B overexamined the correlation between the two methods using

100 min, then 40% to 95% B for 1 min, holding at 95% B

this data.
We also examined the properties of the PA and UC meth-for 3 min, then back to 5% B over 1 min, and ﬁnallyre-equilibrating at 5% B for 10 min. Electrospray ionization

ods using total ion chromatogram, diagrams, and volcano

was performed in positive-ion mode.
The instrument was operated in the DDA mode. Xcalibur

plot, mainly to characterize the recovered peptides. The
volcano plot was also used to examine both methodsʼ agree-

4.1.50 (ThermoFisher Scientiﬁc) was used to record

ment rate and characteristics of the proteins detected.

peptide spectra. The full scan was acquired from 350 to1800 m/z with a resolution of 17,500, automatic gain con-

Volcano plots were created with a fold change value (log2)
greater than or equal to 1.5 and a p-value of 0.05＞. The

6trol (AGC) as 3×10 , and maximum injection time as

methodʼs relative standard deviation (RSD) was determined

60 ms. MS/MS scans were performed with a resolution ofand reproducibility was discussed. RSD was calculated by

535,000, AGC target as 1×10 , and maximum injection timeﬁnding the mean and SD of the PSMs listed in Table 1.

as 60 ms. The 10 highest intensity precursor ions wereWe performed Go Ontology (GO) analysis on cellularcomponent categories using proteins detected by both meth-isolated using the quadrupole analyzer in a window of
2.0 m/z and fragmented by higher energy collisional dissoci-ods for PSMs with a mean of 4 or higher. The analysis used
11,12).
the DAVID open-source program, v2024q1

ation (HCD) fragmentation with normalized collisionenergy (NCE) of 27%. Multiply-charged peptides were

Results

chosen for MS/MS experiments. Dynamic exclusion time
was set to 20 s.

Table 1 shows the protein data obtained by both methods,

Data analysis

using the detection data for the content described in
MISEV2018 as protein content-EV characterization, and

UniProt Homo Sapiens (TaxID＝9606) protein sequence
database downloaded on 09/24/2022 and cRAP for contam-

summarized for those with PSMs of 4 or higher. Using

inants (http://www.thegpm.org/crap/) were used. MS/MS

PSMs, a correlation coeﬃcient of R＝0.765 was obtained
for both methods, resulting in an overall positive correla-

spectra were interpreted, and peak lists were generated

tion in Fig. S1. In addition, results for all proteins listed inusing Proteome Discoverer 2.5.0.400 (ThermoFisher

MISEV2018 are represented in Table S1.

Scientiﬁc). Searches were performed using SEQUEST

Fig. 1A shows the total ion chromatograms. Although the(ThermoFisher Scientiﬁc). Search parameters were set assame samples were treated using PA and UC methods, eachfollows: enzyme selected with two maximum missing

method showed a diﬀerent proﬁle.

cleavage sites, a mass tolerance of 10 ppm for peptide

Fig. 1B show the results of the volcano plot for eachtolerance, 0.02 Da for MS/MS tolerance, ﬁxed modiﬁcation

method. Fig. 1B shows the markers CD9, CD63, CD81,
and Flotillin-1, which were considered particularly import-of carbamidomethyl (C), and variable modiﬁcation ofoxidation (M) and N-terminal acetylation. Peptide identiﬁ-

ant in EVs. The PA method identiﬁed common proteins

cations were based on signiﬁcant Xcorr values (high conﬁ-

believed to be contained in EVs near the origin. Table S2dence ﬁlter). Peptide identiﬁcation and modiﬁcation

presents the list of proteins with quantitative information

information returned from SEQUEST were ﬁltered at aused in the analysis.
Table S3 summarizes the number and percentage of pro-false discovery rate (FDR) of 1% using the Percolator nodeof Proteome Discoverer to obtain conﬁrmed peptide identi-

teins with diﬀerent reproducibility based on the RSD values

ﬁcation and modiﬁcation lists of HCD MS/MS. The
LC-MS/MS data generated in this study have been depos-of PSMs in proteomics using PA and UC methods. About

ited in jPOST repository database (project ID, JPST001807,

smaller RSD value than 30%, whereas about 20% of the

80% of the proteins detected with the PA method have a37

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

Table 1.

Protein content by LC-MS/MS
PSMs

Description

Integrin alpha-2Integrin beta-3
HLA class I histocompatibility antigen, C alpha chain
Integrin alpha-6Integrin beta-1
HLA class I histocompatibility antigen, A alpha chain
HLA class I histocompatibility antigen, B alpha chain
Integrin beta-4 OS＝Homo sapiens
Syndecan-4 OS＝Homo sapiens
CD9 antigen OS＝Homo sapiens
Receptor tyrosine-protein kinase erbB-2
Charged multivesicular body protein 1b
Annexin A7 OS＝Homo sapiens
ADP-ribosylation factor 6
Flotillin-2
EH domain-containing protein 2
Transforming protein RhoA
Flotillin-1
Annexin A13
Charged multivesicular body protein 2b
Arrestin domain-containing protein 1
Charged multivesicular body protein 1a
EH domain-containing protein 3
Vacuolar protein sorting-associated protein 4A
Charged multivesicular body protein 3
Annexin A4
Tumor susceptibility gene 101 protein
Charged multivesicular body protein 4b
Vacuolar protein sorting-associated protein 4B
Charged multivesicular body protein 4c
Annexin A3
EH domain-containing protein 4
Annexin A1
Programmed cell death 6-interacting protein
Charged multivesicular body protein 2a
EH domain-containing protein 1
Putative annexin A2-like protein
Annexin A2
Syntenin-1
Annexin A11 OS＝Homo sapiens
Glyceraldehyde-3-phosphate dehydrogenase
Actin, cytoplasmic 1
Tubulin beta-3 chain
Tubulin beta-8 chain
Tubulin beta-2B chain
Tubulin alpha-4A chain
Tubulin alpha-3E chain
Tubulin alpha-3D chain
Tubulin beta chain
Tubulin alpha-1A chain
Tubulin alpha-1B chain
Tubulin alpha-1C chain
Tubulin beta-4B chain
Tubulin beta-2A chain
Heat shock protein HSP 90-beta
Heat shock cognate 71 kDa protein
Albumin

Gene Symbol

ITGA2
ITGB3
HLA-C
ITGA6
ITGB1
HLA-A
HLA-B
ITGB4
SDC4
CD9
ERBB2
CHMP1B
ANXA7
ARF6
FLOT2
EHD2
RHOA
FLOT1
ANXA13
CHMP2B
ARRDC1
CHMP1A
EHD3
VPS4A
CHMP3
ANXA4
TSG101
CHMP4B
VPS4B
CHMP4C
ANXA3
EHD4
ANXA1
PDCD6IPCHMP2AEHD1
ANXA2P2ANXA2SDCBPANXA11
GAPDH
ACTB
TUBB3
TUBB8
TUBB2B
TUBA4A
TUBA3ETUBA3DTUBB
TUBA1A
TUBA1B
TUBA1CTUBB4B
TUBB2A
HSP90AB1
HSPA8
ALB

(a)

(b)

Mean

S.D.

Mean

S.D.

4.0
6.7
9.0
10.3
13.7
16.3
16.3
18.7
45.3
6.7
22.0
4.0
1.3
1.7
1.7
3.7
4.0
4.0
5.0
5.7
5.7
7.3
8.0
8.0
8.3
9.7
10.0
12.0
12.7
12.7
16.3
18.0
21.3
23.7
26
26.7
50.3
67.7
5.0
11.0
4.0
61.0
2.5
2.7
2.7
3.3
4.0
4.0
4.3
4.7
4.7
4.7
6.0
2.7
32.0
39.3
67.7

2.0
1.2
1.7
0.6
1.5
1.2
1.5
4.9
5.7
1.2
1.7
1.0
0.6
1.2
1.2
1.2
0.0
1.0
1.0
1.5
1.5
1.5
0.0
3.0
3.8
1.5
1.7
1.7
4.0
1.5
0.6
3.0
1.2
1.5
2.6
3.2
3.2
3.1
1.0
2.0
0.0
2.6
2.1
1.5
1.2
1.2
1.0
1.0
0.6
1.5
1.5
1.5
1.0
1.2
5.3
4.7
18.5

3.7
4.5
8.7
9.0
12.0
15.3
18.7
15.0
5.5
5.0
24.3
6.3
4.0
4.5
6.5
5.5
6.7
7.0
13.5
5.0
7.3
9.3
8.3
12.5
12.7
14.3
13.3
19.7
14.7
17.3
12.3
17.0
18.0
34.7
45.7
23.7
42
59.3
10.0
12.0
4.7
49.0
4.0
4.5
7.5
5.0
5.0
5.5
9.0
8.5
9.5
9.5
6.3
8.0
22.0
43.0
12.0

2.3
0.7
6.1
8.0
5.3
11.9
11.0
12.1
2.1
4.0
16.0
4.0
1.4
0.7
0.7
3.5
3.8
5.3
4.9
4.0
4.7
6.4
6.7
4.9
9.3
10.7
9.3
11.0
11.1
8.1
6.4
14.1
8.5
23.2
28.7
22.1
18.1
25
4.2
5.3
4.0
32.8
4.2
4.9
6.4
6.1
5.7
6.4
5.7
10.6
12.0
12.0
6.1
7.1
14.2
27.1
12.2

(a) PA method and (B) UC method. The analysis was performed with N＝3, and the list was made up of PSMs obtained by any of the methods with a mean of 4 or higher (PSMs, peptide spectrum matches; SD, standard deviation).

38

June 2024

Fig. 1.

Medical Mass Spectrometry Vol. 8 No. 1

Comparison of both methods using total ion chromatogram and volcano plot.
A. Total ion chromatogram for each method. (a) PA method, (b) UC method. B. Volcano plot for each method. Marked for proteinsin EVs at NCI-N87. Created using abundance data obtained by LC-MS/MS. The x-axis indicates log2 fold change for each gene,and y-axis indicates -log10 p-value. Red dots indicate proteins with p-values ＜0.05, and green dots indicate proteins with p-values≥ 0.05. The signiﬁcance of diﬀerences was evaluated by t-test, with p-value ＜0.05 being signiﬁcant.

proteins with the UC method.

capture tumor dynamics owing to the heterogeneous

In the GO analysis, EV markers, including exosomes,

distribution of tumor cells imposes challenges to cancer

dominated the highest annotation cluster with enrichment.

diagnosis . Meanwhile, liquid biopsy is a promising

The enrichment scores for the PA and UC methods were

method to address these challenges. In addition, liquidbiopsy results have recently been used to administer com-

14)

206.6 and 233.6, respectively (Table S4 A, B) .

panion diagnostic agents.

Furthermore, information regarding cellular components

We compared the EVs obtained by PA and UC methods
using the LC-MS/MS. Together with the UC method,

obtained in this analysis is described in Fig. S2.

Discussion

Cancer is the leading cause of death worldwide; there-

LC-MS/MS detected proteins that indicate EVs according
to the categories in the MISEV2018. The total ion chro-fore, early cancer detection and appropriate treatment are

matograms obtained using the PA method diﬀered from13)

crucial . Usually, cancer is diagnosed by tissue biopsy;

those obtained using the UC method. In the analysis withhowever, the invasive sampling and inability to accurately

the volcano plot, the common markers that were supposed39

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

to be present in the EV appeared relatively close to the ori-process.

gin. However, their distribution showed diﬀerent patterns.

Liquid biopsy has been reported in urine, saliva, cerebro-

Although the UC method is the standard method for EV
recovery, it simultaneously recovers impurities such as ves-

spinal ﬂuid, and stool other than blood , and is expected to

icles and proteins other than EVs, resulting in lower purity

proteins in the samples used in liquid biopsies are often

22)

be used in actual clinical practice. Free nucleic acids and

8)

23)

of EVs . These factors may have inﬂuenced the diﬀerences

present at low concentrations

and require a large sample

observed between the two methods in the present study. In
addition, EVs contain exosomes, microvesicles, and apop-

volume. In addition, large amounts of reagents and special

totic bodies, and the reactivity of each substance in eachcan be enriched, it is possible to use ordinary processing

equipment are required. However, because the PA method15)

method , including PA method, can signiﬁcantly aﬀect thereagents to handle enriched samples.

results.

We have demonstrated the presence of protein markers in

The proteins detected by both methods are positively
correlated and show a similar trend in detecting EV-related

the characterization shown in MISEV2018 using the PA

markers. Furthermore, we believe the PA method is lessin EVs were recovered in this study. Furthermore, we
believe that a comparative study of this point using meth-method; however, it is unclear to what extent the exosomes

susceptible to contamination by dead cells. The results of
the GO analysis also suggest that this is an eﬃcient methodof recovering EV-related substances. Although the PA

ods other than the UC method should be conducted. There-

method has shown recovery of proteins in EVs, its charac-

analytical methods.

fore, we are currently examining this point using various
The cargo of tumor-derived EVs is consistent with theteristics will need to be clariﬁed through further studies.

24)

genetic content of parent tumor cells . Therefore, thePolyamines are involved in cell growth and survival,
16)

autophagy , and the onset and progression of neurological17)
disease . Additionally, a link between polyamine meta-cargo contained in the EVs has attracted attention as a new
25)

biomarker for cancer diagnosis and prognosis prediction .

18)

bolic pathways and cancer has been reported . Polyamines

Moreover, EVs are stable in body ﬂuids, and the cargo ininclude streptomycin, spermidine, and spermine, which
exist in cells at a level of several millimolar concentra-

the EVs is protected from degradation

are of interest as excellent biological components for liquid

19)
tions . Moreover, polyamines are polycationic mole-biopsy26,27)

20)

1,28). Therefore, EVs. We are currently conducting various studies

cules , which is the characteristic the PA method focuses

focusing on its application in liquid biopsy. We believe thaton.

the PA method can potentially demonstrate a broad utility

This method can recover cell-free nucleic acids and EVs
9)using polyamine-containing solutions , which we believe isby comparing its performance with that of other methods

due to the formation of ion complexes between polyamines

Mass spectrometry is being implemented in various ways

and these substances with negative charges on the surface.
Furthermore, under high salt concentration conditions, sub-in the medical ﬁeld because of its ability for comprehensive

for EV recovery.

analysis and its excellent sensitivity and speciﬁcity. We
believe that this method will be an eﬀective method in clin-stances with small particle sizes and low charge density do
21)

not lead to the formation of coacervate , which may also

ical testing of liquid biopsy samples.

aﬀect not recovering a variety of free proteins.

Conclusions

The PA method does not require special equipment, isrelatively easy to operate, and is low-cost. In addition, a
pretreatment is possible when further downstream puriﬁca-

EV analysis using LC-MS/MS with the SP3 method was
also feasible. The LC-MS/MS results showed that the PAtion or nucleic acid extraction is performed on samples col-method can detect peptides with EV properties.

lected using the PA method. Regarding the reproducibility

Conﬂict of Interestof the PA method, approximately 80% of the proteinsdetected in this study showed RSD within 30%. Maintain-

The authors declare no competing interests.

ing and improving reproducibility is an essential issue in

Additional Information

analysis. We believe that attention should be paid not only

The results of this study are organized in the text or into technical proﬁciency but also to further simplifying the40

June 2024

Medical Mass Spectrometry Vol. 8 No. 1

GB, et al: Single-pot, solid-phase-enhanced sample prepa-

supplementary materials.

ration for proteomics experiments. Nat Protoc 14: 68‒85,

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et al: Secretory mechanisms and intercellular transfer of42Medical Mass Spectrometry Vol. 8 No. 1, 2024
Publication Date: June 25, 2024
Publisher: Japanese Society for Biomedical Mass Spectrometry
6 Nikko-cho, Ichinomiya, Aichi 491‒0938, Japan
Shubun University
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