Shinshu Med J, 72⑶：159～167, 2024

Direct Evidence that 4’-O-methylpyridoxine Induces Hyperactivity andConvulsions due to Pyridoxal Phosphate Deficiency in the Brain
Tomomitsu Sado＊, Tetsuhiro Fukuyama and Yoshiko Nakayama
Department of Pediatrics, Shinshu University School of Medicine

Ginkgo biloba seed (GBS) poisoning is caused by the toxic substance 4’-O-methylpyridoxine (MPN). Gammaaminobutyric acid (GABA) in the brain is suggested to be involved in the convulsion-inducing effect of MPN.
To clarify the direct effects of MPN in the brain on behavior and changes in vitamin B6 and GABA concentration, we intrathecally administered MPN (5-80 nmol) to mice. Some of them developed hyperactivity and seizures. Incidence of behavioral abnormalities were correlated with the MPN dose/body weight. In the analysis of
the brain, mice with hyperactivity shown decreased Pyridoxal-5’-phosphate (PLP) and decreased GABA/Glutamae (Glu) ratio. Administered MPN dose/body weight was negatively correlated with PLP (p＜0.001) and GABA/
Glu ratio (p＝0.004) in the brain. MPN concentration in the brain negatively correlated with PLP (p＜0.001) and
GABA/Glu ratio (p＜0.001), and a positive correlation between concentration of MPN and phosphorylated MPN(4’-O-methylpyridoxine-5’-phosphate, MPNP) was found (p＝0.01). We report for the first time that MPNP is
produced from MPN in vivo and MPN decreases PLP concentrations in the brain, resulting in the decrease of
GABA/Glu ratio and this change leads to behavioral changes. Shinshu Med J 72 : 159―167, 2024
(Received for publication February 9, 2024 ; accepted in revised form March 4, 2024)
Key words : 4’-O-methylpyridoxine, 4’-O-methylpyridoxine-5’-phosphate, gamma-aminobutyric acid,
Ginkgo biloba seed, pyridoxal-5’-phosphate
Abbreviations : BW, body weight ; GABA, gamma-aminobutyric acid ; GBS, ginkgo biloba seed ; Glu, glutamate ;GAD, glutamate decarboxylase ; HPLC, high-performance liquid chromatography ; MPN, 4’-O-methylpyridoxine ;MPNP, 4’-O-methylpyridoxine-5’-phosphate ; PBS, phosphate-buffered saline ; PK, pyridoxal kinase ; PL, pyridoxal ;PLP, pyridoxal-5’-phosphate
seizures6）7）. Research into the mechanism of convulsion

Ⅰ Introduction

by MPN in mice suggests the involvement of gamma-

Ginkgo biloba seed (GBS) is a popular food in Asia.

aminobutyric acid (GABA), which is produced fromIt can be eaten after heating them and are often in-glutamate (Glu) by glutamate decarboxylase (GAD),

cluded as an ingredient in stir-fries and other dishes.

to exert an inhibitory effect on brain activity8）. GABA

However, children have been reported to suffer poi-and Glu are respectively two major inhibitory andsoning after consuming even small amounts of GBS

excitatory neurotransmitters in the central nervous

1）-3）

and are warned against eating them

. Case of GBS

poisoning in adults have been described as well4）5）.

system9）. In complex partial epilepsy patients, decreaseof GABA/Glu ratio in intrahippocampal microdialy-

Present in GBS, 4’-O-methylpyridoxine (MPN) andsates was linked to seizures10）. When GAD generates

MPN-5’-glucoside (MPNG) causes hyperactivity and

GABA from Glu, pyridoxal-5’-phosphate (PLP), an＊

Corresponding author : Tomomitsu Sado
Department of Pediatrics, Shinshu University School of
Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan
E-mail : t_sado@shinshu-u.ac.jp

No. 3, 2024

active form of vitamin B6, is needed as a cofactor11）.
Decreases in plasma PLP concentration and PLP/
Pyridoxal (PL) ratio were found in rats receiving
MPN8）. Lower PLP/PL ratios have been detected in
159

Sado・Fukuyama・Nakayama

Fig. 1 The pathway by which MPN lowers GABA production. PK is needed to convert PL to PLP, and
PLP is needed as a cofactor to convert Glu to GABA. MPN is phosphorylated to MPNP by PK.

the serum and cerebrospinal fluid of patients withMPNP along with vitamin B6 in the mouse brain.

2）

GBS poisoning . The PLP/PL ratio in blood is a bio-Ⅱmarker for pyridoxal kinase (PK)8）, which is involvedin the production of PLP, as well as for the alkalineＡ

Materials and Methods

Chemicals

phosphatase that is needed to produce PL from PLP12）.

MPN-HCl and MPNP were chemically synthesized

In vitro studies have described an inhibitory effect ofas reported previously13）16）. All other reagents used

MPN on PK13）14）.

were of analytical grade.

Based on the above reports, the inhibition of PK

Ｂ

Animalsby MPN is thought to cause decreases in PLP, PLP-

The study was submitted to the Ethics Committee

dependent GAD activity, and GABA concentration,in Shinshu University with the stipulation that ad-

1）
8）
15）

thereby leading to convulsions (Fig. 1). However,ministered MPN doses that do not cause convulsions

reports on the relationships among MPN concentration,

should be used, and that in the event of convulsions,vitamin B6 concentration, GAD activity, and GABA

the mice should be euthanized immediately. Duringconcentration in the brain after MPN administration

intrathecal MPN administration, the mice were anes-

are lacking. The present study on mice intrathecallythetized with isoflurane to avoid causing them pain.

administered MPN was conducted to clarify the direct

All experiments were approved by the Institutional

effects of MPN on neurological symptoms, brain vita-

Animal Care Committee of Shinshu University (Ethicalmin B6, GAD activity, and GABA concentration. We

committee approval number : 021064) in accordance

opted for intrathecal administration because intra-

with the US National Research Council 2011 (Guide

peritoneal and oral administration of MPNs may cause

for the Care and Use of Laboratory Animals). Report-

phosphorylation of MPNs before they enter the intra-

ing of animal testing experiments comply with the14）thecal space , and there is a possibility that individual

ARRIVE guidelines. In this investigation, 4-5-week-14）

differences could be observed. Since Kästner et al.

old male ddY mice were obtained from Japan SLC, Inc

found that PK generated 4’-O-methylpyridoxine-5’-(Shizuoka, Japan). Mean±standard deviation body

phosphate (MPNP) from MPN, we also evaluatedweight was 29.2±3.1 g. All mice had free access to160

Shinshu Med J. 72

Effect of 4’-O-methylpyridoxine administration in brain

food and water throughout the study period.

a Shimadzu LC-10Avp system equipped with a RF-Ｃ

10A spectrofluorometer (Shimadzu, Kyoto, Japan). In

Intrathecal MPN administration
The mice were first anesthetized with isoflurane,vitamin B6 measurements, chromatographic separationand then head position was fixed with positioning

was performed in an Inertsil ODS-3 column (5 μm,

devices. Insertion of a needle into the right lateral ven-

150×4.6 mm, GL Sciences, Tokyo, Japan) at a flow rate

tricle was performed by incision with a scalpel andof 1.0 mL/min and column temperature of 25 ℃. Mo-

advancement of the needle tip 3 mm into the brain

bile phase A was 60 mM disodium hydrogen phos-from a position 1 mm to the right and 0.5 mm caudal

phate containing 400 mg/L ethylenediaminetetraacetic

17）

to the small fontanelle . MPN was dissolved in phos-acid disodium salt adjusted to pH 6.5 with concen-

phate-buffered saline (PBS), and 5 μl of MPN (5-80

trated phosphoric acid. Mobile phase B was methanol.

nmol) solution was injected into the right lateral ven-

The ratio of mobile phases A to B was 90 : 10. For

tricle using a microinjector (5 nmol for 2 mice, 10 nmol

determining the concentrations of vitamin B6 analogues

for 2 mice, 20 nmol for 5 mice, 40 nmol for 9 mice, andapart from the aldehyde types of vitamin B6, PL and80 nmol for 5 mice). In control mice, 5 μl of PBS was

PLP, fluorescence measurement was performed atadministered to 6 animals. The mice were observed

420 nm emission wavelength and 320 nm excitationfor 30 minutes after injection, during which time thewavelength. To identify PL and PLP, fluorescence mea-

experiment was stopped in mice exhibiting seizures.

surement was conducted at 450 nm emission wave-

After either 30 minutes of observation or seizure

length and 380 nm excitation wavelength after deri-

onset, all mice were anesthetized with isoflurane andvatization by semicarbazide. The concentration of

sacrificed by cervical dislocation for harvesting brain

vitamin B6 analogues were calculated using the abso-

tissue. Brain specimens were immediately frozen

lute calibration curve method. Linearity for eachwith dry ice and cryogenically stored at -70 ℃.

compound was confirmed from 6 nM to 900 nM. In

Ｄ

GABA and Glu measurements, chromatographic sep-

Sample preparation
A 10-fold volume of 5 mM Tris-HCl buffer (pH 7.4)

aration was performed in an Eicompak SC-5ODS

containing 1.15 ％ KCl, 0.1 mM EDTA, and homoser-

column (3.0 μm, 150×4.6 mm, EICOM, Kyoto, Japan)

ine as an internal standard was added to each brain

at a flow rate of 0.5 mL/min and column tempera-

sample, which were subsequently homogenized withture of 30 ℃. Mobile phase A was 100 mM disodium

a homogenizer, and stored at -70 ℃ until analysis. A

hydrogen phosphate containing 28 ％ methanol andreaction solution containing semicarbazide and gly-

0.5 ％ tetrahydrofuran after adjustment to pH 6.0 withcine was added to the sample for vitamin B6 concen-

concentrated phosphoric acid. GABA and Glu con-tration measurement. After 30 minutes of reaction tocentrations were determined by fluorescence mea-

produce a semicarbazone derivative, the sample wassurement at 455 nm emission wavelength and 340

deproteinized with perchloric acid and centrifuged.

nm excitation wavelength. GABA and Glu concentra-

The supernatant was then neutralized with potassium

tions were calculated by the internal standard methodhydroxide, centrifuged again, and analyzed by high-using homoserine as an internal standard.

performance liquid chromatography (HPLC) after fil-Ｆ

8）

Statistical analysis

tration . To measure amino acids and GABA, homog-

The relationship between MPN dose/body weight

enized samples were deproteinized with methanol,

and abnormal behavior in mice was analyzed bycentrifuged, and filtered. The samples were injected

Student’s t-test, as were the relationships of behav-

to HPLC 5 minutes after derivatization by ortho-

ioral abnormalities with the concentrations of vitaminphthalaldehyde.

B6 and amino acid analogues. The relationships ofＥ

MPN dose/body weight with vitamin B6 and amino

Chromatographic conditions
HPLC conditions were optimized from a previously

acid analogue concentrations in the brain were eval-reported method8）18）. The HPLC apparatus comprised

uated with Pearson’s chi-squared test. This test was

No. 3, 2024

161

Sado・Fukuyama・Nakayama
Table 1

Relationship between MPN dose/body weight (BW) ratio and abnormal behavior in mice
MPN dosage/ BW (mg/kg)

Group 1 : No hyperactivity, no seizure (n＝8)
Group 2 : Hyperactivity, no seizure (n＝12)Group 3 : Hyperactivity and seizure (n＝3)

0.22±0.18
0.28±0.14
0.50±0.08

Values represent the mean ± SEM of each group. Statistically significant differences were foundbetween group 1 and group 3 (p＝0.007) as well as between group 2 and group 3 (p＝0.013).

also employed to assess the correlations between vi-

able for the analysis are included in this study. In

tamin B6 and amino acid analogue concentrations. All

total, 18 mice brain were analyzed. MPN dose/bodyanalyses were performed using SPSS software (ver-weight had positive correlations with MPN and MPNP

sion 29.0.0.0). A p-value of less than 0.05 was consid-

concentrations (Fig. 2A, B). MPN dose/body weight

ered statistically significant.

was negatively correlated with the GAD biomarker

Ⅲ
Ａ

Results

Reactions of experimental animals

GABA/Glu ratio, PLP concentration, and total vitamin
B6 (PLP＋PL) concentration (Fig. 2D, E, H). In Fig. 2,
two mice that had a seizure within 30 minutes were

After intrathecal injection, the test mice required

excluded. Comparisons between control mice and hyper-

10-15 minutes to awaken from the isoflurane-induced

active mice showed that GABA/Glu ratio, PLP con-anesthesia. Hyperactivity was defined as obvious ab-

centration, and total vitamin B6 (PLP＋PL) concentra-

normal behavior, such as jumping around in a beakertion were significantly lower in the hyperactive groupor spinning in the same direction and was observed(Fig. 3A, C, E). The GABA/Glu ratios of the two micein groups administered 20 nmol or more of MPN.

that suffered seizures were 0.19 and 0.16, respectively,

The percentages of mice that became hyperactive

showing a decreasing trend compared with the control

were 5 nmol : 0 ％, 10 nmol : 0 ％, 20 nmol : 100 ％, 40

group. In the analysis of vitamin B6 and amino acidnmol : 67 ％ , and 80 nmol : 80 ％ . No hyperactivity

analogues in the brain, MPN concentration was sig-

was seen in the control mice. Obvious seizures werenificantly negatively correlated with GABA/Glu ratio

observed in the 40 nmol and 80 nmol MPN adminis-

and PLP concentration while being positively correlat-

tration groups. The percentages of mice displaying

ed with MPNP concentration (Fig. 4B, C, G). GABA/

seizures were 5 nmol : 0 ％, 10 nmol : 0 ％, 20 nmol :Glu ratio displayed a significant positive correlation

0 ％, 40 nmol : 11 ％, and 80 nmol : 20 ％. In terms ofwith PLP concentration and a negative correlation

MPN dose/body weight ratio, the lowest dose thatwith MPNP concentration (Fig. 4H, J). GABA con-caused seizures was 0.39 mg/kg (1.79 nmol/kg). All

centration and PLP/PL ratio showed a significant

mice that suffered seizures were hyperactive imme-

positive correlation (Fig. 4K).

diately beforehand. We observed a significant difference in MPN dose/body weight between mice withⅣ

Discussion(0.50±0.08 mg/kg) and without (0.22±0.18 mg/kg)

MPN intrathecally administered to mice induced

hyperactivity and seizures (p＜0.05). In addition, abehavioral abnormalities, including hyperactivity andsignificant difference was seen between mice with

convulsions, which were closely related to increases

hyperactivity (0.28±0.14 mg/kg) and those with sei-in MPN dose/body weight. By comparable amounts

zures (0.50±0.08 mg/kg) (P＜0.05) (Table 1).

of GBS intake, children with lower body weight may

Ｂ Measurement results of vitamin B6 and amino

experience increased MPN concentration in the brain,acid analogues

which is thought to contribute to the higher propensity

During the process of sample preparation and anal-of GBS poisoning in youths8）.

ysis, only data that are considered sufficiently suit162

The significant difference in GABA/Glu ratio, a
Shinshu Med J. 72

Effect of 4’-O-methylpyridoxine administration in brainp < 0.001
r = 0.753

70
60
50
40
30
20
10
00

00

05

10

15

20

MPN/BW (nmol/g BW)

00

05

10

15

20

MPN/BW (nmol/g BW)

25

60

40
30
20
10
00

00

05

10

15

20

MPN/BW (nmol/g BW)

25

25

PLP+PL

PLP/PL

10
05
00

00

05

10

15

20

MPN/BW (nmol/g BW)

25

30

0 30
0 20
0 10
0 00

00

05

10

15

20

MPN/BW (nmol/g BW)2530

p = 0.004
r = -0.678

0 25
0 20
0 15
0 10
0 05
00

05

10

15

20

MPN/BW (nmol/g BW)

45
40
35
30
25
20
15
10
05
00

25

30

p = 0.178
r = -0.355

00

05

10

15

20

MPN/BW (nmol/g BW)

10 0
90
80
70
60
50
40
30
20
10
00

25

30

p = 0.013
r = 0.606

(H)

20
15

0 40

(F)

30

p = 0.928
r = 0.250

(G)

0 50

0 00

PL (pmol/mg)

50

0 60

0 30

30

p < 0.001
r = -0.758

0 70

(D)

GABA/GluGABA (pmol/mg)

45
40
35

(E)

PLP (pmol/mg)

30

p = 0.282
r = -0.286

(C)

30
25
20
15
10
05
00

25

p = 0.004
r = 0.671(B)

MPNP (pmol/mg)

MPN (pmol/mg)

(A)

00

05

10

15

20

MPN/BW (nmol/g BW)

25

30

BW: Body weight, GABA: Gamma-aminobutyric acid, Glu: Glutamate,
MPN: 4'-O-methylpyridoxine, MPNP: 4'-O-methylpyridoxine-5'-phosphate,
PL: Pyridoxal, PLP: Pyridoxal-5'-phosphate
Fig. 2 Relationship of vitamin B6 and amino acid analogues with MPN dose/body weight (BW) were shown.
Only the mice that could be observed for 30 minutes after MPN administration were analyzed.

No. 3, 2024

163

Sado・Fukuyama・Nakayama

Fig. 3 Vitamin B6 and amino acid analogues in mice with or without abnormal behavior were shown. The graphs
contain the data of control mice. Data was expressed as mean ± SEM. ＊p＜0.05 versus control mice.

biomarker of GAD, between the hyperactive group

Due to ethical considerations, the mice in this investi-

and control group indicated a major influence on thegation were euthanized at the time seizures occurred.

behavioral abnormalities of the mice. We witnessed

As their brains were removed earlier than in othersignificant differences in brain GABA/Glu ratio, PLP

mice, they were excluded from the correlation studies

concentration, and PLP＋PL concentration between

in Fig. 2 and 3. Accordingly, mice without convulsions

controls and hyperactive mice. As the given MPN

were analyzed, suggesting that GABA might be moredose or MPN concentration in the brain increased,

important in seizures than in hyperactivity. Another

PLP concentration and GABA/Glu ratio in the brain

possible explanation for the non-significance of GABA

fell, likely since PK inhibition by MPN reduced PLP

was individual differences in the control mice and the

production and prevented GABA production from Glu

limited sample size ; further research is required onby GAD.

the impact of GABA concentration after MPN admin-

Although we observed a tendency for GABA to de-istration. Considering our results, it can be proposedcline, no significant difference or correlation was found.

that not only a decrease in GABA concentration in

164

Shinshu Med J. 72

Effect of 4’-O-methylpyridoxine administration in brain
p = 0 093
r = -0 408

0.0

1.0

2.0

3.0

4.0

5.0

MPN (pmol/mg)

6.0

6.0

4.0
3.0
2.0
1.0
0.0

1.0

2.0

3.0

4.0

5.0

MPN (pmol/mg)

6.0

7.0

2.5

PLP/PL

2.0
1.5
1.0
0.5
0.0

1.0

2.0

3.0

4.0

5.0

MPN (pmol/mg)

6.0

8.0

0.60
0.50
0.40
0.30
0.20
0.10
0.00

0.0

2.0

4.0

MPN (pmol/mg)

6.0

4.5
4.0
3.5
3.0
2.5

0.05
0.0

1.0

2.0

3.0

4.0

5.0

MPN (pmol/mg)

6.0

4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0

7.0

8.0

p = 0 662
r = -0 125

0.0

1.0

2.0

3.0

4.0

50

MPN (pmol/mg)

6080

p = 0 071
r = -0 435

(F)
10.0
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0

70

0.0

2.0

4.0

MPN (pmol/mg)

6.0

(H)

8.0

p = 0 030
r = -0 653

0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.00

0.10

0.20

0.30

0.40

0.50

MPNP (pmol/mg)

0.60

p = 0 011
r = 0 584

(J)
0.30

GABA/Glu

0.25
0.20
0.15

2.0
1.5
1.0

0.10
0.05

0.0

1.0

2.0

3.0

4.0

PLP (pmol/mg)

(K)
4.5

5.0

6.0

p = 0 011
r = 0 581

4.0

0.00

0.0

1.0

(L)

2.0

3.0

4.0

PLP (pmol/mg)

0.30

5.0

6.0

p = 0 909
r = 0 029

0.25

3.5

GABA/GluGABA (pmol/mg)

0.10

(D)

8.0

p = 0 059
r = 0 453

(I)

GABA (nmol/L)

7.0

p = 0 010
r = 0 700

0.70

3.0

0.20

2.5

0.15

2.0
1.5

0.10

1.0

0.05

0.5
0.0

0.15

GABA/GluMPNP (pmol/mg)

(G)

0.5
0.0

0.20

8.0

p = 0 366
r = -0 226

(E)

0.0

0.25

0.00

8.0

5.0

p < 0 001
r = -0 819

0.30

PL (pmol/mg)

PLP (pmol/mg)

7.0

p < 0 001
r = -0 709

(C)

0.0

(B)

GABA/Glu

4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0

PLP+PL (pmol/mg)

GABA (pmol/mg)

(A)

0.0

0.5

1.0

1.5

PLP/PL

2.0

2.5

0.00

0.0

0.5

1.0

1.5

PLP/PL

2.0

2.5

Fig. 4 The concentrations of vitamin B6, amino acid analogue, MPN, and MPNP in all mice brain was measured. The relationship between MPN and amino acid analogue, vitamin B6, and MPNP were shown in (A - G), and (H) shows therelationship between MPNP and GABA/Glu ratio. The relationship between vitamin B6 and amino acid analogues
were shown in (I - L).
No. 3, 2024

165

Sado・Fukuyama・Nakayama

the brain, but also a lower GABA/Glu ratio, play a

travenously injected into rats8）. These differences arerole in the behavioral changes caused by GBS poison-since MPNP is produced in cells but cannot be ex-ing. GABA suppresses brain activity, while Glu works

ported extracellularly owing to its hydrophilicity. Our

19）

to activate brain activity . Stable global neural func-

results indicate that after MPN enters the spinal cavity,tion is achieved by a coordinated and dynamically reg-part of it is phosphorylated in the brain to becomeulated balance between these excitatory (glutamater-

MPNP by PK.

20）gic) and inhibitory (GABAergic) inputs . ExperimentsⅤon rats have shown that decreased GABA in the brain

Conclusioncauses hyperactivity21）. The present study demon-

Intrathecal administration of MPN to mice induced

strated an importance of GABA/Glu ratio in behav-

seizures and abnormal behavior. MPN concentration

ioral changes involving the balance between excit-

increases were significantly correlated to decreases

atory and inhibitory neurotransmitters.

in PLP concentration and GABA/Glu ratio. In the

We report for the first time the changes in vitamin

pathology of GBS poisoning, lower GABA/Glu ratio

B6 in the brain after MPN administration to animals.

causes the brain to become more excitatory, result-

Thirty minutes after intravenous MPN administration

ing in hyperactivity and seizures. We report for thein an earlier rat experiment, blood PL concentration

first time that MPNP is produced in the brain in a

was significantly increased and PLP concentration

dose-dependent manner upon MPN administration.

8）

was decreased, resulting in an increase in PLP＋PL .

Additional intrathecal MPN administration studies

A marked decrease in PLP/PL ratio was also observed

are needed to understand the chemical changes oc-

in the blood and cerebrospinal fluid of patients withcurring in the brain during GBS poisoning.

2）

GBS poisoning . Those extracellular fluid results

Declaration of competing interest

were due to PL production from MPN in the liver aswell as MPN inhibiting PK8）. However, in the intra-

The authors declare that they have no known

thecal administration of MPN in mice, both PLP andcompeting financial interests or personal relationships

PLP＋PL decreased in the brain, with no increase inthat could have appeared to influence the work re-

PL. This suggests that the metabolic activity from

ported in this paper.

MPN to PL is low in brain, and that PL may have

Acknowledgements

leaked out of cells owing to its good membrane per-Mouse experiments were performed with Aikomeability.
To the best of our knowledge, this is the first inves-Hasegawa at the Department of Pediatric Medicine,

tigation to detect MPNP in vivo. Although MPNP could

Shinshu University School of Medicine, and brain

decrease GAD activity at such non-physiologicallymeasurements were performed at Hokkaido Medical

22）high concentrations as 0.5-3.5 mM , the MPNP con-

University by Haruka Yasunaka, Mao Otani, Toshiki

centration measured in this experiment was insuffi-

Yamashiro, Keiji Wada, Daisuke Kobayashi, and Naoya

cient to inhibit GAD. A significant positive correla-

Hamaue at Hokkaido University of Medical Science.

tion was seen between MPN and MPNP concentrations

The authors would like to express their sincere grat-

in our study. In other animal experiments, MPNP

itude to these individuals.

was not detected in the plasma when MPN was inReferences
1） Mei N, Guo X, Ren Z, Kobayashi D, Wada K, Guo L : Review of Ginkgo biloba-induced toxicity, from experimental
studies to human case reports. J Environ Sci Health C Environ Carcinog Ecotoxicol Rev 35 : 1-28, 20172） Sado T, Nakata S, Tsuno T, et al : Concentrations of various forms of vitamin B6 in ginkgo seed poisoning. Brain
Dev 41 : 292-295, 2019166

Shinshu Med J. 72

Effect of 4’-O-methylpyridoxine administration in brain
3） Kajiyama Y, Fujii K, Takeuchi H, Manabe Y : Ginkgo seed poisoning. Pediatrics 109 : 325-327, 2002
4） Azuma F, Nokura K, Kako T, Kobayashi D, Yoshimura T, Wada K : An Adult Case of Generalized Convulsions Causedby the Ingestion of Ginkgo biloba Seeds with Alcohol. Intern Med 59 : 1555-1558, 2020
5） Miwa H, Iijima M, Tanaka S, Mizuno Y : Generalized convulsions after consuming a large amount of gingko nuts.
Epilepsia 42 : 280-281, 2001
6） Wada K, Ishigaki S, Ueda K, Sakata M, Haga M : An antivitamin B6, 4’-methoxypyridoxine, from the seed of Ginkgo
biloba L. Chem Pharm Bull 33 : 3555-3557, 1985
7） Zhang W, Shi M, Wang J, Cao F, Su E : A comprehensive review of ginkgotoxin and ginkgotoxin-5’-glucoside in
Ginkgo biloba L. seeds. J Food Compost Anal 125 : 105726, 2024
8） Kobayashi D, Yoshimura T, Johno A, Ishikawa M, Sasaki K, Wada K : Decrease in pyridoxal-5’-phosphate concentrationand increase in pyridoxal concentration in rat plasma by 4’-O-methylpyridoxine administration. Nutr Res 35 : 637-642,
20159） Cai H-L, Zhu R-H, Li H-D, et al : Elevated plasma γ-aminobutyrate/glutamate ratio and responses to risperidone
antipsychotic treatment in schizophrenia. Prog Neuropsychopharmacol Biol Psychiatry 34 : 1273-1278, 2010
10） During MJ, Spencer DD : Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain.
Lancet 341 : 1607-1610, 199311） Modi JP, Prentice H, Wu J-Y : Regulation of GABA Neurotransmission by Glutamic Acid Decarboxylase (GAD). Curr
Pharm Des 21 : 4939-4942, 201512） Akiyama T, Kubota T, Ozono K, et al : Pyridoxal 5’-phosphate and related metabolites in hypophosphatasia : Effects
of enzyme replacement therapy. Mol Genet Metab 125 : 174-180, 201813） Mizuno N, Kawakami K, Morita E : Competitive inhibition between 4’-substituted pyridoxine analogues andpyridoxnl for pyridoxal kinase from mouse brain. J Nutr Sci Vitaminol 26 : 535-543, 1980
14） Kästner U, Hallmen C, Wiese M, Leistner E, Drewke C : The human pyridoxal kinase, a plausible target for ginkgotoxin from Ginkgo biloba. FEBS J 274 : 1036-1045, 200715） Leistner E, Drewke C : Ginkgo biloba and ginkgotoxin. J Nat Prod 73 : 86-92, 201016） Harris SA : Chemistry of Vitamin B6. II. Reactions and Derivatives. J Am Chem Soc 62 : 3203-3205, 1940
17） Smith JS, Angel TE, Chavkin C, Orton DJ, Moore RJ, Smith RD : Characterization of individual mouse cerebrospinal
fluid proteomes. Proteomics 14 : 1102-1106, 201418） Talwar D, Quasim T, McMillan DC, Kinsella J, Williamson C, O’Reilly DSJ : Optimisation and validation of a sensitive
high-performance liquid chromatography assay for routine measurement of pyridoxal 5-phosphate in human plasma
and red cells using pre-column semicarbazide derivatisation. J Chromatogr B Analyt Technol Biomed Life Sci 792 :
333-343, 2003
19） Petroff OAC : GABA and Glutamate in the Human Brain. Neuroscientist 8 : 562-573, 200220） Sears SM, Hewett SJ : Influence of glutamate and GABA transport on brain excitatory/inhibitory balance. Exp Biol
Med 246 : 1069-1083, 2021
21） Yokawa T, Shiota K, Takahashi M : Hyper-running activity originating from the hypothalamus is blocked by GABA.
Physiol Behav 47 : 1261-1264, 1990
22） Buss K, Drewke C, Lohmann S, Piwonska A, Leistner E : Properties and interaction of heterologously expressed
glutamate decarboxylase isoenzymes GAD(65kDa) and GAD(67kDa) from human brain with ginkgotoxin and its
5’-phosphate. J Med Chem 44 : 3166-3174, 2001

（2024. 2. 9 received ; 2024. 3. 4 accepted）

No. 3, 2024

167