Presence of tetrahydroisoquinoline and 2-methyl-tetrahydroquinoline in Parkinsonian and normal human brains

Vol. 144, No. 2, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1084-1089

April 29, 1987

PRESENCE OF TETRAHYDROISOQUINOLINE AND 2-METHYL-TETRAHYDROQUINOLINE PARKINSONIAN AND NORMAL HUMAN BRAINS

IN

Toshimitsu Niwa I, Naohito Takeda 2, Norio Kaneda 3, Yoshio Hashizume4,. and Toshiharu Nagatsu 3.

IDepartment

of Internal

Medicine,

Nagoya University Branch Hospital,

Higashi-ku, Nagoya 461, Japan 2Faculty of Pharmacy, 3Department

Meijo University,

of Biochemistry

and

Tempaku-ku,

4Department

Nagoya University school of Medicine,

Nagoya

468, Japan

of Pathology,

Showa-ku,

Nagoya 466,

Japan

Received March 25, 1987

1,2,3,4-Tetrahydroisoquinoline (TIQ) and 2 - m e t h y l - 1 , 2 , 3 , 4 - t e t r a h y d r o q u i n o line (2-Me-TQ) were identified for the first time by gas chromatography-mass spectrometry in the parkinsonian and normal human brains. TIQ~ an analogue of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) , was markedly increased in the parkinsonian brain and could be an endogenous neurotoxin to induce Parkinson's disease. © 1987 Academic Press, Inc.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine state in humans,

monkeys and mice

by the degeneration decrease of dopamine

(I-3).

of the nigrostriatal contents

causes the degeneration

dopaminergic (4).

MPTP is a neurotoxin

dopaminergic

neurons

ion (MPP +) in the brain

the striatum after a single administration

(5). in tissue slices of

to mice (6).

that MPTP and MPP + inhibited tyrosine hydroxylation

*To whom all correspondence

Neurotoxi-

and caused reduction of tyrosine

itself after repeated administration

specially from the striatum,

(I-3).

and

oxidase type B to 1-

We reported that MPTP inhibited tyrosine hydroxylation

hydroxylase

parkinsonian

neurons and by the

city of MPTP is dependent on its conversion by monoamine methyl-4-phenylpyridinium

induces

Parkinson's disease is characterized

in the striatum

of nigrostriatal

(MPTP)

We also reported

in tissue slices in situ,

and that the inhibition by MPTP but not the effect

should be addressed.

0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of M P P + was prevented by a MAO inhibitor or by a d o p a m i n e uptake inhibitor (7, 8).

W e have developed a simple screening system to find a probable compound to

induce Parkinson's disease by the inhibition of the tyrosine hydroxylase system in striatal tissue slices (9).

The results of screening of various

MPTP or

M p p + - r e l a t e d compounds indicated that both pyridinium and phenyl group are essential for the effects and that either N-methyl-1,2,3,4-tetrahydroisoquinoline

(N-Me-TIQ)

or

1,2,3,4-tetrahydroisoquinoline

(TIQ) that is structurally

similar to MPTP and related to dopamine could be one of the candidate of endogenous or environmental

factors that produce Parkinson's disease

(10,11).

In

fact, TIQ was shown to reduce tyrosine hydroyxlase after repeated administration to mice

(10,11).

Kohno et al. (12) have recently reported the presence of TIQ

and I-Me-TIQ as novel endogenous amines in the rat brain by coupled gas chromatography-multiple

ion detection.

W e report here presence of TIQ and 2-methyl-1,2,3,4-tetrahydroquinoline

(2-

Me-TQ) in parkinsonian and normal h u m a n brains by gas c h r o m a t o g r a p h y - m a s s spectrometry. MATERIALS AND METHODS The brain was obtained at autopsy from a patient with Parkinson's disease. The patient was a 68-year-old female suffering from Parkinson's disease for 4 and half years, died suddenly from asphyxia, and the p o s t m o r t e m time was 5 hours. The diagnosis of Parkinson's disease was confirmed histopathologically by the neuronal loss in the substantia nigra and by the observation of the Lewy bodies. The normal control brain Was from a patient w i t h stomach cancer w h o died from asphyxia. The patient was a 81-year-old male, and the p o s t m o r t e m t i m e was 6 hours, and no histopathological abnormalities were observed in the brain. The brains were stored at -80°C until sample preparation. TIQ, 1,2,3,4-tetrahydroquinoline (TQ), and 2-Me-TQ were purchased from Wako Pure Chemical Industries, Ltd., heptafluorobutyric anhydride (HFBA) and pentafluoropropionic anhydride (PFPA) from Gasukuro Kogyo Inc. All other chemicals used were of analytical grade. The brain samples were treated for gas c h r o m a t o g r a p h y - m a s s spectrometry, as described by Kohno et al. (12). The frontal cortex (25 g) was homogenized for 30 sec at 0°C in 0.4 N perchlorie acid (20 ml) containing EDTA (0.1% w/v) and ascorbic acid (0.1% w/v). The homogenate was centrifuged at 12,000 g for 15 m i n at 4°C. The supernatant was transferred to a glass test tube, and the pellet was vortexed w i t h 0.4 N perchloric acid (20 ml) containing EDTA (0.1% w/v) and ascorbic acid (0.1% w/v) and centrifuged again. The combined supernatant was extracted w i t h diethyl ether (20 ml). The aqueous phase was adjusted to pH 11.0 with 6 N NaOH and extracted twice with dichloromethane (20 ml). The organic phase w a s extracted with 0 . 1 N HCI solution (20 ml) containing EDTA (0.1% w/v) and aseorbic acid (0.1% w/v). The aqueous phase was adjusted to pH 11.0 and extracted w i t h dichloromethane (20 ml). The organic phase w a s dehydrated over anhydrous sodium sulfate and the filtrate evaporated to dryness with N 2 stream. The residue was dissolved in ethyl acetate/HFBA (20 91: 20 ~i) or ethyl acetate/PFPA (20 pl: 20 pl), and derivatized at 70°C for 30 min. For identifica-

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Vol. 144, No. 2, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

tion no c h e m i c a l w a s a d d e d as an internal standard, b u t for s e m i q u a n t i t a t i o n a constant a m o u n t of TQ w a s added to the s u p e r n a t a n t of the h o m o g e n a t e as an internal s t a n d a r d . For gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y (GC/MS), a S h i m a d z u gas c h r o m a t o g raph (GC-gA)-mass s p e c t r o m e t e r (9020-DF) w a s used. Gas c h r o m a t o g r a p h y w a s performed on a HiCap-CBPI-M25-025 f u s e d - s i l i c a c a p i l l a r y c o l u m n (25 m x 0.2 m m I.D.), f i l m thickness 0.25 p m (Shimadzu), w i t h h e l i u m (23 cm/sec) as a c a r r i e r gas. The s a m p l e s w e r e i n j e c t e d s o l v e n t l e s s w i t h a m o v i n g - n e e d l e type injector. The i n j e c t i o n t e m p e r a t u r e w a s 250°C, t e m p e r a t u r e p r o g r a m f r o m 130 to 190°C at 3°C/min; s e p a r a t o r t e m p e r a t u r e 280°C, ion source t e m p e r a t u r e 250°C, e l e c t r o n i m p a c t i o n i z a t i o n (EI) e n e r g y 70 eV, t r a p current 60 ~A, a n d a c c e l e r a t i n g voltage 3 kV. C h e m i c a l i o n i z a t i o n (CI) m a s s s p e c t r o m e t r y w a s p e r f o r m e d w i t h i s o b u t a n e as a r e a c t a n t gas. CI e n e r g y w a s 200 eV and e m i s s i o n c u r r e n t 200 pA. The o t h e r c o n d i t i o n s w e r e the s a m e as for El. RESULTS Fig.

I shows

mass

chromatograms

(a), a n d the HFB d e r i v a t i z e d Parkinson's

disease

(b) and

2-Methyl-I, 2, 3, 4tetrahydroquinoline

of the HFB d e r i v a t i v e s

extracts from

from

frontal

a control

patient

cortex

of 2 - M e - T Q

of a p a t i e n t

and T I Q with

(c).

1, 2, 3, 4 - T e t r a h y d r o i s o q u i n o l i n e

l

/

mlz 343

x 1)

328

xl) xl)

210

xl)

329

16

Parkinsonism Peak 1

Peak 2

l

,,

l •" < - , ~ - , - - J ' ~ - , ~ - ~

~

__~.~._J~

~

~_..--.._/k__.~

9

I'0

1'I

'2--

I'3

14

15

343

x

- ~

329

x 1

~-.~--.

328

x 1

343

x

1

329

x

1

328 210

x 1 x 1

1

16

Control Peak I

~

6

9

I'0

1'1

12

13

14

15

16

Time (min) Figure 1. Mass chromatograms of the HFB derivatives of 2-methyl-1,2,3,4tetrahydrequinoline (2-Me-TQ) and 1,2,3,4-tetrahydroisoquinoline (TIQ) (a), the HFB derivatized extracts from parkinsonian frontal cortex (b) and normal frontal cortex (c).

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Vol. 144, No. 2, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1O0 ]

a)

32

~ C H 3 I

COC3F7

>..,

M +

u~

328 343

5o~ % 50

100

lO0-

m/z 200

150

250

300

350

250

300

350

b)

>-, C

50 4-, ~3

.

50

~igu{e 2. in Fig.

it s h o w e d

same

spectrum

result

showed

spectrum

identical

son's d i s e a s e

zation

200

Peak

on GC

(Fig.

I was

(a), and peak I (b)

I in Fig.

cortex

ion at m / z 344,

[M+H] +,

identified

was

identical

of the a u t h e n t i c

2 was

in Fig.

since

EI m a s s

compound.

identified

The

confirmed

of the c o n t r o l

identical

of a p a t i e n t by using

confirmed patient,

patient

330,

[M+H] +,

EI m a s s compound.

with

Parkin-

PFP derivatization

by using but

CI m a s s

as TIQ since it

of the a u t h e n t i c

cortex

also

3 (b).

ion at m / z

I) a n d a l m o s t

of the p a r k i n s o n i a n

1087

as 2 - M e - T Q

I (c).

Peak

on GC (Fig.

of T I Q w a s

cortex

CI m a s s

quasimolecular

in the frontal

The d e t e c t i o n

2 (b).

I (b) is s h o w n

329.

patient

in Fig,

I) and a l m o s t

of the HFB d e r i v a t i v e

a n d of a c o n t r o l

in the f r o n t a l

343.

an intense

time

of 2 - M e - T Q

in the f r o n t a l

quasimolecular

2 i n Fig.

ion at m / z

retention

I (b) is s h o w n

of the HFB d e r i v a t i v e with peak

3) to those

of HFB.

confirmed

time

of peak

the m o l e c u l a r

(Fig.

an i n t e n s e

2 also showed

The d e t e c t i o n

instead

.

I in Fig.

retention

obtained

of peak

spectrum

.

ion at m / z

2) to those

was

EI m a s s

indicating

.

of p e a k

the m o l e c u l a r

(Fig.

spectrum

.

150 m/z

I revealed

identical

spectrum

.

El mass spectra of the HFB derivative of 2-Me-TQ

of p e a k

indicating

.

I.

EI m a s s spectrum

.

100

PFP derivati-

could

b y using

not be

PFP derivatiza-

Vol. 144, No. 2, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

100 t

~N-COC3F 210

a)

7 M +

329

104 m

cg3

117

g

5oi

........ T ......... ~ .Ji

50

100

1 oo

150 m/z

200

150 m/z

200

30o

"3 o

b)

c

°

,1, .... 1.........J ,I II, II 50

Figure 3. F i g . 1.

tion,

due to the l o w e r

with

ion m o n i t o r i n g ,

of the p a r k i n s o n i a n

patient

that of the control

patient;

TQ w a s

concentration

that of the p a r k i n s o n i a n

By s e l e c t i v e

less than

250

300

350

El mass spectra of the HFB derivative of TIQ (a), and peak 2 (b) in

probably

compared

100

I ng/g

similar

between

patient,

as

patient.

the c o n c e n t r a t i o n

w a s found

of T I Q in the frontal

to be m a r k e d l y

approximately

in the control

of T I Q in the control

brain.

10 ng/g

increased

the p a r k i n s o n i a n

brain

as c o m p a r e d

in the p a r k i n s o n i a n

In contrast,

the c o n c e n t r a t i o n

(2 ng/g)

cortex

and the n o r m a l

with

brain

and

of 2 - M e brain

3

ng/g ). DISCUSSION The d i s c o v e r y tal d o p a m i n e r g i c Parkinson's

of MPTP, neurons

disease

xins s i m i l a r

which

is a n e u r o t o x i n

and p r o d u c e s

We have s c r e e n e d

M P T P or M P P + b a s e d on the i n h i b i t i o n slices

(7-9)

endogenous

Parkinson's

m i g h t be c a u s e d by s o m e

to MPTP.

and have

neurotoxins

reported (10,11).

specific disease

endogenous

the c o m p o u n d s

of t y r o s i n e

1088

TIQ w a s

(I-3),

suggests

or e x o g e n o u s structurally

hydroxylation

that T I Q or N - M e - T I Q In fact,

for the n i g r o s t r l a -

neurotosimilar

in striatal

could be a c a n d i d a t e

found

that

to r e d u c e

to tissue

for the

tyrosine

Vol. 144, No. 2, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hydroxylase in the striatum after repeated peripheral administration to mice (10,11). brain.

Kohno et al. (12) have recently found TIQ and I-Me-TIQ in the rat 6,7-Dihyroxy-1-Me-TIQ

was also found in the rat brain (13).

In the present study, we have confirmed for the first t i m e the presence of TIQ and 2-Me-TQ in the brains from a parkinsonian patient and a control patient. 2-Me-TQ is a novel a m i n e that has never been identified in animals.

The level of

TIQ was m u c h higher in the parkinsonian brain than that in the normal brain, whereas the level of 2-Me-TQ w a s similar b e t w e e n the parkinsonian brain and the normal brain (Fig.

I).

We could not detect in the parkinsonian or normal brain

I-Me-TIQ that was found in the rat brain by Kohno et al. (12).

Although further

studies are required, the present results suggest the possibility that TIQ which is structurally related to MPTP and dopamine and has an in vivo effect similar to that of MPTP

(10,11) could be a candidate for the endogenous neurotoxin to

produce Parkinson's d i s e a s e . REFERENCES I. 2. 3. 4.

5. 6.

7. 8. 9. 10. 11. 12. 13.

L a n g s t o n , J.W., B a l l a r d , P., T e t r u d , J.W. a n d I r w i n , I. (1983) S c i e n c e 219, 979-980. B u r n s , R.S., c h i u e h , C.C., M a r k e y , S.P. and Ebert, M.H. (1983) Proc. Natl. Acad. Sci. USA 80, 4546-4550. Heikkila, R.E., Hess, A. and Duvoisin, R.C. (1984) Science 224, 1451-1453. Ehringer, H. and Hornykiewicz (1960) Klin. Wschr. 24, 1236-1239. M a r k e y , S.P., J o h a n n e s s e n , J.M., Chiueh, C.C., Burns, R.S. a n d H e r k e n h a m , M.A. (1984) Nature 311, 464-467. Hirata, Y. and Nagatsu, T. 1986) Neurosci. Lett. 68, 245-248. Hirata, Y. and Nagatsu, T. 1985) Brain Res. 337, 193-196. Hirata, Y. and Nagatsu, T. 1985) Neurosci. Lett. 5_~7, 301-305. Hirata, Y. and Nagatsu, T. 1986) Brain Res. 397, 341-344. H i r a t a , Y. and N a g a t s u , T. 1985) S h i n k e i K a g a k u 24, 169-171. Nagatsu, T. and Hirata, Y. 1986) Europ. Neurol. $I-159, I-5. K o h n o , M., Ohta, S. and H i r o b e , M. (1986) B i o c h e m . B i o p h y s . Res. C o m m u n . 140, 448-454. N e s t e r i c k , C.A. and R a h w a n , G.C. (1979) J. C h r o m a t o g r . ~64, 205-216.

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