PHARMACOLOGICAL STUDIES UPON THE PRODUCTION OF GLUTAMIC ACID AND GLUTAMINE IN THE BRAIN*

PHARMACOLOGICAL GLUTAMIC

STUDIES

ACID

AND

UPON

GLUTAMINE

SHOJIRO Department

of Pharmacology, Received

for

THE

PRODUCTION

IN THE

OF

BRAIN*

TAKENAKA

Kyoto Prefectural

publication

Medical College, Kyoto

November

30,

1953

INTRODUCTION Weil-Mahlherbe

(1) reported

which can be absorbed

in 1936 that

and assimilated

in the

glutamic brain.

acid is the Since

only amino

that time there

acid

has been

developed the clinical application of glutamic acid to brain diseases. For instance, it is reported that the glutamic acid intravenously infused can relieve or cure hypogly cemic

coma (2) ; when

administered

orally, it shows a striking

curative

effect on a

patient of intelligence deficiency with mongolism (3, 4), idiot, underdevelopment of brain and so on (3), relieves the fit of epilepsia, and promotes the psychological and intelligent growth of children (3). Moreover, some reports refer to important physiological actions of glutamic acid such as to effect the convulsion on the central nervous system (3, 5) or to activate the choline-acetylase and so on (3, 6). On the other hand, as to the process produced,

more

knowledge

is remained

glutamic acid intravenously or glutamine in the brain. acid content,

ranking

of which glutamic blank.

Schwerin

acid and

glutamine

et al. (7) stated

that

are the

injected did not result in an increase of glutamic acid Brain, anyway, is an organ richest in the glutamic

next to kidney.

For example,

brain contains

50 times as much

glutamic acid, and 10 times as much glutamine as blood has (7). Thus we come to the question from what substance and how is this great deal of glutamic acid and glutamine formation

produced in this organ. Herein my exprimentation is directed process of them from the pharmacological standpoint.

to pursue

the

METHODS The

experiments

were carried

out in vivo and in vitro.

(1) Experiment in vivo : Via a vein of the tail, rats weighing around 90 g sodium dl-glutamate or sodium a-ketoglutarate was injected and then those rats were decapitat ed in an hour and in 3 hours, respectively, Then

the

determination

Some

part

Area

of the

of this Japanese

of the

description

has

Pharmacological

to be taken off the brain

amounts

already

of glutamic

been

Society

read

on the

acid

7th

and

cortex

immediately.

of glutamine

Regional

Meeting

was

of Kinki

carried

out on the cortex extirpated.

The

determination

micromethod To

of glutamic

of chromatography

assay

glutamine

the term "total

the method

glutamic

of the naturally lyzing glutamine.

was

performed

by Prescott

of Bessman

acid" in the following

occurring

glutamic

The

a-ketoglutaric

acid

Neuberg

and Ringer's

method

2)

acid

worked

used

according

and Waelsch et al. (9) was

description

acid and

to the chemical (8).

employed.

represents

the one which is produced

in my experiment

was

(10) in our laboratory,

synthesized

by hydro

according

M.P. 113'C and acidity

to

98%.

Ex heri went i n vi tr) : A)

The slice preparation

cortices

in aerobic condition :

(1TC) (11) in Warburg's

37.5°C. After the solution for the

the

incubation

in the

following

to the mixture, the further minutes,

decapitation,

side-arm

addition

4 ccm

determinations acid

were referred

to Fujita's

and

was

of pyruvic

a-ketoglutaric

(14) and Umbreit's

acid

C) Krebs

acid and

10

The 2

a-ketoglutaric

glutamine.

were conducted apparatus,

for

amount.

The

according

techniques

and

(15;, that is, in the main chamber

buffer ; in the inner cup a filter paper

and the gas space was filled out with 0,

gas.

For

below 0°C, accord

el al. (15)

The slice preparation and Henseleit's

was put in the inner from

after

centrifuged

glucose assay Hagedorn-jensen's method (16) was employed. B) 11omogenation : The homogenation was always performed ing to Timbreit

at

was added

an ad which,

acid and

the Warburg's

and 2.7 ccm of phosphate

by 0.3 ccm of 10'x; KOH,

acid,

of glutamic

(13). In operating

0.3 ccm of substrate

aid

with emery,

trichloroacetic

bath

equilibrium,

in the flask to be shaken

was added upto 6 cc in the whole

for the, estimations

for

of pyruvic

to Shimiz u's method

wetted

water

cerebral

in water

thermodynamic

with the solution

down with a pestle

of 0.5 ccm of the

provided

other

determinations

mixed

the

Then 0.5 ccm of 10%' trichloroacetic

which was ground

to the supernatant

the

remarks

was

to get

rats'

in the phosphate

flask (12). The flask was immersed for 15 minutes

90 minutes.

ccm from it was acid,

After

were taken out to be made into slices, which were suspended

buffer

free

Besides,

the sum total

in anaercbic

solution cup.

The

condition :

(17) was used gas

space

For

and 1-2 was

anaerobic

treatment

g of wet yellow

filled by N2+5%

C02,

the

phosphorus completely

02-gas. RESULTS AND DISCUSSION I.

dl-Glutamic into a vein tirpated

acid,

of the

Administration

of glutamic

1.2 mg per 1 g of body

tail.

for determinations

After a certain of glutamic

time

weight elapsed

acid of a normal the cerebral

acid and glutamine

(Table

rat, was

infused

cortex

was ex

1).

TA'P-LE 1.

Neither cerebral

Administration

increase

cortex

after

nor

of glutarr is acid into a vein of the rat's

decrease

acid

or glutamine

is observed

in the

the infusion.

In order to investigate attempted in the following aerobically

glutamic

tail (mean value)

sliced brain

this elaboration in vivo the direct observation experiment in vitro, using the brain cortex itself.

cortices

were

dipped

in the solution

containing

was The

5 mg of dl

glutamic acid, and then shaken according to Warburg's method. After separating the dipping solution from slices, the glutamate and glutamine contained both in the solution

and slices were estimated TABLE 2.

From the experiment a great

part

stated

in the cerebral

These facts inferred,

normal

glutamic

acid

above it is concluded : The brain acid that is initially

cortex

consumes

added into the conical

solution.

Glutamic

acid is scarcely

Schwerin

et al. (7) reported

flask, accu

cortex.

coincide

II. the

with

in the dipping

well

too, from the results

After

2).

Incubation

(69 %) of the glutamic

and 31% is found remaining mulated

(Table

injection

with

what

by Friedberg

Administration of

and Greenberg of a-ketoglutaric

a-ketoglutarate,

rat, via a vein of the tail, its content

1.2 mg

can be

(19). acid

per I g of

in the brain

and

body

was estimated

weight

of a

in the same

manner

as (1) (Table TAFLE

3.

The increases approximately

3). Administration

of glutamic

a-ketoglutaric

a-ketoglutaric

into

a vein

of

the

after the infusion

rat's

tail

are very striking,

to the time elapsed.

the experiments

acid

acid

acid and glutamine

proportionate

Discussion : From of

of

above mentioned

in vivo brings

forth

the

it is found that the infusion

increases

of glutamic

acid

and

glutamine in the brain cortex, while the glutamic acid infused is never accumulated, though it spreads over the brain tissue. From these facts it will be inferred that the accumulation

of glutamic

acid and glutamine

in the cerebral

cortex

a-ketoglutaric

acid, which is an intermediary product of glucose metabolism, plays an important and the accumulation is not caused by glutamic acid proved hematogenously. On the other hand, the conversion and kidney. which

Dewan et al. (20) and

of glutamic Taking

can

never

acid

account

enter

the

into

of that

other workers

a-ketoglutaric fact, the author

brain tissue

in the

(7, 21-26)

acid in some infers

role,

reported

organs,

the

that the glutamic

that liver acid,

form as it is, can participate

in the

glutamic acid formation in the brain tissue when provided hematogenouly in the form of a-ketoglutaric acid, the conversion of which is carried out in some other organs,

the

liver

administered

and

in the

high

mental

conditions

administered

brain

(7) stated liver,

it is inferred

not cause the

accumulation

that

that

muscle,

the

particularly, the glutamic

and formation

glutamic

especially

acid

in kidney

under the

experi

acid hematogenously of glutamic

acid and

because the conversion into a-ketoglutaric acid in such organ very slow and small. Thus the effect of glutamic acid in the

seems

indifferent

to the accumulation

process

of glutamic

acid in

cells themselves.

Recently kidney by from

et al. in the

So, in my experiments,

of the author's

could

practice

Schwerin

is accumulated

concentration.

glutamine in brain, as the kidney was clinical

kidney.

intravenously

it was

reported

sucrose

by Hogeboom

all kinds

of enzyme

and

by

that

that the cyclophorase

Green et al. (27), of which

this

system

preparation

properties

preparation,

resemble

et al. (28) with a super-high involved glutamic

extracted

speed

is completely

isolated

centrifuge,

in C.A.C. (citric acid cycle) acid

from the

the mitochondria after

oxidized

contains Krebs

under

(29)

aerobic

condition,

but

accumulation

under the presence of glutamic

This fact well coincides in brain

of a-ketoglutaric

acid occurs, regardless with my results

tissues

has already

and accumulation

of glutamic

acid might

afore-mentioned

we know

From the portant role brain tissue. acid in the which

been

the condition,

and, moreover,

afficined.

Thus

formation intended

of glutamic

acid the

to investigate

III. is glucose

shaking

arm, according and glutamine

the

formation

Incubation

(0.093.x) in the

or anaerobic. of cyclophorase

that

the formation

this system.

Krebs

(29) plays

aerobically

experiments

processes

excluding

were

of glutamic

numerous

an im

The same estimations

Incubatirl

with

flask,

which

serves

gluc)3e

in aerobic

were carried out on samples

5.

Incubation

with

result

as a substrate.

N~H4C1 in the side

glucose

which

in anaerobic

glutamate

cond.iti)n

were shaken

5). TABLE

acid and

with glucose

conical

two kinds of flasks, with and without

4.

performed,

influences

to Warburg's method (12), glucose, pyruvate. a-ketoglutarate, were estimated on each sample (Table 4). TABLE

ly (Table

out through

C.A.C. after

following

glutamine as functions of brain cells themselves, ing from other tissues than the brain.

After

the abundance

it is inferred

be carried that

aerobic

the

in producing and accumulating glutamic acid and glutamine in the For further studies on roles of a-ketoglutaric acid, glucose and pyruvic

were

A load

acid as well as of ammonia,

condition

anerobical

As shown above, are

scarcely

changes

observed

of pyruvate,

compared

with

a-ketoglutarate, the

blind

glutamate

test,

and glutamine

regardless

to the substrate,

glucose only or glucose plus NH4C1, and, to the manner of shaking, aerobical and anaerobical. Though it is considered that glucose is the main substrate supporting the functional

activity

of the brain

(30) it is not likely to participate

in this

system.

Discussion : For the fact that glutamic acid can be produced from a-ketoglutaric acid in brain, which is an intermediary product in the glucose metabolism, while from glucose, stated,

that

glycolysis

it is never

yielded, we have

glucose decomposition by Embden

suggestion,

might take an unknown

(32) and Meyerhof IV.

an interesting

Incubation

(33) in brain

pathway

as Baker (31)

different

from the

tissues.

with a-ketoglutaric

acid

a-Ketoglutaric acid, a component participating in the .formation and accumulation of glutamic acid and glutamine, is an important member of C.A.C.. In the following description

'as to a-ketoglutaric

acid

of it and

"low

to 0.003 M.

ruvate,

concentration"

glutamate,

(1)

and glutamine

Administration

a-Ketoglutaric

added

As shown

6.

above,

Incubation

from

consumed

case added is found

as total

synthesis

of glutamine.

refers

to 0.03 M

of a ketoglutarate,

out in the following

manner

py

:

as a substrate.

The

comparison

was

NH4C1 (0.03M), glucose (0.01M), and NH4C1 (0.03M)+ glu (Tabled). with

a-ketoglitaric

a-ketoglutaric

NH4C1 from the

in the case with a-ketoglutarate

concentration"

estimations

acid at high concentration

glutamic alone.

acid

acid

glutamine, but pyruvate is not. Particularly, is found as total glutamic acid. In the

The

(0.03M) was loaded

cose (0.01M) from the side-arm TABLE

term "high

were carried

of a-ketoglutaric

acid

made with the cases

the

there

at

high

are

concentration

recovered

7.2% of the a-ketoglutaric side-arm,

acid.

This

Moreover,

glutamate

12.8 °o of the a-ketoglutaric recovery

is 78 °o larger

this increment

and

acid consumed

than

acid that

is chiefly due to the

When glucose is added from the side-arm, is found

recovered

as total

glutamic

9.8 % of the a-ketoglutarate

acid.

than it is when alone, and the glutamine

In this case, too, the

synthesis

is as striking

consumed

recovery is larger

as in case of NH4C1

addition. When

NH4C1 plus glucose are added from the side-arm,

the recovery

of a-keto

glutaric acid amounts to 13.5°0' as total glutamic acid. The a-ketoglutarate recovered is larger than in the cases when NH4C1 only or glucose only is added and, in particular, 88 % larger than in the case of a-ketoglutarate alone. In those which show a large recovery, the glutamine synthesis is very remarkable. Discussion : siderable

By the

amount

and it increases in the

with a-ketoglutaric acid is produced

when NH4CI is added.

formation

anaerobic

incubation

of total glutamic of glutamic

experiment

with

acid.

from

a-ketoglutaric

acid alone, and

acid increases

very strikingly.

the

saline

Warburg

hand,

Dickens

solution

liberate

(33) reported

that

of the brain

cortex

Consequently

and

a small

when

a considerable the liberation

tissues to a considerable

Cohen amount

the

where

ammonia

Ammonia further

brain,

retina,

tamine. Though to the ammonia,

glucose

addition

when

brain

amount

of ammonia.

of ammonia

amounts

of glu

slices dipped In this

in

respect,

to 1.4 ccm from

1g

can be produced in cerebral

suggest that a large supply of in cerebral tissues. Krebs (37)

have a system

from a-ketoglutaric

to promote

to synthesize

glutamine

acid to glutamic

preferentially

about

is an

reactions.

,

the

acid and

formation

of glu

essential

of glutamine and

When

glutamate

into glutamine

was

other stages. of the glutamine

synthesis

is found

as well

In this

endothermic

And Cathcart

metabolism.

of glucose

of ammonium

NH4C1 is added.

were absolutely

synthesis

kidney

is added the increase

of glutamine

the

seems

was known

synthesis

protein

and

in which the conversion

case

with

that

in the

the fate of glutamine involved in this process is ill-defined yet, as Krebs (37) stated that there appeared to be a cycle of ammonia in

as in the

hydrates

is added, the production

acid and glutamine

the synthesis and

one step, but nothing

energy-giving

that

acid is yielded

is utilized.

to glutamine,

When

(34) reported

extent.

promotes

nervous tissues

alone,

role of ammonia

of glutamic

(35) stated

Moreover, the results from my experimentation ammonia favors the production of glutamic acid that

a con

in 1 hour, while 0.07 ccm from the liver, 0.7 ccm from the kidney.

it seems that glutamic

noted

an important

ammonia

and Greville

that

even when a-ketoglutarate

indicates

Krebs

the kidney

tamic

On the other

This

acid it is found

cases

respect , noted Krebs (37) that the reaction and therefore depends upon

(38) put forth the hypothesis

for the endocellular glucose

is added

synthetic

that

the carbo

proccess in connection

for the purpose

to give

energy

is to be promoted . Furthermore, by the simultaneous NH4C1 more striking increase of glutamine synthesis may

,

be caused than (2)

by the addition

Incubation

of them respectively.

with a-ketoglutaric

acid at low concentration

a-Ketoglutaric acid (0.003M) as a substrate was incubated. between the case with NH4CI (0.03M) was added (Table 7).

TABLE

7.

Incubation

with

a-ketoglutaric

acid

at

low

Comparison

was made

concentration

When a-ketoglutarate alone is loaded, the recovery rate amounts to 9,0 / as total glutamic acid, in contrast to 7.2 °o in case of high concentration. In case of NH4CI addition, from the side-arm, also, the recovery rate is 15.2 /, though 12.8 / in case of high concentration. Moreover, this rate is 63 % higher than the case loaded by a-ketoglutarate alone. To be noted, glutamic acid is decreased, while glutamine shows a striking increase in this case. Discussion : The a-ketoglutaric acid administered at low concentration is recover ed with higher percentage than that at higher concentration. By the addition of NH4CI the recovery rate is far higher, and the glutamine synthesis becomes far striking. When ammonia is added in the larger amount (0.03M) of NH,4Cl for the low concentration of the substrate (0.003M),the glutamic acid produced is very little, while glutamine amounts to a remarkable quantity, as Krebs (37) pointed out. This fact is practically interpreted that all the glutamic acid produced is readily converted into glutamine. V. Incubation with pyruvic acid Pyruvic acid is well-known as a starting member of C.A.C. and, also, as an important compound of the lowest class in a-ketonic acids. I have learned that a-ketonic acid is an important factor which participates in the glutamic acid forma tion in the brain. Here, pyruvic acid which is situated in the class one step lower than a-ketoglutaric acid is necessarily a subject to be studied on how it behaves in the glutamic acid formation, I believe. For the convenience of comparing with a-ketoglutaric acid, 0.03m of pyruvic acid is called by the term "high concentration" and 0.003m by "low concentration" in the following description. In my experiment pyruvate, a-ketoglutarate, glutamate and glutamine were measured as following : (1) Incubation with pyruvic acid at high concentration Pyruvic acid (0.03M) was loaded as a substrate. Tn? case of pyruvic acid

alone was compared

with

TABLE

As shown are recovered. amounts

above,

8.

the

Incubation

from

pyruvic

In case of pyruvate

to 6.1% as total glutamic

the case of a-ketoglutarate When

case NH4C1 was.added

NH4Cl

at high

is added

10.1%

with

pymvic

acid,

'acid

to it (Table

at r igh

a-ketoglutarate,

alone,

the

acid.

This

recovery

8).

concentration

glutamate of the

rate is a little

and glutamine

pyruvate lower

consumed

than 7.2% in

concentration. out

of the

pyruvate

consumed

is found

as total

glutamic acid. This is a little lower than 12.8% in the case of a-ketoglutarate, but 65% larger than in the case of pyruvate alone. This increase is -derived from the increased synthesis of glutamine, as well as in the case of a-ketoglutarate loading . (2) incubation with pyruvic acid at low concentration Pyruvic acid (0.003M) was loaded as a substrate. Comparison the case of NH4C1 addition TABLE

9.

(Table

Incubation

was made between

9). _ with

pyruvic

acid

at

low

concentration

The case of pyruvate alone shows higher -rate of recovery, 7.9% as -total glutamic acid, as compared with 6.1% in the case at high concentration. Whereas it. is a little lower than the rate (9.0 %) in the case -of a-ketoglutarate at low concentration alone. In case of NH4C1 addition from the side-arm, the recovery rate amounts.to 12.4 %, though 10.1 °o in the case at high concentration, and 57 % larger than in the case of pyruvate at low concentration alone. Moreover, this increase is mainly due to the promoted glutamine synthesis. The rate is a little smaller than 15.2.% in the case of a-ketoglutarate alone.

Discussion :

In the

incubation

with

a-ketoglutaric

acid and

with

pyruvic

acid,

generally speaking, the two acids go along nearly,the same course b)th in case of a-ketoglutarate or pyruvate alone and in case that NH4C1 is added, but the former shows a little better

rate of recovery

in case of a-ketoglutarate rate

or pyruvate

than the high concentration.

tamine

synthesis

is always

In the incubation and glutarnine, glutamate

for total

glutamic

the low concentration

than the latter.

shows a better

In case that NH.1CI is added, the

And

recovery

remarkable

glu

found.

with pyruvic

acid there

are produced

but in the loading test of a-ketoglutaric

and glutamine,

acid

while pyruvate

a-ketoglutarate,

acid there

is not observed.

That

glutamate

are observed

only

is to say, the course

(pyruvic acid-; a-ketoglutaric acid-- glutamic acid--* glutamine) is possible, but the course (a-ketoglutaric acid--* pyruvic acid) is impossible or, if possible, it might be an intracellular taric

course.

It is supposed

acid corresponds

courses

are not proved

mentioned

that

the course from pyruvic

to C.A.C. by Krebs.

It is, however,

at all in the loading

acid to a-ketoglu

very interesting

test of glucose

that such

which has been already

(III). VI

Addition

of arsenite

(1) In case of incubation with c -ketoglutaric acid a-Ketoglutaric acid (0.003M) is loaded as the substrate, (0.005M) is added (Table

to which sodium

arsenite

10).

'TABLE 10

.

Addition

of a-ketoglutaric

acid

and

arsenite

The rate of recovery as total glutamic acid becomes better by adding aresenite. It is an increase of 700. Especially in this case the production of glutamic acid is increased,

quite different

from the case where

NH4C1 or glucose

Discussion : Still et al. (39), and other workers of 0.0017M arsenite, a-ketoglutarate

the oxidation

of glutamate

; Krebs (37) reported

kidney as the substrate,

arsenite

that,

inhibited

is added.

(24, 40) reported was largely

when strongly

glutamic

that, in the presence

arrested acid

the process

was

at the stage of loaded

in the

in which the ammonia

produced by deamination, combining with the remaining glutamic acid changed into glutamine ; Green et al. (41) reported that arsenite inhibited cyclophorase to absorb 02 and, at the

same

time,

inhibited

phosphor

to be taken.

Accordingly,

I might

say, it is because a-ketoglutaric glutamic decreases taric

the

reversible

reaction

of the

acid is tended to be advanced

production

one-sidedly

of glutamic

by arsenite

acid

acid is inhibited to change to glutamine that in my experiments glutamine inspite of the increase of glutamic acid when arsenite is added to a-ketoglu

acid ; or the supply

to glutamine

of enery

is considered

which is necessary

is added (Table

acid

to change

to which sodium

arsenite

(0.005M)

11).

TABLE

As shown

for glutamic

to be made insufficient.

(2) I n case of incubation with Pyruvic acid Pyruvic acid (0.003M) is loaded as the substrate,

above,

11.

when

Addition

of pyruvic

pyruvic

acid

acid

and

and

arsenite

arsenite

are

added,

a-ketoglutarate,

glutamate and glutamine, are scarcely recovered. This fact is strikingly from the case in which a-ketoglutaric acid and arsenite are added. Discussion : acid, but glutamic may be because of the

from

and the produced

production

Glutamic

acid

increases

when

acid is not produced of the

fact that

of a-ketoglutaric

part of C.A.C. by Krebs, ketoglutaric acid through

when

pyruvic acid

arsenite arsenite

is added

pyruvic

to a-ketoglutaric

is added to pyruvic

acid is arrested from

different

by arsenite

acid.

and yet, as Kritzman (42) said, C.A.C. by oxidation, so arsenite

But

acid.

It

in the process

this

process is a

pyruvic acid becomes a is supposed to arrest the

oxidation. Judging

from what I stated

above,

it is supposed

that a-ketoglutaric

acid is not

produced from pyruvic acid in the anaerobic condition and consequently glutamic acid is not produced either. So I tried the following experiments in the anaerobic condition. VII.

Incubation

with a-ketoglutaric

acid and Pyruvic acid in anaerobic condition

(1) Incubation with a-ketoglutaric acid in anaerobic condition I compared the case in which a-ketoglutaric acid (0.003M) was loaded as a substrate in the substrate

anaerobic (Table

condition 12).

with

the case where

NH4C1 (0.03M) was

added

to the

TABLE 12. Incubatian

In the

anaerobic

with a-ketoglutaric

condition,

total

of a-ketoglutarate

alone

and

markable

increase

when

compared

Especially

the

synthesis.

production

When

glutamic

acid is recovered

at 29.3 % when acid

from

15.2 % in the

is more

the

remarkable

side-arm,

20'o more than in the case of a-ketoglutarate increase

NH4C1 is added,

with 9.0 % and

of glutamic

NH4C1 is added

in spite of the remarkable

acid in anaerobic

alone.

of glutamic

total

in the

anaerobic (Table

condition

with

the case

at 24.4 % in case which

shows a re

aerobic than

glutamic

condition.

the glutamine acid is proved

In this case glutamine

decreases

acid.

(2) Incubation with Pyruvic acid. in anaerobic condition I compared the case in which pyruvic acid (0.003M) was substrate

condition

where

NH4C1

incubated

(0.03M) was

as a substrate added

to the

13).

TABLE

13.

Incubation

with

pyruvic

acid

in anaerobic

condition

In the anaerobic condition, a-ketoglutarate. glutamate and glutamine are hardly recovered from pyruvate, regardless to the presence of NH4C1. This is quite dif ferent from the case of the a-ketoglutaric acid stated in (VII, 1). Discussion : In the anaerobic condition, glutamic acid is produced remarkable from a-ketoglutaric acid, and, when NH4Cl is added glutamic acid shows a more remarkable increase, but, on the contrary, glutamine decreases. In case of pyruvic acid, however, glutamic acid and glutamine are not observed, regardless to the presence of NH4C1. This is just the same as the case in which arsenite is added in the aerobic condition. In other words, in the anaerobic condition or by arsenite, the production of glutamic acid from a-ketoglutaric acid is proved more than in the case of the aerobic condition,

but the

production

2), a-ketoglutaric

of it from pyruvic acid is produced

acid is proved at all.

vic acidnot produced from a-ketoglutaric such a course of the production (pyruvic

acid by oxidation

Moreover,

from pyruvic acid in the aerobic acid.

of glutamic

a-ketoglutaric

Judging

as stated

from these points I can suppose

acid _and glutamine

acidammonia-~ glutamic

from pyruvic acid as acid arnmonia+energy -~

glutamine). But, recently it is noticed that ATP has an important part in synthesis and it is clarified that this energy-rich phosphate bonds have action upon the metabolism of living tissues. So I have studied tamic acid formation is influenced by ATP, AMP, pyrophosphate following : VIII.

Addition

the glutamine an important how the glu and DNP as

of ATP (adenosione triphosphate)

As ATP does not percolate intact cells used homogenate in the following experiments

according :

to Nachmansohn

(I) In case of incubation with a-ketoglutaric acid I compared the case in which homogenate was used to a-ketoglutaric as the substrate the side-arm

with the case where

(Table TABLE

In homogenate ATP is added Increases instance tion than glutamic (3)

in (V,

condition, but pyru

et al. (44), I

acid (0.003M)

ATP (0.004M and 0.0065m) was added to it from

14). 14.

total

Addition

glutamic

to a-ketoglutaric

of

ATP

acid acid

and

a-ketoglutaric

is proved

than in the

acid

to

at a higher

homogenate

rate

case of a-ketoglutaric

in case

when

acid

alone.

of 18 % by ATP (0.004M) and 30 % by ATP (0.0065M) are observed. In this total glutamic acid is proved at a higher rate by ATP of high concentra by ATP of low concentration. acid has a tendency In case of incubation

to decrease,

In case of adding but glutamine

with pyruvic acid

ATP, the accumulation increases

remarkably.

of

I studied the

case

on the

that ATP

TABLE

case that pyruvic

acid (0.003M) was used as a substrate

(0.004M and 0,0065m) was

15.

Addition

of ATP

and

added to it (Table

pyruvic

acid

and on

15).

to homogenate

In homogenate, a-ketoglutaric acid and total glutamic acid are proved at a higher rate in the case where ATP is added to pyruvate than in the case of pyruvate alone. Total glutamic acid shows increases of 26% fox :ATP (0.004M) and 53 % for ATP (0.0065M). And ATP of high concentration shows much higher rate than ATP of low concentration. Especially, in this instance the remarkable glutamine synthesis by ATP seen, but, on the contrary, glutamic acid decreases. Discussion : Speck (45) suggested that on the synthesis of glutamine the ATP might play an essential role in the amide formation ; the utilization of the energy of ATP in the synthesis of amide linkages seems to be a reaction of general impor tance ; Elliot (46) stated that glutamine was formed when the experiment was incubated with glutamate ammonia, and ATP, the synthesis of glutamine is carried out ; Krebs (37) said that the glutamine synthesis requires energy ; Epelbaum et al. (47) and other workers (48, 49) stated about the existence of adenosine triphosphatase in the brain. Accordingly when glutamine is synthesized from a-ketoglutaric acid or pyruvic acid in homogenate, such high energy phosphate bonds as ATP may play an important role. In my experiments, too, total glutamic acid increases remarkably when ATP is added in case of both pyruvic and . a-ketoglutaric acids. Especially, glutamic acid decreases contrary to the increase of glutamine. This is, perhaps, because glutamic acid changes to glutamine by means of the high energy of ATP. In the case where I used pyruvic acid, a-ketoglutaric acid also increased by ATP and this . is, perhaps, because high energy phosphate bonds-have specific influences on the 'pyru vate metabolism. According to Schwarz and Dibold (50), ground brain cells show a more. remarkable production of ammonia than intact cells. This may be the reason why the homogenate shows a more remarkable production of total glutamic acid or glutamine than the slice in a-ketoglutarate

or pyruvate, .

IX. Addition of AMP (adenylic acid) I studied on the case where a-ketoglutaric acid (0.03M)as a substrate was used and on the case when AMP (0.0045M)was added to it (Table 16). TABLE

16.

In homogenate AMP

is added

this increase

Addition

of AMP

total glutamic

Discussion : It is said that,

than in the if glutamic

for the

as stated

my experiments,

diphosphate)

in (IV, 1), and

too, I noticed that X.

By control

alone.

And

the

Mg

can be replaced

accordingly

glutamine

Addition

of

pyrophosphate alone.

with

system , and According to Speck

glutamine

increases

by AMP.

In

a little by AMP.

of pyrophosphate

a-ketoglutaric

Incubation

by AMP.

increased

TABLE

17.

of this

body as AMP decomposes in the liver, glutamine synthesis increases by addition

17).

addition

of a-ketoglutarate

phosphorylation

(0.1M) was added (Table

of a-ketoglutarate

in case in which

of glutamine . acid is accumulated by cyclophyrase,

(1) In case of loading of a-ketoglutaric acid a-Ketoglutaric acid (0.003M) was loaded as pyrophosphate

to homogenate

more increase

case

oxidative

(45)° and Embden et al. (52) such a purin kidney, etc. and produces ammonia. The of ammonia,

acid

to be ch*,efly due to the increase

and AMP (51) are necessary ATP and ADP (adenosine

a-ketoglutaric

acid shows a little

to a-ketoglutarate

is considered

and

, I can

the

hardly

acid

substrate,

and

find

to which

sodium

pyrophosphate

any

difference

from

the

(2) In case of loading of Pyruvic acid Pyruvic acid (0.003M) was loaded as the phate (0.1M) was added (Table TABLE

By the

addition

control of pyruvate

18.

with

pyrophosphate

phosphate

sodium

pyrophos

pyruvic

acid

and

I can

hardly

pyrophosphate

find

any difference

from

the

alone.

Discussion : When a-ketoglutaric inorganic

to which

18).

Incubation

of

substrate,

disappears.

acid is oxidized by the cyclophorase According

preparation,

to Ochoa (53), this is recovered

as labile

phosphoric esters (after 7 min. at 100°C with N-HC1), but according to Kornberg (54), inorganic phosphate or pyrophosphate is formed from these esters or they undergo transition

from one to another,

and according

to Binkly

undergoes

complete

by phosphatase

in the

hydrolysis

and Olson (55), pyrophosphate brain

tissue

and

changes

into

phosphate. Greig and Munro (56) stated that this inorganic pyrophosphate has no influences on the metabolism of brain slices. In my experiments, too, the glutamic acid formation a-ketoglutaric

is scarcely

influenced

and pyruvic XI.

(1)

by the

Addition

was added from the side-arm

19.

in case of both

acid

acid (0.003M) was incubated

TABLE

of pyrophosphate

of DNP (a-dinitrophenol)

In case of loading of a-ketoglutaric

a-Ketoglutaric

addition

acids.

(Table

Incubation

as the substrate

to which DNP (0.0002M)

19).

with

a-ketoglutaric

acid

and

DNP

Total glutarnic acid decreases by the addition of DNP, compared 'of a -ketoglutarate alone . Especially glutamine seems to decrease. (2) I n case of incubation with pyruvi c acid Pyruvic acid (0.003M) was loaded as the substrate added (Table

to which

with the control

" DNP (0.0002M) was

20). TABLE

20.

Addition

of pyruvic

acid

and

DNP

Total glutamic acid decreases by the addition of DNP, compared with the case of the control of pyruvate alone. Especially glutamine shows a remarkable decrease. In this instance, I can scarcely find any influence of DNP on the course from pyruvic acid to a-ketoglutaric acid and, further, to glutamic acid, but DNP is considered to have a remarkable influence on the course to glutarnine. Discussion : Loomis and Lipmann (57), and Elvehjem et al. (58) said that DNP' prevented phosphorylation without affecting the oxidation, and Judah (59), Case and Mcllwain (30) stated that DNP inhibited phosphorylation affecting respiration only slightly. That is to say, the action of DNP is considered to prevent the utilization of the energy which is supplied by respiration and glycolysis, inhibiting the formation of high energy phosphate bonds. Accordingly, in my experiments, the reason why the glutamine formation decreases by the addition of DNP in case of both a-ketoglutaric and pyruvic acids may be that as 1) the utilization of the energy which is supplied by respiration and glycolysis is prevented, 2) the formation of energy rich phosphate bonds is inhibited and 3) the supply of energy is prevented in the process of the glutamine -formation. Now, I have studied how glutamic acid and the formation of glutamine influenced by ATP and by the phosphate related with it. Though ATP increases the glutamine synthesis remarkably, AMP and inorganic pyrophosphate which are degradation products of ATP do not show such a remarkable action. Namely, it is supposed that the action of ATP in the glutamine synthesis is not by the components but by the supply of the energy from high energy-rich phosphate bonds in ATP. And this is proved by the fact that if the process of . phosphorylation is :inhibited by DNP, the glutamine synthesis is-prevented. If glutamic acid is formed from a-ketonic_ acid but not from glucose, as mentioned

above, the supply-sources are considered to be decomposition products of glucose which are produced in other organs or intermediate products in the course from fat to carbohydrates, or I can suppose such a course that amino acid changes into a-ketonic acid by transamination (3, 21, 25, 42, 53, 60, 61) or by oxidative deamination (3, 39, 40, 62, 63) which goes into the brain with the circulation of blood and changes into glutamic acid. If, as Weil-Mahlherbe say, it is partly ascribable to the' accumulatioa`' of glutamic acid and glutamine that the administration of l-glutaric acid per os has' a certain effect on the brain function, it is supposed that the glutamic acid which is' absorbed in the digestive tract is stored in the liver, kidney etc., which decomposes, into a-ketonic acid gradually and goes into the brain with the circulation of blood 'k to be resynthesized to glutamic acid and glutamine. SUMMARY The observations

have been carried

about

the action of glucose, pyruvic acid and

a-ketoglutai is acid on the formation and accumulation of glutamic acid and glutamine in the brain tissue and about the influences of various chemicals in the formation mechanism,

and obtained

When I inject tamine

in the

increase.

the following

glutamic

brain

acid

do not

into a rat

increase,

but,

intravenously,

glutamic

when I inject

acid

a-ketoglutaric

and

glu

acid, they

It is the same with the case in vitro .

Glucose

forms

in an anaerobic

glutamic

acid and glutamine

in the aerobic

condition,

each of them.

In both

a little

increase

in case of arsenite

by

condition

nor

and

pyruvic

changes

further

acid scarcely

to glutamic

acid

to glutamine

by

and

in glutamic

by DNP . However, a-ketoglutaric acid

condition, forms

by ATP

changes

decreases

pyruvic acid becomes

changes

increase

any remarkable

and glutamine

added or in an anaerobic

acid, while

acid and glutamine

when NH4C1 or glucose is added to

shows a 'remarkable

and I do not find

it is supposed that

oxidation,

increases

glutamine

by pyrophosphate, being

glutamic

Consequently cyclophorase

in an aerobic

and pyruvic acids produce glutamic

and glutamine cases

by AMP,

acid and in glutamine increases

neither

condition.

In vitro both a-ketoglutaric

C.A.C.

results :

either. a-ketoglutaric

the

action

by the

energy

acid through

of ammonia from

and

energy-rich

phosphate bonds. It is also supposed that the glutamic acid taken orally is absorbed in the body organs and decomposed into a-ketonic acid gradually, which goes into the brain

with

the

circulation

of blood to be resynthesized

to glutamic

acid

and

glutamine. I wish ce and, and

at

to express the same

M. Kawakami

my heartfelt time, will

gratitude

the incessant

be gratefully

to Dr. S. Kosawa

assistance acknowledged.

by Assistants

for his

cordial

S. Yoshie,

guidan

A. Kimura

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42)

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43)

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46)

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