A NEW EXPERIMENTAL METHOD OF DIURETICS USING MICE AND ITS APPLICATION TO SOME DIURETICS

A NEW

EXPERIMENTAL AND

ITS

METHOD

OF DIURETICS

APPLICATION

TO

YASUNAO Department

of Pharmacology, Received

for

SOME

USING

MICE

DIURETICS

OGAWA

Faculty

of Medicine,

publication

August

Kyoto University

31,

1953

In the experimental detection of urinary increase by diuretics, dogs and rabbits have chiefly been used, and in some cases, cats, rats, and frogs were also

employed.

Rabbits

are

accuracy.

The

metabolism, assumed

o f these

used,

pointed

of reports

and

using

the

given

other

(1)

and

are

Marx

most

obtained

results.

obtained

are likely

is the most

the

satisfactory

results

procedures

(4) is noteworthy

actions

animals

have

experimental

a dog,

by Bonsmann diuretic

of experimental

methods

but

out by Bonsmann

an experiment,

in examining

number

none

use of anesthesia

as was

that

the series that

However,

most frequently

to affect water

(2).

It has

suitable (3).' in that

accurate

been

However,

it has

results

by using

lack

pointed

from

mice simply

a

out larger

and

econo

mically. In

1930, Gibbs

urination

(5)

and Molitor

(6) compared

for the examination against

urination,

three.

experimented

After

of the and

inhibitive

obtained

1934, Bonsmann

and

of Gibbs

Bonsmann

experiments

using

2) 3)

with

differences,

made

with

the

groups

as against

an individual

characteristics

of diuretic

need

not be fixed,

and

can

be

observed

under

differences

advantageous

out on a and

large

obtaining

in finding

the

number

more

of animals,

accurate

relationship

thereby

results. between

lethal

and

doses. Experiments

However, tion when group

animal

can be carried

individual

It is more

were

as the

each of

experiments

individual

which

points

substances

from

of urination.

Experiments

effective

out

animals

pituitary

results

on

Glaubach

mice:

conditions

decreasing

Glaubach,

pituitarins

as experimental

similar

carried

of

determination.

of posterior

to decrease

the following

An experimental

natural

4)

ahd

gave

action

others,

the experiments

action

a mousy

approximately

in a group, in order

mouse.

inhibitive

any quantitative

a dog, a rat, and

of mice, several

1)

the

with mice, but did not reach

at a

can be carried

Bonsmann

forced, definite

adopted

by suppression period

out simply the method of the

(approximately

and

economically.

of determining

bladder

in an

4 hours),

the amout

individual

and

measuring

of urina

mouse the

of total

a

amount.

The

accurate

experimental

periodical

present

experiments

were

results

amount

of

urination

undertaken

would

probably

of

in

mice

on be

the

assumption

obtained

a natural

that

by

more

following

the

state.

METHOD As

a fundamental

mouse.

All the

would

be different

mice

of animals

average

weight

The

control was

cylinder filter the

two

urine

was

from

the

method

identical

as

was

feed,

time

of

a control

Special

examined

but

the

the

with

amount

experiment,

in every

care

was

conducted

with

8 cases,

Before

beginning

to cause

so

experiment

taken

accurately

at

laid

the

to

an

taken

that

the

same

equalize

the

to

keep

individual

of water

the

experimental

the

of

given

animal

placed

the

was weighed

and

In order

level.

those the

weighed,

moisture high

including experiment,

a previously

of urine.

a fairly

to within

the

and

30 minutes,

amount

paper,

constant

was

every

as the

filter

urination,

of which

changed

taken

approximately made

used lots.

were

in the bottom was

on

to the

animals.

pressed,

paper

fed

the

condition.

experiments

their

animal

was

were

in both

in a healthy

and

were

according

number

mice

experiment,

the

experimental

filter

glass

paper.

This between

evaporation room

weighing

of the

in. a

difference

to prevent

The

diuretics

bladder

of the

of

was filter

kept paper

10 trig.

RESULTS A)

Natural

Some water

evaporatio;'z of moisture from filter Paper was dropped

the amount

of moisture

(cf.

1).

Table

disregarded even when TABLE 1.

It was

in the case the

amount

Natural

that found

into the filter evaporated that

of water of water

evaporation

paper,

naturally

the amount below was

10)mg.,

over

of moisture

placed during

in the 30 minutes

of natural while

100 mg..

it was

and

was examind

evaporation

Since

from filter paper

cylinder,

only

could below

it is rare

(Room temperature

be 10%

in actual 15°C)

experiment there

that

was

and

it

found,

was

B)

over no

amount

the smallest, amount

decreased

Urination

i)

of

the difference better

and

urine.

The

only

hydrous

salt dieresis

the present

results,

error.

mouse

(Average

hour

of the experiment.

after

of events

the

start,

(Fig.

and

1).

value of 20 mice in mg.)

of saline and gluc-)se solutions solutions

received the

isotonic with

method

amount

of urine

micturition that

diuretic

of following

solutions which

high

action, micturition

given

and glucose

were

tonic

and

solutions,

was indicated

periodically

solution, with

solution

was observed,

of the solution

and high tonic

isotonic

and

amount

In both saline

of following action,

0.2cc./10g. of the respective

in the

than

as well as hydrous

experimental

0.5-1.0

39 minutes

in the first 30 minutes

to the beginning

the injection.

obtained

diuretic

every

of urine

course

increase

between

method

result

weighing,

experimental

determined

natural

5% saline

greater

after

of activities

show

the

solutions

being

by the present

prior

injection

each mouse

2.5 hours

total shows

saline

urine

within

the

to

experimental

was

during

the

after intravenous

experiment,

mice

urination

vein, and a conspicuous

amount

urinated

an

2, the amount

of normal

5 °o and 20 °o glucose

In every

urination in

within

the greatest

urination

Physiological

in the tail the

in Table

indicating

Natural

after

evaporation be

in 20 normal

was

later,

elapse

natural would

due to forced

of urine

TABLE 2.

would

of normal mouse

of urine

The

ii)

it

As shown

was

C)

of

that

urination

over 2.5 hours.

again

minutes

effect

assumed

Natural

The

30

than

by the

is assumed solution,

clearly

periodically,

to

which

different

by

and proved

its significance. Kamon

(7) showed

that

intravenous

injection

of abut

1/10 the

weight

of

physiological saline solution in a rabbit resulted in extreme micturition and the amount of urine rapidly increased. About 8 hours after the injection, the amount corresponding

to or larger

than

the amount

injected

was

ci.npletely

excreted,

the amount experiments individual

being

the greatest

also gave mouse,

similar

in the case

FIG. 2.

Urination

at 1 hour after results,

as shown

of saline

solution,

after

FIG. 3.

Urination

after

FIG. 4.

Urination after (every mouse)

i. v. injection

injection.

The

present

in Fig. 2 and are

of saline

shown

3.

in Table

solution

i. v. injection

of glucose

solution

i. v. injection

of 20,o glucose

solution

series

of

for

an

Data 3.

Only

average

values

are

shown

for

other

glucose shown in Table 4. State injection of 20% glucose solution TABLE 3.

TABLE 4.

D)

Urination

Urination

Diuratic

Potassium was

after

after

intravenous

intravenous

experimental

in the

case

of

of urination in an individual mouse is shown in Fig. 4 as a reference.

by

the

injection

in mg.)

injection

of saline

of glucose

results,

solutions

as

(Amount

of urine

solutions

(Amount

of urine in mg.)

diluted

50 times,

actio.i of Potassium acetate Acetate

administered

Solution

orally

water

orally.

The

total

amount

of urine

while

animals

JP (ca. 34 %') was the control

receiving

compared

animals

the acetate to the

control.

received solution

and 0.1cc /10g.

the same showed

Bonsmaan

amount

1.8 times and

others

of the (8)

obtained

experimental

value

0.008g.120g. of potassium stronger acetate

diuretic (Table

TABLE 5.

of

1.5 times

acetate,

while

effect by the oral 5 and

Urination

after

injection

experiment

indicated

of 0.0014g./20g.

of far

of potassium

oral administration

5.

Urination

of potassium

after

oral

acetate

(Amount

administration

of urine

in mg.)

of

acetate

action of Euphylline

i)

Subcutaneous

ii)

Subcutaneous

followed

subcutaneous

present

administration

potassium

Diuretic

the

the

Fig. 5).

FIG.

E)

by

injection

of 0.lcc./10g. of 0.5%o Euphylline

injection

by subcutaneous

of 0.lcc./10g.

injection

of 0.lcc./10g.

of

(Table

physiological

6 and Fig. 6)

saline

of 0.5% Euphylline

solution

(Table

7 and

Fig. 7). In the case of (ii), the ministration

of the

diuretic

saline

solution

in order

FIG. 6.

was

to supply

Urination injection

given

3!) minutes

water,

because

after sabcataa3aas of Euphylline (i)

before of the

the ad powerful

diuretic

action

of Euphylline.

of physiological at I hour thereafter. in the

saline

after

compared

acetate,

in (i) and 2.4 times

These total

sodium series

The

of 0.02-0.08cc.

the highest

the diuretic

were

but the total

TABLE 6.

TABLE 7.

greatly Urination

Urination

affects after

after

total

using

the value

subcutaneous

still apparent

18-20g.

mice

in (i)

out over a period

In the present of urine

injection

with

when was

1.7

theophylline

3 .2 times of 4 hours,

during

in (ii). but the

giving an average

series of experiments

obtained

smaller

2.5 hours was 0.44g.

This may indicate

of Bonsmann's

injection

of urine

and

of 6 mice was only 0.1-0.5,c.,

amount

subcutaneous

was amount

clearly as in the

injection

of 3 times

carried

did not appear

administration,

level rapidly

to the control .

out experiments

in a group

action

micturition

in (ii), compared

values

comparatively

before

in the

in the case of (i) and 0.47g. in the case of (ii). evaporation

decreased

urination

increased

per mouse for 4 hours.

mice were used,

reached

of urine

and

increase

and obtained

of urine

amount (ii),

but the

of experiments

amount

the same amount

Urination after subcutaneous of Euphylline (ii)

(9) carried acetate

received

due to forced

FIG. 7.

and

case the control

of (i), however,

with the control.

Bonsmann

The

in (i) and

30 minutes

case of potassium times

solution.

injection

In the case

initial

In this

that the natural

method.

of Fuphylline

of Fuphylline

(i) (Amount

(ii) (Amount

of urine in mg .)

of urine in mg .)

F)

Diuretic

A market

action of mercurial product

salicylallylamide

diuretics

containing

and sodium

10r,

acetate,

of

and

mercuric

compound

3% of theophylline

(Salyrgan)

was

diluted

of

50 times

and 0.1 cc.,10 g. of this solution was subcutaneously administered. As shown in 'T'able 8 and Fig . 8, the results showed a striking effect of the amount of water taken by the mice which were used. While the amount of natural urination in a controlled

normal

mouse

averaged

one-half

of the amount

increase

of 2.6 times over TABLE 8.

shown

Urination

that after

(Amount

The diuretic

action

Diuretine

Urination

after

The

in the order indicated

total

to that

after

caffeine

is said

drugs the

and theophylline and

content

drugs

and theophylline, indicated

The diuretic the initial times

(theophylline

nucleus

equal,

and 0.97%,

shown

in Table

9 and

action became stronger

but the course course

of urination

of diuretic

effect,

mice was less than that of the control action

30 minutes.

stimulating

are

an orderly

to

were used and 0.08cc./lOg.

that the diuretic

Diuretine

results

was compared

and sodium salicylate,

Euphylline

of purine

of the respective

from caffeine-administered

to be 5-6

an

th-obroynine, and theophylline

of urine indicated

during

below

showed

of

used were caffeine

salicylate),

theobromine,

may be due to the vasocentral

being

and theophylline

injection

The

administration.

of Diuretine

subcutaneous

subcutaneously.

some difference.

but the micturition one hour

amount

of caffeine,

of Salyrgan

theobromine,

to make

solutions

of each was administered

diuretics

theophylline

The

and sodium

In order

injection

action of caffeine,

of caffeine,

0.8;x, and 0.5;,,' aqueous Fig. 9.

and

method.

(theobromine

ethylenediamine).

2.5 hours, the

in mg.)

Com arison of the diuretic

the use of the present

during given

subcutaneous

Salyrgan

G)

104 mg. 2, those

of the control.

of erine

FIG. 8.

only

in Table

action

of Euphylline

was also inferior

It is assumed

that

of these

that of theobromine,

drugs.

such

effects

The toxicity

and that of theophylline

of

about

8 times

that

of theobromine.

and

theophylline

and

the

central

affected

the

In the

administered nerve

action

vasodilation

present

seems

case,

therefore,

to be slightly

of these

of kidney

drugs and

larger

appeared

retarded

the amount than

strongly

the

of caffeine

the amount

appearance

suitable

, which may have of the diuretic

action...

FIG.

9.

Comparison

caffeine,

Ach (10) compared diuretic values,

administered, three

the action

drugs

Although

from a few

some doubt

znd

cannot

effect

of

theophylline

drugs

in rabbits

However,

of different

periods

as to the

results amount

were

Comparison (Amount

H)

Excretion

In order of administered

of the diuretic

were

The

amounts

effects

ratio

administered

on

of these

of 1:1 .40:1.5. the foregoing

actions

accion of caffeine , theo'~)ro:n ine

average

different

in the

point, it is assumed that a more accurate comparison of their by the selection of a suitable amount to be administered. TABLE 9.

and showed their

these and

be said to be accurate.

experimental

remains

diuretic

of 1 : 1.45 : 2 .42.

examples

so the results in the present

the

of these three

effect to be in the ratio derived

of

theobromine

can be made

nd theophylline

of urine in mg.)

of administered

to confirm

iodine into the urine

the accuracy

iodine into urine

of the present

experimental

of mouse was quantitatively

method, the amount determined

.

Sodium

,

iodide

was

subcutaneous used were after

every

the method The

used as the iodine injection, slightly urination,

and 0.1cc. of its 1% aqueous

making

larger

ones, weighing

and the iodine

of Bodo (11) adapted

results

the amount

obtained

of administered 19-25g..

content

in every

in Table

was

given

iodine $40,ug.

The filter

paper

urination

to microanalytical

are shown

solution

by

The mice

was

changed

was determined

by

procedure.

10 and Fig.

10, from

which

it

can

be seen that the excretion of iodine occurs 11 minutes after the injection of sodium iodide. iodine after

is

About

excreted

30;% of the

during

the injection;

6 hours

approximately

25

% of the amount injected being ex creted during 2.5 hours i.e. between 0.5 and 3 hours, cretion very

after

of iodine small,

presence detected

injection.

after

and

that

in

all

of iodine (less than

Ex

becomes cases

the

is only barely 2 ,ug.) by the

present method of determination 24 hours after the injection. As is clear from

Fig. 10, the

excreted

and the amount

in parallel

in which

According

of

17/x, and

iodine

of

experiments

rabbits,

of

later

50%.

Oral

of sodium (13), showed

present

hungry metabolism

in

15% excre

administration,

30% 6 hours it was

iodide

out by Greenbaum

tion 3 hours after In the

after

3 hours

later

carried

and Raiziss

1.5 hours

was 9/,

6 hours

administration

water

the excretion

to the

administration

became

for

and Fujii (12) on the total in an individual mouse, the

excretion

ments,

except

is at its height.

Hirata iodine

about

of iodine

of urine are

relationship,

the period iodine

amount

and

after. series

found after

of

experi

that

the

mice

4

hours

and

became

sluggish,

resulting

in distinct

results,

the

decrease

present

of

the

experimental

amount

method

of

micturition.

seems

to

From

possess

a

the

fair

above

amount

of

accuracy.

FIG.

10.

Excretion

As can be seen clearly offers

a satisfactorily

the periodical action short

The examples must

amount

diuretics.

iodine

the period

present

2.5-3

in the latter

of the difference special

caution

of a suitable state

in the

was

taken amount

by the present

of a diuretic

compared

earlier,

administered.

of the

conditions used,

drugs

to the

consideration

of O.lcc./1Og. of 0.5% 1..7 times in the former

control , showing drug administered.

of the

according

special

In the case of Euphylline,

to the

for the health experimental

method

is comparatively

determined

but, as described

of the drugs

amount

experimental

hours.

the diuretic effect in the case of the administration Euphylline (Table 6) and of 0.O8cc./lOg. (Table 9) was and 2.6 times

urine

of experiment

within

to be administered

and others

to the amount

the

action . At the same time, by it can also enable examination of the mode of

Moreover,

of the drugs

the

into

of diuretic

effect can be detected

of Bonsmann

be paid

indication

of urination,

and a diuretic

administered

from the foregoing,

accurate

tracing

of various

of

the sensitive effect However, from the

of the mouse used, and the adoption

observation

of urination

method offers the means

of measuring

in a

natural

the potency

and , accurately, in a short period of time, with the added advantage of knowing the mode of action of such drugs . The present series of experiments was carried out on an individual mouse as a fundamental

simply

study,

of mice, the method of a new diuretic

but by the use of a group can be fully adopted

in a simple

manner.

consisting

as a method

for

of a definite potency

number

determination

SUMMARY 1.

A new experimental

mice was 2.

method

elaborated

allows

tion in mice and, by the periodic can be obtained

diuretic 3. was

of a diuretic

quantitative

tracing

accurately.

determination

of the amount

At the same

of natural

of urine,

urina

the action

time, th4 mode of action

effect of saline, glucose, potassium

observed.

the course

At the same time, the

of diuresis

specific

acetate,

Euphylline

characteristics

of a of the

Excretion

immediately

after

diuretic

in

was observed.

of administered

iodine

the administration

administered

and Salyrgan

of each

4. Comparison of the diuretic action of caffeine, theobromine, indicated that they apparently possess different diuretic effects.

amount

in

can be determined. Diuretic

5.

action

examined.

The method

diuretic

for the determination

was found

in a

normal

of sodium

to be excreted

mouse

iodide, into

and theopbylline

was

and about

the

urine

found

to occur

30% of the total

6 hours

after

ad

ministration. 6.

By the application

diuretic

action

of the present

can be made simply

experimental

and accurately,

method,

with a large

examination

REFERENCES 1)

BONSMANN

Arck. exper. Path. u. Pharmakol.

2) 3)

MARX: Ibid. 173, 526 (1933) WALTON : Arch. internat. pharmacodyn.

4)

BONSMANN AND BRAKLAGE : Arch. exper. Path. u. Pharmakol.

5)

GIBBS : J. Pharmakol. 40,

6)

GLAUBACH, AND MQLITOR:

7)

KAMON : Folia

164, 596 (1932)

8)

BONSMANN AND MULLER-NEFF : Arch. exper. Path. u. Pharmakol.

9)

BONSMANN:

46, 97 (1933)

Arch. exper. Path. u. Pharmakol.

Ibid. 175, 322 (1934)

ACH : Ibid. 44, 319 (1900)

11)

BODO : Biockem.

12)

HIRATA AND FUJII : Aichi Med. J. 38, 810 (1931)

Z. 160, 386 (1925)

GREENBAUM AND RAIZISS:

166, 243 (1932)

japon. 7, 184 (1928)

10)

)

174, 453 (1934)

129 (1930)

pharmacol.

J. Pharmacol.

13 407 (1927) 30,

of a

number of animals.

179, 77 (1935)