THE

JOURNAL

Copyright

OF HISTOCHEMI5TRY

© 1976 by The

THE

DISTRIBUTION

MASAHISA

Society,

TAKASHI

Department

of Anatomy,

pp. 591-600, Printed

KIHARA,

MASAHITO

Osaka

for publication

4,

Inc.

OF ‘4C FROM [U-’4C]GLUCOSE WHOLE-BODY AUTORADIOGRAPHY

SHIMADA,

Received

Vol. 24, No.

AND CYTOCHEMISTRY

Histochemical

Medical

July

23, 1974,

WATANABE

College, and

IN

in revised

City,

form

USING

KIYOHISA

AND

Takatsuki

MICE

Osaka,

November

1976

in U.S.A.

KURIMOTO

Japan 11, 1975

Tissue distribution of radioactive carbon from [U-’ 4C Iglucose in the mouse in vivo was studied by whole-body autoradiography. The mice were frozen with Dry-Ice-acetone at 0.5, 1, 5 and 30 mm, 1 and 24 hr and 1 and 3 weeks after intraperitoneal injection of [U“C ]glucose. Whole-sagittal sections of the frozen mouse, obtained by using a microtome in a cryostat, were dried in a cryostat and autoradiographed. The resulting dry autoradiographs are called untreated autoradiographs in the present work. The sections were then fixed in cold 6% (w/v) HCIO4, dried at room temperature and again autoradiographed. Autoradiographs that have undergone this process are referred to as treated autoradiographs. In both untreated and treated autoradiographs, within 1 min following injection of the labeled glucose, the abdominal cavity had the highest autoradiographic density. At 1 hr, density became highest in Harder’s, sublingual and duodenal glands, large intestinal mucosa and tongue, and after 3 weeks, no autoradiographic density was present. When toneally

[U- ‘4C ]glucose

was

into

most

a mouse,

chemical fraction cold acid-soluble stances)

40%

30 mm.

of the

‘4C

At

fraction and approximately as ‘4C0,, while nearly acid-insoluble 20% lated for

at

a later

more

acid-soluble from the

a

In

the

the

10).

and

stayed By

in the

the

mouse

autoradiography

this

method,

tissue

and

acid-insoluble

distribution whole-body

of ‘4C from autoradiography, information technique MATERIALS

Twenty-four adult weighing approximately

‘4C

was

There on

8). By of

from

literature

the [U-

has been the tissue

[U-’4C]glucose although in the

from

investi(7,

“C

could be observed. study undertaken

to this

of

there

by is

relat-

driven

Leitz-sledge

a cryostat

containing

25

microtome

1300

in

(OT/MSH, Bright Instrument Co., England) (15) at -15#{176}C.The resulting sections adhered to the tape and were dried in a cryostat (1S#{176}C) at atmospheric pressure. The dry sections were then brought into contact with x-ray film (Softex HS film,

METHODS

albino male mice 20 g were injected

NaC1

pure.

a hydraulically

(2-5). AND

(w/v)

The mice were killed in groups of three at intervals of 0.5, 1, 5 and 30 mm, 1 and 24 hr and 1 and 3 weeks after injection. They were anesthetized with ethylether for 0.5 mm, except the two groups of mice killed at the 0.5- and 1-mm intervals, which were frozen without anesthesia. Each mouse was frozen with Dry-Ice-acetone (approximately -75#{176}C)for 2.5 mm. A viscous mixture of 6% (w/v) carboxymethyl cellulose was used to mount the frozen mouse on the microtome block. Before sectioning, Scotch tape (Type 810, Minnesota Mining and Manufacturing Co.) or cotton tape (Salotape RI-SO for whole-body autoradiography; Hisamitsu Pharmaceutical Co., Japan) was applied to the surface of the trimmed block as described by Ullberg (7, 8). Sagittal sections, 20 IL thick, through the whole body were obtained by using

fraction

distribution

“C ]glucose no systematic

ically

the

disappeared only 2.9%

fate

by whole-body

ing

tissues

time,

in vivo

using

fragmentary

in the

acid-soluble

work, in the

about assimi-

that

gated

acid-soluble

injection,

was

0.5 ml of 0.9%

ILC

in the sub-

Therefore,

with

of [U-’4Cjglucose. [U-’4C]glucose with a specific radioactivity of 5.0 mC/mmole was obtained from the Daiichi Pure Chemicals Co., Japan. The radiochemical purity of [U-’4Cjglucose was determined by autoradiography of paper chromatograms, using two different solvent systems: A, n-butanol: pyridine:water (6:4:3, v/v/v) and B, phenol:water (5:1, v/v). The R, values of [U- ‘4C Jglucose in solvents A and B were 0.43 and 0.35 at 20#{176}C, respectively. In each system, the compound was found to be more than 99% radiochem-

the

acid-soluble

‘4C had practically At 8 hr after injection,

present

]glucose

(9,

week.

of the ‘4C remained (9, 10). [U-’4C

in

[U-14C]glucose

period

than body.

in

40% was eliminated 20% was in the cold

fraction

‘4C from

of the

“C

1 hr after

was

toneally

intraperi-

of the

of the whole mouse was fraction (water-soluble

within

about

injected

(dd strain) intraperi591

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592

SHIMADA

Fuji

Photo

thin

polyester

Co.,

Japan).

film,

To

prevent

6 IL thick

ET

chemography,

(Daia

Foil,

transferred

to

a glass

slide

by

immersion

in

a

xylene solution. After exposure for 1 week, the autoradiographs were developed and fixed; an autoradiograph processed in this manher will subsequently be referred to as an “untreated autoradiograph.” Upon completion of dry autoradiography, the same sections were fixed in a MYOCARDI

of cold 6% (w/v) HC1O4, which was changed three times at 5-mm intervals. Then the sections were rinsed in four 5-mm changes of distilled water. After having been dried at room temperature, they were again autoradiographed according to the method previously described. An autoradiograph resulting from this procedure will be designated as a “treated autoradiograph.” In the treated autoradiograph the labeled carbon was incorporated only into the cold perchloric acid-insoluble substances, whereas in the untreated autoradiographs “C was distributed in both the cold acid-soluble and -insoluble substances.

solution

Mitsubishi

Jushi Co., Japan), was inserted between the x-ray film and the section (1). For comparative purposes in studying various tissues and organs, some sections were routinely stained wmth hematoxylin and eosin, periodic acid-Schiff or toluidine blue. For these histologic preparations, cotton tape was used because the section could be easily

AL.

Autoradiographic

treated

density

and

of

untreated,

crodensitometer

(PDM

Konishiroku

Photo

the

was -5, Co.,

autoradiographs,

measured

Sakura

with

a mi-

M icrophotometer,

Japan).

Throughout

this

UM SPLEEN(RED

PULP)

0 2.5 .4

.8

1. 0. TI ME 0

L1

Wi

1hs

mm ABDOMINAL

ws URINE

CAVITY

BROWNFAT _ain 0.5

SUBMAXILLARY ws

hs

1

5

30

1 24

AND

PAROTID

GLAND

mm

hs

ws

0

tJ’IJI.Jw 1.

1.6

1.2

1.2

0.8

0.8

0.4 0

0.5

-

3.5

L.

0.4

TIME -

0

-

30,jj

Li hs

Rim

HARDER’S

gru

,.o.S ws

mm

GLAND

SUBLINGUAL FIG.

rVM

1 S__.Q.1_._.4,, hs

ws

GLAND

1A

1. Autoradiographic density of each tissue and organ of the treated and untreated autoradiographs after intraperitoneal injection of [U-’#{176}C]glucose. .D_ , autoradiographic density of the section dried in a cryostat (untreated autoradiograph); .1.1, autoradiographic density of the section dried in a cryostat and then fixed in cold 6% (w/v) HC1O4 (treated autoradiograph). AD., autoradiographic density; hs, hours; ws, weeks. Three mice were used for each survival time. Mean values are expressed in autoradiographic density/0.5 x 5.0 mm’, and error bars give S.D. FIG.

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AUTORADIOGRAPHY

paper sity” black

the terms “autoradiographic density” refer to the transmission specular and white images (autoradiographs),

intensity

of the

to the

concentration

To rated whole

blackness

investigate into

being

of the

the

cold

mouse,

a

directly

OF

or “dendensity of with the proportional

“C.

rate

at

which

‘#{176}C was

acid-insoluble

incorpo-

substances

Geiger-Mueller

of

counter

the

(MCL-6A,

Metro Electro, Co., Japan) was used. First, the untreated autoradiographs of the whole-body sections were counted in three specific regions, namely, the head, the chest and liver and the bowel. Then the same

sections

which

had

been

fixed

with

(w/v) HC1O4 (treated autoradiographs) counted in the same three areas.

cold

were

6%.

again

Autoradiographic treated autoradiographs

density

organ, at intraperitoneal

specific

shown

in Figure

labeled

glucose,

highest

density,

of untreated for each tissue

and and

had

low

graph.

0.5

1

30

mm

0.5

after

abdominal

while

showed

in

head

the

highest

1

24

autoradiographs. graph,

In

Harder’s

ventricle

have

with

vary

gland,

cortex (Fig.

2).

liver

the tongue,

and skeletal At 5 mm,

graph,

the

O.S

3

blood

3f

1

1.( 11!

when

gray

matter, mucosa,

salirenal

Harder’s

1

‘

0 0.4

0.

0.8

0.(

0.6 0.4 0.2 0

0.4

ill

0.2

I:TIME

i

i

mm

24,

0 1

hs

DUODENAL

j

TIME

.

-

0.5

j

-

15

3.Q,..

24,

mm

ws

GLAND

LARGE

.1

hs

3,

ws

INTESTINAL

MUCOSA

SKIN(DORSAL) CORPUS

CALLOSUM

Rim

‘#{246}s 1

5

-

30 .

1

-

hs 24 -

ws,

,

1

3

.4

104

d. 8 0.6 0.4 0.2

TIME 0

IL

‘1

5 mm

CEREBRAL

39,1

24, hs

_1 3. ws

NEOCORTEX

TONGUE FIG.

left

density

ws

2

-u.-1--

0

of the

high

cavity,

hs

5

of

muscle (m. rectus capitis) in the untreated autoradio-

abdominal

1

still

types autoradio-

and

cerebral intestinal

no

autoand liver

both

untreated

relatively

the

cavity

in

the

gland,

did

mm 1

had

autoradio-

abdominal

density

of

showed

untreated

the

is

injection

cavity

the

the

1 mm,

_

‘QIILI;T 1.0

the

periods following [U- ‘4C ]glucose,

LUNG .JI.L

5

1. At

density At

TESTIS Rifl

survival injection of

density in either the treated or untreated radiographs. Blood of the left ventricle

compared

RESULTS

593

‘4C-GLUCOSE

lB

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gland

THYROID

MESENTERI

GLAND

mm ..b.L isi#{246} 1 24

t

‘14)4

LJj1I&”

-

mm 5

5ci

1 3

C

SKELETAL MUSCLE Ui. RECTUS CAPITUS) mm hs ws mi sb iTh 13

LYMPHNODES hs

3b

1

ws

m

21

l(L4

0.8

.4

0.5

11

0.615’

.8

0.6 0.4 0.2

0.51

.Q!_i mm RENAL

#{163}_Z.4- .1 3, hs ws CORTEX

BONE

S

301

inin

-

24 hs

TIME 1 3. ws

LI VE R MARROW(RIB) 0.61

sin Os

0.6

S’--

hs

ws

TI1

15

‘

1.1 I,J1.LIi -

0tM

[0.8

14

0.5

0.5 iin SMALL

1 5

30

1 24

t1114 TIME

0.2.1

1

S sin BLOOD(LEFT

30

24 1 3 hs ws VENTRICLE)

1

‘1 . ‘w%

-

INTESTINAL

MUCOSA

FIG.

1C

Large

intestinal mucos

-

.

-

-

:.

‘-

a

-

#{149}1

1

.!\.\

Har’

--

Tongue

--.-

Blood

-

Abdominal FIG.

Note

2.

the

Whole-body high density

autoradiograph of the abdominal

(untreated) at 1 mm after cavity, liver and blood.

intraperitoneal

594

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injection

cavity of [U-’4C)glucose.

AUTORADIOGRAPHY

and

urine

this

same

tongue, intestinal

displayed time

the interval,

greatest

density,

cerebral

liver, renal cortex, mucosa and blood

gray

duodenal all showed

OF

but

at

cantly

more

matter,

organs

examined.

gland, signifi-

radioactive graph, the Bone marrow (Rib

FIG.

Note

3.

the

Whole-body high density

autoradiograph the sublingual

of

(untreated) gland, large

(treated with of the cerebral

at 1 hr intestinal

cold gray

density

than At

glucose, highest

30

in the density

other

mm

tissues

after

and

injection

untreated occurred

of

autoradioin Harder’s

Spleen

after

intraperitoneal

mucosa,

Large FIG. 4. Whole-body autoradiograph [U- “C Jglucose. Note the low density density of the pancreas.

595

‘4C-GLUCOSE

tongue

injection and

intestinal

of [U-’4Cjglucose.

cerebral

gray

mucosa

6% HC1O4) at 1 hr after intraperitoneal matter compared with that of Figure

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matter.

injection of 3 and the high

596

SHIMADA

ET

AL.

Testis

Large 5. Whole-body

FIG.

Note

the

high

density

autoradiograph

(untreated)

of the

gland

1001

Harder’s

at 24 hr after

and

intestinal

intestinal

intraperitoneal

injection

mucosa

of [U- ‘4C Jglucose.

mucosa.

-

-

501

-

TIME

0%

‘rr

i

0.5

1

0 FIG.

bowel. area,

6. The

rate

Conditions a circular

HEAD.

incorporated

duodenal

rate

of “C

gland,

and urine, while observed in the skeletal muscle

relatively cerebral and bone

autoradiograph,

Harder’s

gland,

tongue

and

showed

high

density.

large

at each

large

cold

counter: 2.0 cm

survival

distance in which

time

intestinal

J

is a mean

BOWEL

substances

value

1 hr

autoradiograph, in Harder’s gland, urine,

all

of the

of three

Within

duodenal

1w

head,

chest

from the section to the counter, the head, chest and liver and bowel

mucosa

mucosa

24Hs

acid-insoluble

high density was also gray matter, pancreas, marrow. In the treated gland,

1H

CHEST AND LIVER,

into

intestinal

i

3OMIN

5MZN

of the Geiger-Mueller area having a diameter

respectively. The from one mouse.

gland,

of “C

I

noted nal

mice.

of the

gland,

tongue, but in the mucosa

Twenty

injection,

large

relatively cerebral and

bone

sections

in

highest sublingual

and

the

intestinal

marrow

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were

mucosa

(Fig.

taken

was found duodenal

density matter,

and

untreated

density gland,

intense gray

liver

1.0 mm; counted were enveloped,

and

was

small 3).

also

intestiA similar

AUTORADIOGRAPHY

OF

597

‘4C-GLUCOSE

FIG. 7. Enlarged autoradiograph (untreated) of the salivary gland at 30 mm after intraperitoneal [U-14C]glucose. Note the high density of the sublingual gland and the low density of the submaxillary

density

pattern

existed in the

graph,

but

cerebral

had

low

density

pancreas unfixed gland

the

and retained

same time nal mucosa, showed

density 3-week

noted

At

density

following

detected

type

that

of

the

Harder’s at the

5). in

any

density

organs

autoradiograph.

density

two

end of the had been

autoradiographic

in

the

pattern

autoradiographs at 1 week

density

between

or It

in both became similarity

the the

tissues should

two

still

Autoradiographic

Results

in

the

abdominal

and

then

mm,

from

after urine

faded

radioactive incorporated

a maximum 24

hr

that

most

in the

cold

30 mm amount

(Fig. of

6). “C

acid-insoluble sub(mean values of the bowel) was approxi-

of the amount that into cold acid-soluble 1 week

within

within

was

within the

incorporated into cold stances of the whole mouse head, chest and liver and

At 24 hr and

out

showed

[U-’4C]glucose

substances after injection,

mately half incorporated

injection and lung density

reaches

investigation

of

gland.

autoradiographic

at 0.5

of our “C

acid-soluble At 1 hr

following

had been substances.

administration

of

glucose, all of the ‘4C had into the cold acid-insoluble

been sub-

stances. DISCUSSION

The density

30 mm in the

5 mm and disappeared injection (11).

recog-

nizable.

mm

blood,

visible

level at following

in

similar of the

within whereas 5

the

be

treated

was

at

In

became

of the

autoradiographic By the glucose

it appeared

24 hr.

both

low density was oband testis; in the other

and untreated at 24 hr, and patern

in

and

injection,

was no longer visible. period after the labeled be

of the

hr,

(Fig.

studied,

no

24

cavity disappeared of [U- 14C ]glucose,

urine

gland, tongue, intestiand bone marrow also

high

organs

and density

autoradiographs, highest density,

administered,

either

4).

of autoradiographs, in skeletal muscle and

the

fixed the

1 week

tissues

could

whereas (Fig.

the sublingual lymph nodes

autoradio-

matter

high

a markedly

After types served

(12),

was

treated

gray

injection

during

untreated

autoradiographs

the

study

present

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clearly

produced show

that

the

598

SHIMADA

ET

AL.

Large

Small 8. Enlarged autoradiograph Note goblet cells

FIG.

“C from dominal

[U- “C Jglucose cavity

injected initially

appeared

and blood of the while there was

left no

ventricle evidence

isotope

uptake

density

of the

blood

became

the

same

time,

liver.

At

cerebral the

gray

white

and Pepeu’s was frozen

injection gray the

of could matter

white

(6,

played

Within

to that

the higher

of

than

This

consistent

brain In both

is more the

30

and

mm

uptake

fact

that

vascular

unfixed 1 hr

and after

and duodenal mucous cells, in

with

increased

16).

contrast

in the

in which the intravenous

to the

of the

the

it was from

(3), after

the

to

the

density

of the

which

are

intraperitoneal

parotid

and

submaxillary

of

serous

Vrba

et

al.

(14)

salivary gland tion (incorporation

had

7).

stances)

of

all other

tissues

Our gland

present having

cells

glands, in

the

mouse

that

reported

that

they

work suggests that a high assimilation

of ‘4C in the

histochemical graphic

organs

cells dine

was

with

examined. the rate

and

tissues

large

mucin intestinal

having

coincided

and

evident. it was

stained with periodic blue (Fig. 8). There

produce the

tissue

investigation, density

the

in comparison

organs

salivary in their

recent study might be the sublingual Large intestinal mucosa also had intense sity in both types of autoradiographs, and zation

of

a very high rate of assimilainto cold acid-insoluble sub-

[U-14C]glucose and

injection

mucosa.

up

the

low

after

made

than

glands, all disthe

intestine at 30 mm of the large intestinal

fixed injec-

mucosa

mucosa

(Fig.

of the

density

are

attributed

density,

1 mm,

equal

findings 0.5 mm

Harder’s, sublingual are composed of high

into the abin the liver

observations

“C-glucose. be

autoradiographs, tion, which

was These

of the large high density

within 0.5 mm, of radioactive

head.

autoradiographs

Cassano cat brain ‘4C

matter

matter.

untreated

of

in the

intestinal

(untreated) showing the

[U- ‘4C]glucose.

intestinal

are mucosa.

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high

with

the

acid-Schiff were goblet abundantly

gland. denlocali-

From

our

obvious

that

autoradiolocations and cells present

It is noteworthy

of toluiwhich in to

AUTORADIOGRAPHY

OF

599

‘4C-GLUCOSE

*9

9

4

Abdominal 9. Enlarged

FIG. intraperitoneal

graph

autoradiograph

injection

in the shape

mention

that

density graphs The into creas,

the

tissues

relatively

high

after data

rate

gland

of the

sublingual

cosa, skeletal pared

of “C

and

high

gland,

to emphasize large amounts

of the

intestinal

labeled

gland

rapid

rate

cosa,

bone

Within glucose,

and

pan-

marrow the

of the of the

abdominal

cavity

abdominal

cavity

mm abdominal

or in tissues

proliferation

mately the

and comIt is assimare

and after

testis) injection cavity

having

(intestinal

the

high-

99%

of

at

and

the

1 mm after autoradio-

autoradiograph.

dry

the

was

This

“C rated

9).

porated

water

conclusion into

These

be

on

because

of

and rinsing, in this paper. in the

results cavity

of

shape

imply might

acid-insoluble

of a

that be

the

incorpo-

substances. from the Geigerof the “C in the

had remained regardless 6% (w/v) perchloric acid

of and

rinses. that

cold

approxito

of autoradiographs

appeared

into

cavity with

distilled

that known

fixing earlier

to data obtained counter, about 15%

abdominal the fixation Our

types

abdominal

rapidly

According Mueller

in

being

a preliminary

disappeared

used described cavity

(Fig.

in the

after

In

confirmed

tape

both

abdominal

density

HC1O4.

[U- 14C ]glucose

adhesive

Furthermore,

no

6%

it

the methodology which has been

the

of radioactive

cold

granule

a mu-

(14). showed

treated

had

with

examination,

mu-

marrow, testis density when and tissues.

in the

autoradiograph

and after

intestinal

density

treated

Harder’s

here that organs of [U-’4C]glucose

pancreas)

of cell 0.5

HCIO4)

density

occurrence was not an artifact caused by insufficient fixation or rinsing, because urine having the highest density at 5 mm in the unfixed

those which either secrete proteins or protein complexes acting on organs outside of their origin (sublingual gland, duodenal gland, Harder’s

6%

est

mucosa

to Vrba 1 week

glucose,

tongue,

high

autoradioincorporation

is similar 24 hr and

lymph nodes, bone muscle had high with other organs

important ilating

cold the

having

fixed

substances

injection (13). At

administration

organs

and

acid-insoluble

sublingual

gland,

and

unfixed mucin.

at 1 hr Winter’s

with Note

of a granule.

in both all produce cold

(treated

[U-’4C]glucose.

of

cavity

the

acid-insoluble

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amount

of “C substances

incorof

600 the whole mouse was about half of the of “C incorporated into cold acid-soluble stances at 1 hr after injection of labeled supports

the

investigation

findings

of

Vrba

and

SHIMADA

ET

amount subglucose

6.

We

greatly help

in

indebted the

to Ms.

preparation

Winter’s

Dorothy of this

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ACKNOWLEDGMENT

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AL.

Vrba

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MK,

Richter

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