STUDIES ON THE MODE OF ACTION OF
SYNTHETIC THYROPROTEIN

By
Wallace Friedberg

A THESIS
Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science
in part ial fulfillment of the requirements
of the degree of

D O C T O R OF PHILOSOPHY

Department of Physiology and Pharmacology

1953

STUDIES ON THE MODE OF ACTION OF
SYNTHETIC THYROPROTEIN

By
Wallace

Friedberg:

AN ABSTRACT
Submitted to the S c h o o l
State College
in partial

of G-raduate Studies of Michigan

of A g r i c u l t u r e
fulfillment
of

DOCTOR

Department of

the

of the requirements

degree

of

OF PHILOSOPHY

P h y s i o l o g y and Pharmacology
1953

Approved

and Applied 3clence

STUDIES ON THE MODE OF ACTION OF SYNTHETIC THYROPROTEIN
By
Wallace Friedberg
Studies were
knowledge

conducted with

of the mode of a c t i o n of synthetic t h y r o p r o t e i n in

the living
lodlnated

body.

The t h y r o p r o t e i n use d was 1-131 labelled

casein.

The first ex p er i me n t w as a c om p ar a ti v e

of the d i s t r i b u t i o n
t is s ue s of

cf l o dl n a t e d ca se in and Iodide

the rat.

administered,

R a d l o l o d i d e and lodlnated

I nt r av e n o u s l y ,

h u n d r e d m i n u t e s af t er
f i c e d and

ti ssue

of l o d l n a t e d
k id n e y ,

the view of i n c r e a s i n g our

s a mples w e r e taken.

stomach,

and

that

iodine) w a s
the

s tu d ie d

The

in the liver,
intestine,

sl i gh t ly

The highest
it was more

lodlnated casein

than
(1-2^
In

shown to co n centration lodlnated

l o dl na t ed c asein was not as c o ncentrated

but more

than

Slightly

spleen,

s t im l lr l y and wa s found to concentrate

same t i s s u e s w h i c h w e r e

casein.

sacri­

found In the liver,

small Intestine.

of the iodide.

One

H i g h e r 'concent r at i on s

c o n c e n t r a t i o n w a s found in the liver, where
10 times

casein were

the a n im a l s we re

c a se i n than iodide w e r e

lung,

In v a rious

to two croups of rats.

the i nj e ct i on s

study

c o nc e n t r a t e d

in the st omach and large

the l od l n a t e d casein.

The m e t a b o l i c b r e a k d o w n of in travenously a dm i nistrated
lodlnated

c a s e i n was

s tudied with a view

the m e t a b o l i t e s and d e t e r m i n i n g

toward characterizing

their relative

conc entrations

2
over a time
considered

series.

The alkali wa s he d

to c ontain

thyroxine-llke

the

of a tissue

I odine w a s

that w h i c h w a s

like

that w h i c h

sample.

Iodine.

The free

could be extracted before

Co m bined t hy r oxine-like

e x t r ac t ab l e after the free thyro xlne-

iodine had a l re a dy b ee n e xt r a c t e d and the tissue sample

hydrolyzed

in 2N sodium hydroxide.

v/ere l £ , 12,

24,

thyroxlne-like
casein,

and 72 hours.

The time i nt e rvals

The re s ul t s in dicated

and

released

c as e in a v a i l a b l e

of t h y r o x l n e - l i k e

in o r de r to c h a r a c t e r i z e
to d e t e r m i n e

to the plasma.

to the liver and

I od i n e v/ere found

O ra l ly a d m i n i s t e r e d

intervals

lodlnated

the plasma

casein ^ae

studied

iodine in the plasma.
12, and 48 hours.

the t h y r o x i n e - l i k e iodine a p pe aring

at a l l time
free b u t a n o l

of co m bi n ed
i nt e rv a l

interval
the c o nc e n­

The

More

time

than 50

in the plasma,

I nt e rv a ls a f t e r oral a d mi n is t ra t io n , w a s in the
so luble

form.

However,

small a m o u n t s of t hy r ox l n e - l i k e
are a b s o r b e d

in rats

into the plasma,

the r e l a t i o n s h i p be tween the time

st u d ie d v/ere 1^,

level

to be interdependent.

the iodine ab sorbed

thyroxlne-like

p er c en t of

that

The amount

a f t e r oral a d m i n i s t r a t i o n of the t hy roprotein and
t ration of

studied

iodine ^as h y d r o l yz e d from the lodlnated

in the liver,

of l o d l n a t e d

a nd

thyrox ln e -l i ke

iodine w a s

hydrolysis

butanol extract was

fr o m the

iodine,

I nt e st i n a l tract.

thyroxine-llke

( l£- hour).

the data sup^est that

iodine was

form,

The hichest percent

found at

This may r e p r e se n t

or some larpe metabolite.

in a combined

the first

the Intact

time

protein

3
The i n f l u e n c e of p l a s m a lodlnated caeein on urine r a d i o ­
a ct i v i t y w a s

st udied by m e as u r i n g the decreases

urine radioactivity
to be r a p i d l y
activity

in

the dog.

lodlnated Cnsein was found

cleared f r o m the p la s ma while the urine r ad i o­

showed a slov/, but steady,

decrease.

tion of the r a d i o a c t i v e m a te r i a l in the urine,
after

In plasma and

A ch aracteriza­
25 minutes

the i n t r a v e n o u s a d m i n i s t r a t i o n of iodine-131

i o d i n a t e d ca s e i n r e v e a l e d
a ct i v i t y

it to be undlalyzable.

in the urine may represent

pro t e i n or

some

lar°-e metabolite.

the intact

labelled
The r ad i o­

iodinated

acknowledgements

The author w i s h e s to express his ap p r e c i a t i o n to
Dr. E. P. R e in e ke

of the D e p a r t m e n t

of Physi ology and

Pharmacology for his g ui d an c e a nd advi ce throughout the
course of these

invest igations,

of this manuscript.
Alfredson,

and during the p r ep a ra t io n

He also w ishes

to thank Dr.

B. V.

head of the Departm en t of Physiology and P h a r m a ­

cology, for the use of the facilit i es
Many thanks are due Miss L.
Mr. W. P. Baker,
all presently

Dr.

C.

Feng, Mrs.

C. Lee, and Dr.

or formerly

and Pharmacology.

of the department.
E. F.

Monkus,

L. F. Wolterlnk,

of the Departm e nt of Physiology

Their assistance and suggestions during

the course of the experi m en t s re ported herein w e r e

invalu­

able.
Finally,

the a u th o r w i shes to express his thanks to

the Michigan A g r i c u l t u r a l Experiment Station for support of
the project u n d e r w h i c h this wo rk w a s

carried out.

TABLE OF CONTENTS
I N T R O D U C T I O N ..........................

1

REVIEW OF THE L I T E R A T U R E .................................. 3
Formation of lodlnated Protein w i t h High Th yroidal
A c t i v i t y .............................................. 3
The Quantitative E x tr a ct i on of Thyroxine from the
Hydrolysate of lodlnated Protein
................
4
The Metabolism of lodlnated P r o t e i n .................... 9
Iodine-containing Compounds in lodlnated Casein. . . . 12
EXPERIMENTAL
lb
The Distribution of lodlnated Casein and a Comparison
with Iodide in the R a t ............................. lb
Preparation of R adioactive lodlnated Casein . . . .
15
Injection and Tissue Sampling Procedure ............ 1 6
Radi oactivity M e a s u r e m e n t s ........................... 18
R e s u l t s ................................................ 18
The Distribution of Slightly lodlnated C a s e i n ........ 19
R e s u l t s ................................................ 22
The Metabolism of lodlnated Casein in the R a t ........ 25
Intravenous A d m i n i s t r a t i o n ...........................25
Preparation of Radioactive lodlnated Casein . . . .
26
Rat Thyroidectomy Procedure .......................... 26
Injection and Tissue Sampling P r o c e d u r e s .............. 27
Collection of Blood and Liver Samples ..............
2?
Fractionation of Plasma, and Liver S a m p l e s ........ 29
Extraction of unhydrolyzed p l a s m a ...................29
Extract ion of unhydrolyzed liver ................. 29
Hydrolysis of the Liver and Fractionation of the
H y d r o l y s a t e ....................................... * 3 °
Alkaline Washing of Butanol Extracts (Hydrolyzed
and Unhydroly z e d ) ............................... 3°
R e s u l t s ................................................31
Combined thyroxine-like iodine in the plasma . . 31
Free thyroxlne-like iodine in plasma ............ 31
Percent free thyroxine-like iodine of the total
t hyrcxine-1 ike iodine, in p l a s m a .......... .31
Combined thyroxine-like iodine in l i v e r ........ 32
Oral A d m i n i s t r a t i o n ..................................32
R e s u l t s .......................
3°
The Disappearance of lodlnated Casein from Plasma and
the Appearance of its Metabolic Products in the
41
Urine, in the Dog ................................
41
Decrease in Plasma and Urine Radioactivity. . . .
42
R e s u l t s ................................ * ...........
45
Radioactive Materi al in Urine .....................
45
R e s u l t s ............................................

DISCUSSION

.

k6

SUMMARY.

51

LITERATURE CITED

53

APPENDIX
. I. The D i s t r i b u t i o n of Iodinated Casein in the
R a t .............................................
The D i s t r i b u t i o n of Iodide in the Rat . . .
II.
The D i s t r i b u t i o n of Slightly Io dinated Casein
III.
in the Rat
The M e t a b o l i s m of I od i na t ed Casein in the Rat
IV.
I nt r av e no u s A d m i n i s t r a t i o n ..............
Oral A d m i n i s t r a t i o n .......................
Sta n da r d Rat Blood Values and Computation of
V.
Correct i on Fa c to r s ..........................
VI.
C or r ec t io n for Physical De c ay ..............
C o r r e c t i o n for Se l f- ab s o r p t i o n
............
VII.
The D i s t r i b u t i o n of R a d i o a c t i v i t y in the
VIII.
E lood 100 Mi n ut e s af ter the Intravenous
A d m i n i s t r a t i o n of Io dine-131 Labelled
I o d i n a te d Casein ............................
The D i s t r i b u t i o n of R ad i o i o d i d e between
IX.
Plasma and R e d Cells ........................
X. k Kilo "Hoppert" Stock R a t i o n ..............

56
56
60
6k

68
70
72

78

80
90

INTRODUCTION
Investigations c a r r i e d out by Reineke,
'45,

*43) a n d others

(Frieden a nd Winzler,

et al

(1943,

1948) have s hown

that pa r en t al l y a d m i n i s t e r e d iodinated c asein exhibits the
physiological proper ti e s

of thyroxine.

activity has been a t t r i b u t e d to

This biological

thyroxine f or m e d in the

casein during its in vitro iodination.

Of pa r ti c u l a r interest

is the fact that the t h y r o x in e in iodinated casein is in
strong co m bi n at i on a n d hours of hydro l ys i s w i t h strong a c i d
or alkali

is ne c es s a r y for

its liberation.

In contrast,

thyroxine norma ll y f o u n d in the b l oo d is only loosely bound
to the serum proteins.
The q u e s t i o n arises:
30 exert a biologi c al

Does the

iodinated protein per

effect or is it first me t a b o l i z e d with

the release of thyroxi ne?

Exp eriments were designed to trace

the pathway of this p r o t e i n - c o m b i n e d hormone,
the rat.

princi p al l y in

The p roblem has been d ivided into the following

classif i o a t i o n s :
1.

The d i s t r ib u ti o n of iodinated casein in c omparison
with

2.

iodide

in the rat.

The d i s t r i bu t io n of s l i g h t l y iodinated casein in
the rat.

3.

The m e t a b o l i s m of iodinated casein in the rat.

2
4.

The

d i s a p p e a r a n c e of

and

appearance

urine,

in th e

iodinated

of its m e t a b o l i c

has

been

development

the

techniques used

reviewed

in order

may be p r o p e r l y evaluated.

summarize

the available

i o di na te d protein.
1.

The

in the

trace

of

the

experiments

It a l s o

serves

i n f o r m a t i o n r e l a t e d to th e
Specifically,

formation

to

in these

that t h e y

of

product

dog.

T he l i t e r a t u r e
of

casein from plasma

to

metabolism

the r e v i e w w i l l

iodinated protein with

so

consider

high

thyroidal activity.
2.

The quantitative
t he

hydrolysate
metabolism

will
of

of

3.

The

4.

Iodine-containing

It is h o p e d t h a t
contribute

the

e x t r a c t i o n of t h y r o x i n e f r o m
iodinated protein.

of i o d i n a t e d p r o t e i n .
compounds

experiments

in

described

toward an understanding

iodinated protein.

i o d i n a t e d case in.
in t h i s

paper

of t h e b i o c h e m i s t r y

R E V I E W OF THE
Formation

of

LITERATURE

Iodinated Protein with High

Thyroidal Activity
Blum and Vaubel

( 1 89 8 )

iodinating albumin.
ated when

they were

When

iodine

activity are

Abelin
insoluble
exhibits

(1934)

all

medium with

p r o c e d u r e the

it w a s

protein held
Present-day

iodinated proteins with high

b a s e d on t h e s e

e a r l y f in d i n g s .

the h y d r o l y s a t e

t he b i o l o g i c a l

sodium

the a c i d a s

c l e a r l y d e m o n s t r a t e d that

f r a c t i o n of

liber­

inhibited the

in f i r m c o m b i n a t i o n .

f o r t h e f o r m a t i o n of

thyroidal

protein,

to neutralize

i o d i n a t e d b y this

7 percent

me t h o d s

able

for

that h y d r i o d i c acid,

By b u f f e r i n g the a q u e o u s

bicarbonate

about

They found

iodine c o m b i n e d with the

reaction.

formed.

s t u d i e d the conditions

of

properties

the a c i d -

iodinated protein

of t h e

thyroid

hormone.
Direct

proof

that

thyroxine

protein hydrolysate was
becker

in 1939.

These

f r o m t he h y d r o l y s a t e
Reineke,
investigations

et al

investigators

temperatures

Von Mutzen-

isolated thyroxine

i o d i n a t e d c as e i n .

(1942,

'43,

effort

high thyroidal activity.
elevated

in i o d i n a t e d

obtained by Ludwig a n d

of

in a n

is p r e s e n t

'43)

c a r r i e d out

to p r o d u c e

extensive

iodinated casein with

T hey r e p o r t e d that

i n c u b a t i o n at

(30— 70 ° C . ) a n d the p r e s e n c e

of s m a l l

4
amounts of o x i d e s of m a ng a ne s e,
s i g n i f ic a nt
rial.

increase

The a m o u n t of

caused a

in the b i o l o g i c a l p o t e n c y of the m a t e ­
io d in e e m p l o y e d a n d the pH of the m e d i u m

were a l s o f o u n d to i n f l u e n c e
thyroprotein.

as a catal yst,

the b i o l o g i c a l a c t i v i t y of the

C o u r r i e r a n d c o w o rk e rs

(1949)

the i n c u b a t i o n at e l e v a t e d t e m p e r a t u r e s
the t h y r o x i n e content

but does

contend

does not

that

increase

i n cr e a s e the a b s o r b a b i l i t y

of the i o d i n a t e d c a s e i n f r o m the g a s t r o i n t e s t i n a l tract.
T he Q u a n t i t a t i v e

E x t r a c t i o n of T h y r o x i n e

the H y d r o l y s a t e

fro m

of I o d i n a t e d P r o t e i n

P r e s e n t - d a y a s s a y m ethods

involving a quantitative

e x t r a c t i o n of t h y r o x i n e have t h e i r o r i g i n in the w o r k of
Kendall

(1915).

isolate,

The o bj e ct

in p ur e form,

of h i3

i n ve s t i g a t i o n s was to

one or mo r e of the c o n s t i t u e n t s

the t h y r o i d g l a n d w i t h p h y s i o l o g i c a l activity.
a p r e l i m i n a r y study of the nature

of

Ca r rying out

of the iodine co mbination,

he o b s e r v e d that s u s p e n d e d or d i s s o l v e d d e s i c c a t e d t h y r o i d
lost les3
against

r u n n i n g water.

h yd r o x i d e
however,

t h a n 5 p e rc e nt

in alcohol,

of its t o t a l

iodine w h e n d i a l y z e d

F o l l o w i n g h y d r o l ys i s w i t h sodium
75 percent

of the

iodine was dialyzable;

it s till r e m a i n e d in o r g a n i c co mbination.

The

h y d r o l y s a t e c o u l d be s e p a r a t e d into two f r a c t i o n s a c c o r d i n g
to s o lu b il i ty :
percent a c i d

a b ou t

50 percent

insoluble.

Kendall

b e i n g a c i d soluble, a n d 50
(1915) f o u n d the acid-

insoluble f r a c t i o n to be of an a c i d i o nature,

e asily soluble

5
in dilute alkali or ammonia, a n d reprec ipitated by a n y acid.
Further p u r i f i ca t io n of the a ci d -insoluble f r ac t i o n was a c ­
co mplished by extracting it with petroleum ether to remove
fatty acids a n d sulfur.
effective

The re s ulting dark brown powder was

in the treatment of myxedema;

subnormal pulse an d

temperature were r a i s e d to normal, an d metabolism was increased.
Symptoms of h y p e r t h y r o i d i s m could be excited by administering
the p reparation to normal dogs.
Kendall

Continuing his

studies,

(1919) h y d r o l y z e d fre3h t h yroid glands in sodium

hydroxide and obtained a clear alk a l i n e filtrate containing
almost all the iodine.

A c i d i f i c a t i o n of thi3

sulted in the separa ti o n of a fine flocculent

solution re­
precipitate

weighing 0.1 percent of the total weight of the fresh glands
and con taining about 23 percent of the total iodine.

After

further p u r i f ic a ti o n of this material he was successful in
isolating thyroxine.

Harington a n d Randall

a chemical assay for thyroxine

(1929) devised

in the thyroid,

based on the

q uantity of ac i d- i n s o l u b l e iodine in the hydrolysate of the
gland.

Specifically,

thy roid material was hydrolyzed for 4

hours in N/l sodium h y dr o xi de a n d the filtrate adjusted to
pH 5 with 50 percent sulfuric acid.
iodine w a s consi dered to be thyroxine

The acid-insoluble
iodine.

While the meth od of Harington and Randall
3 imple technically,

(1929) was

it was shown b y Leland and Foster (1932)

that only about half of the acid-insoluble iodine goes to
make u p thyroxine.

The latter investigators based their

6
method on the e xt r ac t io n of thyroxine with butanol after
alkaline hydrolysis of the gland.

Desiccated thyroid was

hydrolyzed for 13 hours w i t h 2N sodium hydroxide and the
cooled h y drolysate w a s extracted with butanol.

The alcoholic

extract was further p u ri f ie d by shaking it w i t h N/l sodium
hydroxide.

In a series of 52 human, thyroids the mea n th yrox­

ine iodine was 25 percent of the total iodine.
experiments

Recovery

indicated that the amount of thyroxine destroyed

during the a l kaline hydrolysis was not more than 15 percent
of the total thyroxine.
With a view t ow a rd increasing the accuracy of the tech­
nique of Leland
method.

et al,

Blau

He found that,

(1933) made revisions in the

by shaking an aci d suspension of thyrox­

ine with butanol, u s u a l l y 100 percent

of the suspended

thyroxine pa s se d into the organic phase
tion.

in a single extrac­

Therefore, he a c id ified the alkaline hydrolysate of

thyroid gland,

extracted it with butanol, and then w a sh e d

the extract w i t h an alkaline solution consisting of 5 percent
sodium carbonate in 4N s odium hydroxide.
procedure,

With this modi fied

Blau was able to obtain thyroxine values in t h y ­

roid material which we re

10 to 20 percent higher than those

obtained by the earlier method of Leland and Foster (1932).
Further

investigations led Blau

(1935) to conclude that

the chief shortcoming of the Leland and Foster (1932) tech­
nique was the amount of thyroxine destroyed during the
hydrolysis.

After boiling crystalline thyroxine for 13

7
hours w i t h d r i e d a n d d e f a t t e d beef
h y d r o x id e ,
acidified

only about
so l ut i on .

85 p e r c e n t

muscle

in 2N sodium

c o u l d be e x t r a c t e d f r o m

Following the

l e a d of H a r i n g t o n

who u s e d b a r i u m h y d r o x i d e as a h y d r o l y t i c a g e n t
t io n of t h y r o x i n e

from thyroid gland,

material w i t h ' a n 8 p e r c e n t
pared with a n

equivalent

and

able.

loss

activity.

r e n d e r e d the t h y r o x i n e
of t h y r o x i n e was
(1945) w e r e

more r e a d i l y extract-

only about

interested

1 0 percent.

in a

pigs.
might

iodinated

(1935)

m et h o d ,

was

be a t t r i b u t e d to

non-thyroxine groups

barium hydroxide

c i p l e to B l a u 1s.
closely with
of g u i n e a

the h y d r o l y s i s

modified,

but

similar

ef f ec t

in p r i n ­

agreed

metabolic stimulation

butanol

p r e p a r e d t he same as

produced a metabolic

in 40 p e r c e n t

resulting thyroxine values

Furthermore,

of

for 30 hours an d used a

t h o s e d e t e r m i n e d by the

pigs.

a t e d ca se in,

The

the h i g h c h e m i c a l

in peptide c o m b i n a t i o n w i t h

* 8HgO)

extraction technique,

stimulation

incomplete hydrolysis

T h e y c a r r i e d out
(Ba(OH)g

content

considerably higher

It s e e m e d to t h e m that

t he th y ro x in e .

butanol

o he m ic a l p r o ­

iodinated casein with high thyroidal

tha n a b i o l o g i c a l a s s a y b a s e d on the m e t a b o l i c

analysis

rate of

T h e y f o u n d th a t t he a p p a r e n t t h y r o x i n e

a c c o r d i n g t o B l a u 1s

of g u i n e a

When com­

c o n c e n t r a t i o n of s o d i u m h y d r o x i d e he

hydrolysis

cedure for a s s a y i n g

in the i s o l a ­

s o l u t i o n f o r 6 hours.

th e b a r i u m h y d r o x i d e a c c e l e r a t e d t h e

R e i n e k e et al

(1925),

B l a u b o i l e d the t h y r o i d

f o u n d t h at

Th e

the

for

equivalent

extracts

of the

the c h e m i c a l

iodin­

t e ch n i q u e ,

t o the t h y r o x i n e

8
content d e t e r m i n e d c h e m i c a ll y.

Taurog and Chaikoff

(1943)

m od i fi e d B l a u 's p r o c e d u r e b y h y d r o l y z i n g the t h y r o i d in 2N
sodium h y d r o x i d e for

15 hours.

W h i l e the s o d i u m h y d r o x i d e

caused g r e a t e r d e s t r u c t i o n of t h y r o x i n e than d i d barium
hydroxide,

thyroxine

values w e r e

less

va r iable w i t h the

former al k al i .
Rec e nt

i n v e s t i g a t i o n s r e ve a l

s e v e r a l s h or t c o m i n g s

of the

butanol e x t r a c t i o n t e c h n i q u e f o r t h y r o x i n e determi n at i on s .
Hird and Trikojus

(1948)

r e p o r t e d the p r es e n c e

ninhyirin-positive materials
iodinated casein hydrolysate,
f i e d as thyroxine,

in the bu t an o l e x tract
whic^

of

t h e y t e n t a t i v e l y i de n ti ­

triiodothyronine,

T he i r i d e n t i f i c a t i o n w a s

of several

and diiodothyronine.

b a s e d on rf values

o b ta i n e d by

c h r o m a t o g r a p h i c a na l ys i s.
In 1949,

R e i n e k e et a l a p p l i e d the

isotope d i lution

tec h nique t o the d e t e r m i n a t i o n of t h y r o x i n e
casein.

They reported thyroxine

in io dinated

values w h ic h w e r e about 3 5

percent of t h o s e o b t a i n e d by the b u t a n o l e x t r a c t i o n m e t h o d
(Reineke et al,

1945)*

Friedberg and Reineke
graphic

1953) c a r r i e d out c h r o m a t o ­

s tudies on the h y d r o l y s a t e

i od i na t ed casein.

Radioautograms

vealed t h e p r es e nc e of at
like co mpounds.

least

of i odine-131 l ab e ll e d
of the c h r o m at o gr a ms

three

(1933),

re­

i o d i n a te d t h y r o x i n e ­

A f t e r p u r i f y i n g the butanol

B l a u 's a l k a l i n e s o l u t i o n
tr ations

(1951,

extract wit h

th ey found that the c o n c e n ­

of t hese i o a i n a t e d m a te r ia l s we r e not s i g n i f i c a n t l y

reduced.

These t h yr o xi n e- l ik e products w o ul d be included

in the t h yr oxine fraction,

as d e t e r m i n e d by the usual butanol

extraction procedure.

The M et a bo l is m of Iodinated Prot ein
The a n i m a l body responds to synthetic thyroprotein in
a manner si milar to its response to thyroxine.

Investigations

conducted by Kaer (1934) demon s tr a te d that iodinated protein
exerts a thyroxine -like effect w ithout the necessity of a
preliminary treatment.

She u s e d a prepa ration of iodinated

albumin w h i c h had pr e vi o us l y been studied by Barkan and
Leistner

in 1929.

The t h yr o pr ot e in was fed,

intact,

to

tadpoles, a nd the change in their size a n d development noted;
artificial iodinated a lbumin was f o un d to accelerate meta­
morphosis.

KiLer also found the iodinated a l b u m i n to be

effective w h e n f e d to
sometimes,

young guinea pigs; a decrease and,

stoppage of weight ga in was observed.

Lerman a n d Salter

(1939), conduc t in g clinical studies,

found iodinated blood serum protein to be effective for the
relief of myxedema.
solution,

The protein was

iodinated in aramoniacal

according to the procedure of Wormall

(1930).

Four patients with at hy r eo s is were treated and there was
rapid a n a complete recovery with proper dosage; BMP increased
diuresis

occurred, a n d b l oo d cholesterol dropped.

iodinated casein in tadpoles
a nd sheep

(1942), guinea pigs

Assaying
(1945),

(1946), Reineke and Turner found that the parentera!

10
administered material
toxic s id e

effects.

exerted hormonal activity without any
In s u b s e q u e n t

by F r i e d e n a n d W i n z l e r
(Dempsey a n d As t w o o d ,

e x p e r i m e n t s c a r r i e d out

(1948) t h e g o i t e r p r e v e n t i o n m e t h o d
1943) w a s

e m p l o y e d to a s s a y i o d i n a t e d

casein, a n a a p a r e n t e r a l r ou t e of a d m i n i s t r a t i o n w a s al s o
used successfully.
Turner a n d Reineke

(194^)

investigated the

relationship

betw een t he r o u t e of a d m i n i s t r a t i o n of i o d i n a t e d c a sein a n d
its u t i l i z a t i o n by r u m i n a n t a n i m a l s
of t h y r o i d a l a c t i v i t y w a s

the loss

(sheep).

The c r i t e r i o n

in b o d y w e i g h t

during a

tw o-week period.

I o d i n a t e d c a s e i n wa s f o u n d t o be 2 0 times

as e f f e c t i v e w h e n

i n j e c t e d s u b c u t a n e o u s l y as w h e n a d m i n i s t e r e d

orally.

T h e y f u r t h e r r e p o r t e d th at

a d m i n i s t e r e d o r a l l y in d r y form
twice as

in a capsule,

effective as when suspended

or p h o s p h a t e

buffer

i o d i n a t e d casein, w h e n
was n e a r l y

in a s l i g h t l y a l k a l i n e

s o l u t i o n a n d g i v e n as a drench.

When

iod i n a t e d c a s e i n wa s a d m i n i s t e r e d d i r e c t l y

into the a b o m a s u m

of sheep,

increa s e

in o r d e r t o by p a s s the rumen,

z a t i o n was
spec ific

observed.

Thus,

in u t i l i ­

the r u m e n was e l i m i n a t e d as a

s i t e of d e s t r u c t i o n of i o d i n a t e d casein.

Reineke and Turner
of the m a m m a r y g l a n d to

(1944) a l s o

not f o u n d

in the mi lk

d o se s of

r e po r t e d that

of the t h y r o i d h o r m o n e w e r e

of l a c t a t i n g cows w h i c h h a d beer, f ed

i o d i n a t e d casein.

hor m on e a c t i v i t y by f e e d i n g
m i z e d g oats,

s t u d i e d the p e r m e a b i l i t y

iodinated casein and

b i o l o g i c a l l y d e t e c t a b l e am o u n t s

m as sive

no

The milk w as tested f o r

it to guinea pigs,

a n d h u m a n subjects.

t h yroidecto-

11
Ca mpbell et al

(l95o) c o n d u c t e d studies with iodinated

casein l a b e l l e d wi th r a d i o a c t i v e
was fed to l a c t a t i n g

ew9s,

iodine.

the s k i m m e d milk sh o we d a con­

siderable a m o u n t of radioactivity.
sociated w i t h the p r o t e i n portion,
able with butanol.

W h e n this mat erial

About 30 percent was a s ­
the remainder being extract-

T h i r t y p ercent of the a c t i v i t y in the

skimmed m i l k s h o w e d a similar s o l u b i l i t y in a l k a l i n e butanol
as thyroxine;
fraction.

it was,

a n d the plasma wa s f r a c t i o n a t e d 10 hours

57 percent

butanol.

c l a s s i f i e d as the thyroxine

W h e n l a b e l l e d i od i na t ed casein was a dm i ni s te r ed

orally to rats,
later,

therefore,

of the plasma was not e xtractable with

Campbell et al

may represent

(1950) s u g g e s t e d that this f r a c t i o n

the i o d i n a t e d p r o t e i n per se,

butanol s o l u b l e metabolite.

Wh en

or some n on ­

iodinated ca s ei n was in­

jected into the j u gular veins of sheep, 90 percent

of the

r a d i o a c ti v it y was r e m o v e d f r o m the bl o od stream in 7 hours.
As d e t e rm i ne d by r a d i o a c t i v i t y measur em e nt s of sheep blood,
there was

gr e at e r a b s o r p t i o n of io d in a t e d casein following

oral a d m i n i s t r a t i o n t h a n w h e n the material was a d m i n i s te r ed
into the v e nt r al sac of the rumen,

or small intestine; there

was p r a c t i c a l l y no a b s o r p t i o n from the cecum.
Io d i n a t i o n of a p r o t e i n causes changes

in its b io l og i ­

cal properties w hi c h a p p a r e n t l y are not r e lated to its
t hy r cx i ne — like activity.

Wormall

ological c h a r a c t e r i s t i c w h i c h was
proteins.

(19130) obse rved an immun­
specific for all iodinated

The a n t i g e n i c a l l y a c ti v e moiety of heavily iodinated

12
protei ns

is t h e i r d e t e r m i n a n t

iodinated part

acts

diiodotyrOsine

onl y as a carrier.

iodinated proteins h a v e

the

sa me

gr o up ;

Thus,

the n o n -

all h e a v i l y

or a very simi l a r antigenic

specifi c it y .
Another
b rominated,

characteristic
proteins

is

c i r c u l a t i n g pla sma.

of h e a v i l y

their

i o di n at e d,

as w e l l as

rapid disappearance from the

Fine a n d Seligman

(1943,

1944),

studying

halogenated bovine albumin

in dogs,

ical u p p e r

c o n t e n t f o r o b t a i n i n g the s l o w e s t

limit

of

iodine

rate of disappear'.nee
one m o l e c u l e

of

of the p r o t e i n

iodine

present

is 0.2

pe r c e n t ,

assuming

i n c o r p o r a t e d pe r p r o t e i n molecule.

Iodine-containing Compounds
Several

r e p o r t e d th at the t h e o r e t ­

iodine-containing

in i o d i n a t e d cas ein.

in I o d i n a t e d C a s e i n

compounds are

k n o w n to be

Ludwig a n d von Mutzenbecker

(1939)

i s o l a t e d t h y r o x i n e ,m o n o i o d o t y r o s i n e a n d d i i o d o t y r o -

sine.

In 1 9 4 8 P i t t - R i v e r s

hydroxybenzaldehyde.

On

r e p o r t e d the

the b as i s

on i o d i n a t e d c a s e i n h y i r o l y s a t e ,
identified the presence

of

of c h r o m a t o g r a p h i c

Hird and Trikojus

(1948)

of its p o s i t i o n b e t w e e n

t h y r o x in e .

Metabolism studies
indi cate th at

studies

diiodothyronine, and tentatively

identified triiodothyronine by virtue
diiodothyronine and

i s o l a t i o n of d i i o d o -

on rats

diiodothyronine

a c t i v i t y of t h y r o x i n e .

c o n d u c t e d b y G-add urn (1939)

possesses

More r e c e n t l y ,

(1952) h a v e r e p o r t e d that

synthetic

1 /1 5 the b i o l o g i c a l
Gross

a n d P i t t —R i v e r s

triiodothyronine

is 3 to

13
4 times a s a c t i v e as

L-thyroxine.

d u c t e d o n t h i o u r a c i l - t r e a t e d rats.

Their assay was

con­

EXPFFIMENTAL

The

Distribution

of

Iodinated Casein and a

Comparison with

The f i rst

in t h i s

Iodide

series

of

iodinated casein m e t a b o l i s m was
distribution and relative
ated casein
trations,
with

the

in v a r i o u s

determined

Wallace a n d
about the
water.

Brodie

same

concentrated
exceed the
greater

volume

therefore

definitely

concentrations

of

injected iodin­

of

t h e rat.

that

inorganic

Leblond

(1951),

stomach,

of

of

compared

sh o w n by

iodide occupies
the

extracellular

inorganic

iodide

is

in a m o u n t s w h i c h

extracellular

iodinated casein than

establish the

concen­

were

It w a s

and kidney

the

the

Iodide

experiment,

concentrations.

known capacity

concentration

reveal

in the o r g a n i s m as

in l iver,

dealing with

to

in a p a r a l l e l

( 19 3 7)

A c c o r d i n g to

experiments
designed

tissues

iodinated casein

i n t h e Rat

space.

Any

iodide w o u l d

c o n c e n t r a t i n g a b i l i t y of

the t i s s u e .
Female albino
Farms,

N e w C it y ,

rats which were

New

York,

were used

rat s w i t h a n a v e r a g e w e i g h t
venously w i t h
ated casein
of 185 g r a m s

0 . 1 ml.

(approx.
were

purchased from Carworth
experiment.

of 1 5 8 g r a m s w e r e

s o l u t i o n of
3 m g . ).

in the

Nine

i n j e c t e d by the

i o d i n e - 131

injected intra­
labelled iodin­

rats w i t h a n a v e r a g e
same

Ten

r o ut e w i t h

w e ig h t

0.1 ml.

of

15
radioactive
utes a f t e r

1 2
*

iodide.
the

B l o o d s a m p l e s w e r e t a k e n 100 m i n ­

injections,

at w h i c h

t im e the

rats w e r e

sacrificed.

P r e p a r a t i o n of R a d i o a c t i v e
The

Iodinated Casein

method described by Reineke

et al

(1943) was u s e d

w it h o n l y m i n o r m o d i f i c a t i o n s .
One h a l f gm.

of

casein

of s o d i u m b i c a r b o n a t e
were mixed

brass

ca p acity,

fitted snugly

a metal b u s h i n g w a s

vitamin-free),

(C.P.), a n d 18 ml.

in a 120 ml.,

a rub ber s to p p e r ,

(purified,

of d i s t i l l e d w a t e r

h e a v y g l a s s tube.

into the m o ut h of

i n s e r t e d to p e r m i t

at the

In a s e p a r a t e t u b e

the p a s s a g e

radioactive

crystal

drop of c o n c e n t r a t e d
iodide

(1-131)*.

duced a c o l o r

change,

r e l e a s e d f r o m the

iodine

(1-131)

i odide solution,

The

solution

by the

of p o t a s s i u m i o date a n d one

p h o s p h o r i c a c i d to about

A small

of a

end to f a c i l i t a t e stirring.

was p r e p a r e d f r o m a r a d i o a c t i v e
a d d i t i o n of a sm all

Through

the tube,

s t i r r i n g r o d o p e r a t e d by a n a i r - d r i v e n motor.

s ti rring r o d w a s be nt

1 gin.

1 ml.

of r ad i o

iodide c r y s t a l was a d d e d a n d p r o ­

i n d i c a t i n g that

the iodi ne h a d b e e n

iodide.

1 Purchased from Oak Ridge
Tenn.

National

Labo ratory,

O a k Ridge,

^ If the t i s s u e s are t o be c o u n t e d a d a y or t w o a f t e r the
rats a r e sa c ri f ie d , inject a b o u t 2 5 m i c r o c u r i e s per rat.
* T e n to 2 0 m i l l i c u r i e s of i o d i n e - 1 3 1 w e r e u s e d per p r e p a r a ­
tion.
This a m o u n t wa s h i g h f o r the d i s t r i b u t i o n experiment
but n e c e s s a r y for t h e m e t a b o l i s m experiment to be d e s c r i b e d

16
The t u b e c o n t a i n i n g th e c a s e i n w a s p l a c e d in a t h e r m o ­
statically c o n t r o l l e d constant

t e m p e r a t u r e w a t e r b a th a n d

the h e a t i n g u n i t a n d 3 t i r r i n g a p p a r a t u s
being set

t u r n e d on,

for m o d e r a t e l y r a p i d s t ir r i n g .

ture of t h e b a t h h a d r e a c h e d 40°
and the r a d i o a c t i v e
was a d d e d to

iodine

C.,

solution,

th e c a s e i n mixture.

W h e n the t e m p e r a ­

stirring was

a n d the

total v o l u m e
ml.

stopped

p r e v i o u s l y prep ared,

Th e t u b e w h i c h h a d c o n ­

t a i n e d t h e r a d i o - i o d i n e w as r i n s e d w i t h 1 ml.
water

the l at t er

of d i s t i l l e d

r i n s i n g s a d d e d to the c a s e i n mixture.

of w a t e r

in the r e a c t i o n m i x t u r e w a s about

S t i r r i n g w a s r e s u m e d for s e v e r a l

time 0.086 g m s . of

iodine

the c a s e i n mix ture.

(1-127)

d i a l y 2 ed a g a i n s t

mi n u t e s a f t e r w h i c h

c r y s t a l s w e r e a d d e d to

then placed

of 70° C.

'The i o d i n a t e d

in a c e l l o p h a n e

r u n n i n g ta p w a t e r for

Inj e ction a n d Ti s s u e

20

S t i r r i n g w as a g a i n r e s u m e d a n d c o n t i n ­

u e d for 19 h o u r s at a t e m p e r a t u r e
c asein s o l u t i o n w as

The

casing a n d

5 days.

Sampling Procedure

The m a t e r i a l s u n d e r

investigation were administered

i n t r a v e n o u s l y into a b r a n c h of t h e f e m o r a l vein.

The rats

were a n e s t h e t i z e d w i t h

e t h e r a n d the v e i n e x p o s e d by c u t t i n g

the skin o n th e

of t h e u p p e r part

i nside

of a h i n d leg.

i n j e c t i o n w a s u s u a l l y m a d e f ro m a 1 ml.

s yr i n g e w i t h a

three-quarter

n eedle bent

slight

curve.

inch,

26 gauge hypoaermic

F o l l o w i n g the

w i t h m et a l s k i n clips.
jection,

the

i njection,

The

in a

the w o u n d was c l o s e d

O n e h u n d r e d m inutes a f t e r the in­

rats w e r e a g a i n e t h e r i z e d a n d 1— 3 ml.

of b l o o d

17
was w i t h d r a w n f r o m t he

f e m o r a l v e in ,

usually with a 5 m l .

h e p a r i n i z e d s y r i n g e a n d a h a l f - i n c h 34 g a u g e h y p o d e r m i c
needle

bent

ficed w i t h
eration.

in a s l i g h t
ether,

The

b l o o d w a s u s e d to d e t e r m i n e t he

s p r e a d o v e r th e sur face,
room temperature,

of o t h e r t i s s u e s
lung,

small

( e mp t y or c l e a n e d ) ,

or cl e an e d) .

we ighing,

liver,

Wet w e i g h t

was

sp leen,

the

ti s s u e s w e r e

was

the

(empty

balance.**

After

o n the p l a n c h e t w i t h a
a n d s p r e a d o v er
i n s t r u m en t s

in s id e e d g e of the planchet.

tissue s a m p l e s w e r e t h e n p a r t i a l l y d r i e d at r o o m
for s e v e r a l hour s,

(cleaned),

i n te stine

A n y t i s s u e a d h e r i n g to th e c u t t i n g

r u b b e d off on to

included:

on a t o r s i o n typ e

si ng le e d g e d razor b l a d e a n d p i n c h forceps,
the surface.

stomach

a n d l a rg e

on a G r a m a t i c

cut u p

usually over­

kidney,

determined

in a t a r e d p l a n c h e t

by tilting

t a k e n f r o m the ra ts

glut eal m u s c l e ,
intestine

in a t a r e d

s t e e l p l a n c h e t , on a G ra m a t i o

a n d d r i e d at

Samples

or

then sacri­

in p a p e r a n d s t o r e d u n d e r r e f r i g ­

diameter

The b l o o d w a s

the p la n chet,

b al a n ce

rats w e r e

a n d a n o t h e r p o r t i o n w a s w e i g h e d w et

( in s id e )

balance.

night.

wrapped

One p o r t i o n of t h e

hematocrit*
1.9 cm.

curve.

a n d c o m p l e t e l y d r i e d in a n a i r

The

tempera t ur e ,
oven at

* He m a t o c r i t s w er e d e t e r m i n e d on the h e p a r i n i z e d b l o o d in
W i n t r o b e h e m a t o c r i t t ubes w h i c h w e r e c e n t r i f u g e d for 30
mi nu tes at 2 0 0 0 rpm.
** The w e t w e i g h t of b l o o d in the iodide e x p e r i m e n t was c o m ­
p u t e d f r o m th e d r y we i g h t .
T i s s u e wet w e i g h t s in the i o d i n ­
a t e d c a s e i n e x p e r i m e n t were c o m p u t e d f r o m the dry w e ig h t.
The p e r c e n t s o l i d i n e a c h case w a s d e t e r m i n e d e x p e r i m e n t a l ­
ly in rats f r o m the s a m e o r i g i n a l group.

18
90-95° C. for about

30 hours.

in a d e s i c c a t o r u n t i l

Th e d r i e d s amples were s t o r e d

t h e y w e r e rev/eighed to d e te rmine dry

weight.

Radioactivity Measurements
The r a d i o a c t i v i t y in t h e s am p l e s wa s m e a s u r e d with a
thin e n d - w i n d o w G e i g e r - M u l l e r tube

in a l e a d r s h i e l d a n d

record ed by a n e l e c t r o n i c

The r a d i o a c t i v i t y was

scaler.

comp uted as c ounts p e r s e c o n d a n d c o r r e c t e d for phy sical
decay of the

isotope,

self-absorption,

m ea sured by c o u n t i n g a standard.

a n d in strument

Counts per g r a m of p l a s m a

were c o m p u t e d f r o m the c ounts per g r a m of blood.
tations a n d c o r r e c t i o n s are
The

in order that
of di f ferent

The c o m p u ­

s h o w n in the appendix.

d a t a ar e p r e s e n t e d in terms
c o u n t s per s e c o n d
c ounts per s e c o n d

change

of

per g r a m
per g r a m

wet t i ssue
p la s ma

c o m p a r i s o n s might be made b e t w e e n the r es u lt s
experiments.

The data ar e s h o w n in table 1.

Results
Liver was f ou n d to c o n c e n t r a t e the gre atest amount
iod i nated c a s e i n per g r a m of tissue,
a n d p l a s m a b e i n g 5.55.
c a s e i n v e r y ef f e c t i v e l y ;

the ratio b et w e e n l i ve r

Th e sp l ee n a l s o c o n c e n t r a t e d i o di n a t e d
its ratio w a s

3.45.

The k idney h a d

the next h i g h e s t c o n c e n t r a t i o n w i t h a ratio of 1.72.
the t i ssues studied,
ratio of the

of

Of all

m u s c l e s h o w e d the lowest co n ce n t r a t i o n

i o d i n a t e d casein,

0.18.

ied, w i t h the ratios found, were:

The other tissues s t ud ­

stomach 0.89,

lung 0.85,

19
small i n t e s t i n e 0.54, a n d la rge
of iodide

i n t e s t i n e 0.38.

c o n c e n t r a t i o n ra ti o s s h o w s

est w i t h 0.50.

The l o w e s t

in o r d e r of

s p l e e n 0.44,

l i v e r 0.35,

of

ratios s h o w s

large

T he

other tissues

i n t e s t i n e 0.35,

a n d lu n g 0.29.

iodinated casein and
t h e i o d i n a t e d ca s e i n

t h a n iodide

in all t i s s u e s

of the f a ct

that

Bayliss,

0.09.

i o d i d e c o n c e n t r a t i o n ratio,

sm all i n t e s t i n e 0.30,
(figure 1)

the s t o m a c h to be h i g h ­

c o n c e n t r a t i o n r a t i o s w e r e f o u n d in

the muscle, 0.18 a n d c e c a l c o n t e n t s ,
studied,

A comparison

casein

were:

k i d n e y 0.33,

A graphic comparison

iodide c o n c e n t r a t i o n
to be more c o n c e n t r a t e d

studied,

exce pt muscle.

is a large m o l e c u l e

(mol.

In spite
w t . 188,000

19 4 3) w h i c h w o u l d not o r d i n a r i l y be e x p e c t e d to cr oss

cell m e m b r a n e s ,

t hese e x p e r i m e n t s

sh ow

that

it leaves the

blood s t r e a m q u i t e r a p i d l y a n d is c o n c e n t r a t e d p r i n c i p a l l y
in the liver.

The D i s t r i b u t i o n of S l i g h t l y I o d i n a t e d C a s e i n
From the results
c o n c l u d e d that

i o d i n a t e d casein,

t h y r o i d a l ac t iv i t y ,
in the rat;

of the p r e v i o u s

p r e p a r e d so as

rapidly concentrates

p a r t i c u l a r l y liver,

s ee m ed of i n t e r e s t

e xp e ri m en t

sp leen,

iodine

to have

in v ar i ou s ti s su e s
a n d kidney.

to f i n d out w h e t h e r or not

c o u l d be a t t r i b u t e d to the

it ca n be

content

It

this p r o p e r t y

of the protein.

The same p r o c e d u r e p r e v i o u s l y d e s c r i b e d for the st u dy of
i o d i n a t e d c a s e i n d i s t r i b u t i o n w as

c a r r i e d out

ment w i t h the f o l l o w i n g e x c e p t i o n s :

in this e x p e r i ­

nd i od i ne - 1 2 7 was a d d e d

20
T a b l e 1.

Concentration Ratios
and

of

Iodinated Casein

I odide

*T
Tissue
Gluteal

Iodinated Casein
muscle

Iodide

0.18

0. 18

0.68

0.29

5. 65

0. 35

Kidney

1 .7 2

0. 33

Spleen

3. 45

0.44

St o m a c h

0. 89

0 .5 0

Sma l l

i nt e s t i n e

0. 54

0.30

Large

i n te s t i n e

0.36

0. 35

Lung
Liver

i

Cecal contents

0.09

Con cen tr at ion Rat io

(cps per gra tissue/ cps per gm plasma)

FIG.

o

o

M

Y

h

V•r\

0
1

1.

Muscle

CONCENTRATION

xm iiim rm m

Liver

RATIOS

rrrr) T r u m . i ±
Kidney

IODINATED

Tissue

OF

Spleen

2Z2ZZ2SZS&&
Stomach

CASEIN

222Z2ZZ2ZZZZ22222ZZZZZ2ZZZZ

AND

Small intestine
/ I /
/// x

mzz

77IL

IODIDE

Large Intestine

LlU u

/

ua, ( ( / ( /./ / ((m

W
o
O Qt
p H00 3
OP
H- cf
3 CD
P-

M
o
Q*
H*
Q»
O

32
to the c a s e i n o t h e r
small c r y s t a l s

of

t h a n the

iodine

iodide a n d

(about 0 . 0 0 9 5 g . ) in t h e

iodate,

th e r a d i o i o d i n e s o l u t i o n

was n e u t r a l i z e d w i t h a m m o n i u m h y d r o x i d e to a p a l e
color b e f o r e
gm.

s od i um

being a d d e d

bicarbonate

a ve r a g e w e i g h t

to the c a s e i n mixtur e ,

only 0.135

w a s m i x e d w i t h the c a se i n,

a n d the

of th e r a t s w a s 2 5 3 grams.

The p r i n c i p a l d i f f e r e n c e
the

ye l lo w

b e t w e e n th is e x p e r i m e n t a n d

iodinated casein distribution study

tent of tne p r ot o in.
depict..a g r a p h i c a l l y

is the

iodine

con­

The d a t a are s h o w n in tabio 3 a n d
in f i g u r e 3.

Results
me
f ou n d

h i g h *81 c o n c e n t r a t i o n r atios

in the

s p l e e n a n d st o m a c h ,

^3.bo a nd 3 . 3 6 ;

respectively.

also h a d a h i g h c o n c e n t r a t i o n of 1.99.
c o n t e n t s w er e
0.3:f,

l o w e s t w i t h c o n c e n t r a t i o n r atios

respectively.

In the

of c o n c e n t r a t i o n w a s:
l u n g O.vi,

and small

o t h e r t issues

l i v e r 1.15,
i n te s ti n e

a n d the s l i g h t l y

large

and cecal
of 0.33

s t udied,
i nt e st i ne

be t w e e n the

and the d e c r e a s e d
iodinated

casein.

order

1.02,

is tho

in the s t o m a c h a n d lar ge

concentration

the

and

i o d i n a t e d ca s e i n

i o d i n a t e d c a s e i n d i e t r i b u t ions

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

kidney

0.63.

The mo st m a r k n d differonc.-s

slightly

Muscle

The

w^rv

In the liver w i t h

intestine,
the

33
T ab l e 3.

C o n c e ntration Ratios

of S l i g h t l y

iodinated Casein

Tissue

Concentration Ratio

eluteal muscle

0 .3 3

Lung

0. 7 1

Liver

1.1 5

Kidney

1. 99

Spleen

3.55

Stomach

3.36

Small

intestine

0.63

Large

intestine

1. 03

Cecal contents

0. 35

H
P

to

Muscle

CONCENTRATION

Lung

Liver

OF

Tissue

RATIOS

Kidney

SLIG-HTLY

Spleen

Stomach
IODINATED

Small intestine

CASEIN

Large intestine

Cecal contents

35
The M e t a b o l i s m of I o d i n a t e d Ca s e i n in the Rat
Intravenous A d m i n i s t r a t i o n
The m e t a b o l i c b r e a k d o w n of i n j e c t e d
in the rat w a s

i o di n at e d c as e in

s tudied w i t h a v i e w toward

c h a r a c t e r i z i n g the

metabolites a n d d e t e r m i n i n g their r e l a t i v e c o n c e n t r a t i o n s over
a time series.

If the

i o di n at e d c a s e i n w a s m e t a b o l i z e d w i t h

the release of t h y r o x i n e - l i k e io d in e
the thyroidal a c t i v i t y
hand,

if the

of i o di nated

this could a c c o u n t for
casein.

On the other

io d in a t e d p r o t e i n w as m e t a b o l i z e d w i t h o u t the

release of t h y r o x i n e - l i k e

iodine

the

Inference w o u l d be that

thyroxine can ex ert its ho r mo n al a c t i v i t y w h i l e

co m bi n ed in

the protein.
Fema le a l b i n o ra t s w h i c h w e r e p u r c h a s e d f r o m Ro c kl a n d
Farms we r e us e d

in the

experiment.

Four groups

each, w e i g h i n g a b o u t 230 g rams p e r rat, w e re
and inj ected i n t r a v e n o u s l y w it h 0.5 ml.
ated casein s o lu t io n

thyroidectomized

of r a d i o a c t i v e lodin-

( ap p r o x i m a t e l y 10 m g . ).

th yroidectomized in o r d e r to p r e v e n t

of two rats

The rats w e re

the f o r m a t i o n of r a d i o

thyroxine by the thyroid g l a n d , f ro m iodine r e l e a s e d as the
iodinated ca s ei n was metabolized.

Blood and liver samples

were taken from each of two rats at four time periods:
12,

zk,

and 72 hours a f t e r injection.

tionated and c o m p u t a t i o n s c a rried out

The samples w e r e f r a c ­
so that the fractions

could be e x p r e s s e d a s free t h yr o x i n e - l i k e
combined t h y r o x i n e - l i k e

iodine.

1-&,

iodine,

and protein

26
Pr eparation of R a d i o a c t i v e I o d i n a t e d C a se i n
The p r o c e d u r e d e s c r i b e d in the first e x p e r i m e n t w a s
used w i t h the f o l l o w i n g m i n o r m o d i f i c a t i o n s :
iodine s o l u t i o n
ium h y d r o x i d e

(approx.

to a p a l e

the casein m i xt u r e ,
Was

and

the r a d i o ­

15 me. ) w a s n e u t r a l i z e d w i t h a m m o n ­
y e l l o w c olor b ef o re b e i n g ad d ed
only 0.125 gm.

to

of so d iu m b i c a r b o n a t e

mixed w i t h the casein.

Rat T h y r o i d e c t o m y P r o c e d u r e
The rat w a s p a r t i a l l y a n e s t h e t i z e d w it h 1 ml.
percent s o d i u m p e n t a b a r b i t a l
perltoneally.

It w e e

(nembutal),

calcium gluconate was

inject e d

immediately b e f o r e the operat ion,

operation.

The

the trac hea and

as needed,

F o u r - t e n t h s ml.

of

subcutaneous!y,

to pr e v e n t

the p a r a t h y r o i d s are r e m o v e d w i t h the
ether cone w a s used,

intra-

then fully a n e s t h e t i z e d w i t h ether and

tied on its b a c k to a w o o d e n rat board.
10 perc ent

In j e c t e d

of 0.6

tetany,

thyroid gland.

b ecause
An

d u r i n g the c ourse of the

skin w a s cut p a r a l l e l

to and di r ectly over

the l o n g i t u d i n a l m u s c l e s and skin r et r ac t ed

on both sides w i t h two h o o k s made f r o m w ir e p a p e r clips.
Tens ion w a s m a i n t a i n e d w i t h rubber ban ds at t a c h e d to the
sides of the rat board.

The ex p o s e d thyroid gland was teased

from the t r a c h e a a n d b l o o d v es s el s w i t h a h y po d e r m i c needle
ana p in c h forceps.
bleeding?.

W h en

moved a n d the

Small wa ds of c otton were used to control

the b l e e d i n g had stopped,

the cotton was r e ­

skin i n c i s i o n closed w i t h metal

skin clips.

27
The t h y r o p a r a t h y r o i d e c t o m i z e d r a t s w e r e
maintained

on a st a n d a r d d ie t

tion of 1 p e r c e n t

( a p p e n d i x X)

c a l c i u m carbonate.

subcutaneous I n j e c t i o n s

of 10 p e r c e n t

or three t imes e a c h day

for

Injection a n d T is s ue
The i o d i n a t e d
usually b e f o r e

subsequently
with

They w e r e

the a d d i ­

given O.h ml.

c a l c i u m g l uc o na t e

two

several days.

Sampling Procedures

c a s e i n w as

the rat

injected

into the f em o ra l vein,

came out f r o m un d er

the a n e s t h e s i a

a d m i n i s t e r e d d u r i n g the t h y r o p a r a t h y r o i d e c t o m y .

Collection of Bl o od an d L i v e r S a mp l es
At the a p p r o p r i a t e

time the r at s were a n e s t h e t i z e d w i t h

sodium p e n t a b a r b i t a l an d e th e r and

tied to the rat board.

Blood sa m p l e s of ab o ut 5 ml. w e r e w i t h d r a w n

from the a b d o mi n al

aorta, u s u a l l y w i t h a c urved one an d o ne - ha l f inch 20 gauge
hypodermic n e e d l e and a l n ml.
blood w a s c e n t r i f u g e d for
separate

syringe.

The

?>bout 30 m in u t e s at 2000 rp m to

the plasma.

Following
was

heparlnized

the w i t h d r a w a l of the blood

s a c r i fi c ed and a li v er

about 1 gm.

w a s removed.

ml. p y r e x c e n t r i f u g e

sample of

a size es t im a t e d

to w e i g h

This was pl a c e d in a tared 12 or 15

tube and weighed.

was under r e f r i ge r at i on .

sample , the rat

An y storage necessary

IODINATED CASEIN

I n t r a v e n o u s or
oral administration
PLASMA OR LIVER
SAMP LE

BUTANOL
EXTRACT

RESIDUE

Alkali was h
(4N N a O H 4
N a 2 CO^)

Hydrolysis
(2 N NaOH)
BUTANOL
EXTRACT

BUTANOL
PHASE

i

FREE T H Y R O X I N E - L I K E I O D I N E
Alkali wash
(*4N N a O H 4
NapCO^)
BUTANOL
PH ASE

1
C O M B I N E D T H Y R O X I N E - L I K E IODI NE

FIG.

3.

F R A C T I O N A T I O N OF

P L A S M A A N D L I V E R SA M PL E S

29
F r a c t i o n a t i o n of P l a s m a an d L i v e r

Samples*

E x t r a c t i o n of u n h y d r o l v z e d p l a s m a .
was t r a n s f e r r e d
ml.

to a n o t h e r

of b u t a n o l ea ch time.

tube and

One ml.

extracted

of the p l a s m a

twice w i t h 2

At e a ch e x t r a c t i o n the m i x t u r e w a s

stirred f o r 2 m i n u t e s w i t h a ? l as s rod,

s t o p p e r e d an d a l l o w e d

to settle f or 20 or m o r e m i n u t e s at r o o m temper a tu r e.
alcoholic e x t r a c t w a s

s e p a r a t e d by

for 10 m i n u t e s at 2000 rpm.
into a n o t h e r tube and
inside d i a m e t e r ,
temperature
pump,

^

The e n t i r e

ml.

was

ni c ke l plated

ing extract w a s

planchet,

trap,

d r i e d at r o o m

Three

m3,

stopper

the c e n t r i f u g e

The

The li v er w a s m a s h e d
taking several

for 2 minutes.

The

s u s p e n s i o n wa s

s topper w a s r e m o v e d and

Into a n o t h e r

residue e x t r a c t e d a second
combined,

O n e - h a l f ml.

centrifuge

time w i t h 2 ml.

stoppered,
of

and

soaked

in

s ha k en v i g o r ­
the tube

spun in

The a l c o h o l i c
tube a nd the liver
of butahol.

Bo th

stored u nd e r r e f r i g ­

the c o m b i n e d e xt r ac t s wa s

*A s ch e m at ic r e p r e s e n t a t i o n

the

tube w as cl o se d

which had previously been

for 10 m i n u t e s at 20 0 0 rpm.

extract w a s p o u r e d

extracts w e r e

liver.

the p r o c e s s

butanol an d w i p e d d r y , and the
ously 24 times.

The r e m a i n ­

of b u t a n o l w e r e a d d e d a n d m i x e d w i t h

the glass rod

with a r u b b e r

and counted.

stored u n de r refrige r at i on .

in the tube w i t h a g l a s s rod,

eration.

e xt r ac t w a s d e c a n t e d

t r a n s f e r r e d to a 2. 5 c m . ,

E x t r a c t i o n of u n h y d r o l y z e d

liver w i t h

the m i x t u r e

in a d e s i c c a t o r w i t h the a i d of a h i g h va c uu m

and a dry l c e - a c e t o n e

minutes.

centrifuging

The

is s hown in fi gu re 3*

t ra n sf e rr e d

30
to a p la n c h e t ,

d r i e d as d e s c r i b e d e a r l i e r ,

twice-extracted

and counted.

l iver r e s i d u e w a s d r i e d in the

tube at r o o m t e m p e r a t u r e

as a l r e a d y

The

-

centrifuge

described and

saved for

further treatment.

H yd r ol y si s of
Five ml.

the L i v e r a n d F r a c t i o n a t i o n

of the H y d r o l y s a t e

of 2N s o d i u m h y d r o x i d e w e r e s.dded to the dry

liver r e s i d u e

in the c e n t r i f u g e

tube,

an a i r - c o o l e d r ef l ux

condenser w a s a t t a c h e d a nd the tube p l a c e d in a b o i l i n g w a t e r
bath

(approx.

cooled to r o o m

9 9° C . ) f o r

15 hours.

The h y d r o l y s a t e was

t e m p e r a t u r e a n d its p H a d j u s t e d

to a p p r o x i m a t e l y

3.5 by the d r o p w i s e a d d i t i o n of 6N su l fu r ic acid;

B r o m Cres ol

Ireen w a s u s e d as an i n t e r n a l indicator.

The h y d r o l y s a t e w a s

3 ml.

for e a c h e xtraction,

then e x t r a c t e d twice w i t h bu tanol,
by mixing,
half ml.

of

s h ak i ng and

c en trlfugirg as a l r e a d y describ ed.

the c o m b i n e d

chet, d r i e d as d e s c r i b e d

One-

extracts was

t r a n s f e r r e d to a p l a n ­

earlier,

counted.

of the c o m b i n e d e x t r a c t s w a s

and

The r e m a i n d e r

s t o r e d u n d e r r e fr i ge r at i on .

A lk aline W a s h i n g of B u t a n o l E x t r a c t s

( H y d r o l y z e d and

Unhydrolyzed)
T hr e e ml.
an other

of u n w a s h e d b u t a n o l e xtract w a s

tube a n d e x t r a c t e d

tion c o n s i s t i n g of 4N
carbonate.

twice w i t h 3 ®1»

t ra n s f e r r e d

v o l u m e s of a s o l u ­

s od i u m h y d r o x i d e and 5 p e r c e n t

Each extraction involved

c e n t r i f u g i n g 10 m i n u t e s at

2000 rpm.

tion w a s r e m o v e d w i t h a t ap e re d g l a s s

sh aking

to

2k

sodium

times,

and

The a l k a l i n e w a s h sol u­
tube, a t t a c h e d

to a

31
gr eased 5 ml.

syringe w ith rubber

the t w i c e - e x t r a c t e d b u t a n o l w a s
dried as d e s c r i b e d e a r l i e r ,

tubing.

O n e - h a l f ml.

transferred

of

to a pl a nc h et ,

a n d counted.

Results
The d a t a are p r e s e n t e d
cally In f i g u r e s 4-7.

in t able 3 and d e p i c t e d g r a p h i ­

The b a s i c d a t a are

shown In a p p e n d i x

IV.
Combined
In figure 4,

t h y r o x i n e - like Iodine
combined

in the p l a s m a .

thyroxlne-like

cleared f r o m the plasma;

b e t w e e n the

iodine wa s r a p i d l y
!■£• hr.

intervals there w a s a 99 p e r c e n t d ec r ea s e,
Interval on l y a very

sm a ll a m o u n t

As s h o w n

and

24 hr.

time

and by the 72-hr.

of t h y r o x l n e - l i k e

iodine

was found in the plasma.
Fr ee

thyroxlne- like

like Iodine

Iodi ne in p l a s m a .

concentrations

in the p l a s m a

only a 21 p e r c e n t d e c r e a s e b e t w e e n
vals.

A large decrease,

and 72 hours.
lir a n d 24 h o u r s
that free

(Figure 5)

p lasma fr o m some o ther

showed

constant

o c c u r r e d b e t w e e n 24
concentration between

and the large d ro p a f t e r 24 h o u r s

thyroxine-llke

thyroxlne-

the 1-jj? and 2 4 h o ur i n t e r ­

of 87 p e r c e n t ,

The r e l a t i v e l y

The free

Indicates

Iodine wa s b e i n g r e l e a s e d

to the

tissue or t i s s u e s and the am o u n t

sup­

pl ied d e c r e a s e d and f e l l b e l o w the pl a s m a turnover.
Percent
like Iodine.,

free
in

t h y r o x l n e - 111e

the p l a s m a.

iodine Is c o m p u t e d by m e a n s

1 odlne of the

The p e r c e n t

total t hy r ox l ne -

free t h y r o x l n e - l i k e

of the f o l l o w i n g r el ationship:

free
free

t h y r o x l n e - l i k e I o d i n e ______________
t h y r o x i n e - l i k e i o d i n e -f co m b i n e d
t h y r o x l n e - l i k e iodine

As shown in F i g u r e
proportion of
was free.

6, at

the t o t a l

the l £ h o u r

7 percent a t l£ h o u r s

24- and 7 2 - h o u r

intervals.

t h y r o x l n e - like
increase

bined t h y r o x i n e - l i k e
vale

(Figure

7).

bined t h y r o x l n e - l i k e

not equal

The
the

t ur n o v e r ,

the

only

of c o m ­

the 1-^-and 2 4-hour

inter­

there was a 91 p er c en t

72 hours.
i n di c a t e s

that

iodine had been

supply

This hi gh

There w a s

in the li ver c o n c e n t r a t i o n s

Cn the other hand,

sharp d e c l i n e

decreased.

iri l i v e r .

io d i n e be t we en

decrease b e t w e e n 24 a n d
The

i odine rose from

iodine was m a i n t a i n e d at

iodine

small

in the p lasma

to 75 p e r c e n t at 12 hours.

thyroxlne-like

an 18 p e r c e n t

iodine

thyroxine-like

percent of f r ee

Com b in e d

time in t e r v a l a v e r y

thyroxine-like

The p e r c e n t f r ee

x 100

of c om b in e d

the rate at w h i c h c o m ­
supplied

to the liver

thyroxine-like

an d the liver

iodine did

c o n c e n t r a t i o n s could

not be m a i n t a i n e d at the l ev el of the l£- and 2 4 - h o u r i n t e r ­
vals.
Oral A d m i n i s t r a t i o n
A recent report
io dine-131

labelled

on the a b s o r p t i o n
i o d i n a t e d c as e i n

indicates that a large
alimentary

tract

a mo u n t

of rats and

c h a r a c t e r i s t i c s of

(Campbell et al,

of the iodine a b s o r b e d from the
sheep is combined as

casein or some b u t a n o l - i n s o l u b l e metabolite;
a d m i n i s t r a t i o n a b ou t
insoluble.

1950)

iodinated

10 hours after oral

50 ,^ of the p l a s m a r a d i o a c t i v i t y is butanol-

33
T a b l e 3.

M e t a b o l i s m of I o di n a t e d

Casein,

Intravenous Administration

Time
(hr. )

1*

Rat
No.

c.p.s. per ml. p lasma
Free T h y r o x i n e Combined Thyroxinelike Iodi ne
like Iodlne

1
2

20.9
3^.0
27 . 4

av.
24

72

5
6

30.5
12.7
av.
21.6
d e c .# 21.1
2.64
3.20
2.92
86.5

7
8
av.
dec.

av.

407.6
310.9
359.2

av.
dec.

3.68
3.76
3.72
98.9

av.
dec.

0. 80
0. 80
0. 80
78.4

c. p. s . p e r erm. liver
li

1
2
av.

24

72

5
6

66. 9
58.1
62.5

av.
dec.

49. 6
38.9
44. 2
29.2

av.
dec.

12.9
11. 8
12.3
72.1

4

7
8

197.5

231.0
av.

214.2

300 .0
206. 7
a v . 253.3
i n c r . 18.2

25.2
22. 0
av.
dec.

* Percent d e c r e a s e fr o m p re v io u s a v erage count.

23.6
90.6

10

-

Plasma

Radioactivity

(cps

per

ml.

100 -

1.0

0.1

-

k
5
Rat No1
2
3
Time (hr. )
li
2k
72
FIG. k . COMBINED THYROXINE-LIKE IODINE IN PLASMA

per
(cps
Radioactivity

-

Plasma

10

1- ,
Time ( h r . )
Rat No.
FIG. 5.

2

2h

3
4
6
7
F REE T H Y R O X I N E - L I K E IODINE IN PLASMA

60

-

50-

c
o
o 40<D
CL.

30-

20

-

10^

Rat No.
1
Time (hr. )
FIG. 6.

100

-

E
br;
U
a>
ft

co
ft
o
•H
>
■H
O

OS

Sio

T3

aS

PC

u

0)
>

1 ~{

ft

Time (hr. )
l£
2^
72
Hat No.
1
2
3
^
5
6
FIG. 7.
C O M B I N E D T H I R O X I N E - L I K E IODINE IN L I V E R

It se e m e d

of i n t e r e s t

fraction a nd d e t e r m i n e
interval a f t e r

to f u r t h e r

the r e l a t i o n s h i p b e t w e e n

oral a d m i n i s t r a t i o n

of i o d i n a t e d

the c o n c e n t r a t i o n of t h y r o x l n e - l i k e
The e x p e r i m e n t w a s

tions:

the time
ca s e i n and

iodine in the plasma.

each.

The m e t h o d s of

e x p e r i m e n t w e r e u s e d w i t h the f o l l o w i n g e x c e p ­

Two ml.

volumes

of i o d i n a t e d

c as ei n w e r e a d m i n i s t e r e d

by stomach tube w i t h the a i d of a p l a s t i c
a hypodermic

this

c o n d u c t e d on 3 c r o u p s of 2 male

albino r a t s w e i g h i n g a b o u t 3 37 g r a m s
the p r e c e d i n g

characterize

syring e,

the b lood

tube

s a mp l es w e r e

connected

to

ta k en at 1-jjr, 12

and ^8 hours.
Results.
ment

(table

k)

Although

the i n d i v i d u a l r e s u l t s

were generally

erratic,

that the b u t a n o l - i n s o l u b l e m a t e r i a l
after oral a d m i n i s t r a t i o n
thyroxine-like

iodine

The p e r c e n t

free

means of the f o l l o w i n g

they

8 shows

iodine

in the p l asma,

the free,

suggest

e n t e r i n g the b lo o d

of i o d i n a t e d

stream

casein c o n t a i n s

in c o m b i n e d form.
thyroxlne-like

iodine w a s

co mputed by

relationship,

________ free t h y r o x l n e - l i k e iodine_____
free t h y r o x l n e - l i k e iodine + c o m b i n e d
t h y r o x i n e - l i k e iodine
Figure

of the e x p e r i ­

that more

than 50 p e r c e n t

at the time

butanol-soluble,

form.

,

of the t h y r o x l n e - l i k e

intervals

studied, w a s in

39

Tab l e 4.

M e t a b o l i s m of I o d i n a t e d Casein,
Cral Administration

c.p.s. per ml. P l a s m a __________
F r e e 'fhyroxineC o mb i ne d T h y r o x i n e li ke Io d i n e
like Iodine

Time
(hr.)

Rat
No.

i£

1
2

14. 4

1.84
5.52

3
4

24.1
17. 6

3. 28

12
48

5
6

2.32

3*60
2* 08

3.60
0. 96
1. 12

Time ( h r . )
1^
12
^8
FIG. 8.
P E R C E N T F R E E T H Y R O X I N E - L I K E I O D I N E OF THE
T O T A L T H Y R O X I N E - L I K E IO D I N E IN P L A S M A

^1
The D i s a p p e a r a n c e
and

of I o d i n a t e d

the A p p e a r a n c e

C a s e i n f ro m P l a s m a

of Its M e t a b o l i c

Products

in the U r i n e , In the Dog
The d e c r e a s e s
dog, f o l l o w i n g

the i n t r a v e n o u s

iodinated c a s e i n ,
influence

In p l a s m a an d u r i n e r a d i o a c t i v i t y

were

administration

compared

of p l a s m a i o d i n a t e d

in o rd er

casein

in the

of r a d i o a c t i v e

to d e t e r m i n e the

c o n c e n t r a t i o n on u r in e

radioactivity.
The r e l a t i v e

molecular

a pp e ar i ng in d o g u r i n e
of r a d i o a c t i v e

Immediately

iodinated

the urine r a d i o a c t i v i t y
Decrease

si7,e of the r a d i o a c t i v e m a t e r i a l
following

the

injection

c asein was d e t e r m i n e d by d e t e r m i n i n g
b e f o r e and a f t e r dialysis.

in P l a s m a and U r i n e R a d i o a c t i v i t y

A male

do g w a s u s e d

urine r a d i o a c t i v i t y .

to c o m p a r e

decreases

in p l a s m a and

It w a s kept u n d e r a n e s t h e s i a by the

i n t r a v e n o u s a d m i n i s t r a t i o n of 3 p e r c e n t

sodium p e n t o b a r b i t a l ,

as needed.
Radioactive

i o d i n a t e d c a s e i n w a s p r e p a r e d by

cedure d e s c r i b e d
ment,
ml.

except

(approx.

in the i o d i n a t e d c a s e i n d i s t r i b u t i o n e x p e r i ­

that

sa m p l e s

ized test

tu b e s

the

hu b

the p e r i o d of d i a l y s i s w a s 52 hours.

10 rag.) w a s a d m i n i s t e r e d

and blood

nula was m a d e

the p r o ­

of a b o u t

10 ml.

were

f r om the c a n n u l a t e d

into the

fem oral vein

c o l l e c t e d in h ep a ri n -

c a ro t id artery.

fr o m a 16 g a u g e h y p o d e r m i c n eedle

h a d b e e n r e m o v e d w i t h wi r e

Cne

cutter s.

The c a n ­

fro m w h ic h
The

straight

k2

p or tion w a s

extended

several

cm.

by m e a n s of t i g h t - f i t t i n g

plastic t u b i n g a n d p l u g g e d w i t h a w i r e
the d i s t a n c e b e t w e e n

the m o u t h

s li g ht l y

of the p l a s t i c

longer than

e x t e n s i o n and

the point of the h y p o d e r m i c needle.
At the a p p r o p r i a t e
removed a n d a b l o o d

time

sample

i n t e r v a l s the w i r e p l u g was

taken.

The p l a s m a w a s

se parated

by c e n t r i f u g a t i o n , an d r a d i o a c t i v i t y m e a s u r e m e n t s w e r e made
on ml.

portions with a

tube and a

scaler.

The

s h ie l d e d t h in e n d - w l n d o w G e i g e r - M u l l e r
time p e r i o d s

studied a n d

the p e r c e n t ­

ages of the r a d i o a c t i v i t y at the f i r s t pe r i o d are

shown in

table 5 .
Urine

samples were

ureter w i t h

the aid

measurements

p ercentage

of a p l a s t i c

of 1-J- ml.

Just described.

collected directly from

the ri g ht

c a t h e t e r and r a d i o a c t i v i t y

p o r t i o n s w e r e made w i t h

The d a t a are s ho wn

the a p p a r a t u s

in table 6 in terms of

of r a d i o a c t i v i t y at the first

interval.

R es u lt s
Intravenously administered radioactive
leaves

i o d i n a t e d ca s e i n

the b l o o d p l a s m a at an e x t r e m e l y r ap i d rate.

When

co mpared w i t h the b l o o d p l a s m a r a d i o a c t i v i t y at 1 minute,
per cent was

c l e a r e d f r o m the p l a s m a by the 17 - mi n u t e

After the f i r s t large d e cr e as e ,
at a m uc h sl o we r rate.
intervals

i nt erval

fur ther c l e a r a n c e occurred

B e t w e e n the 17-ml nu t e and 3 ^ 2 — minute

there was only a 6 p e r c e n t

plasma r a d i o a c t i v i t y .

83

further r e d u c t i o n in

*43

Table

5.

Decrease

Time after
admin, ( m i n . )
1

in B l o o d P l a s m a R a d i o a c t i v i t y

c.p.s.
per ml.
25 .62

P e rcent of
first s ample

100

17

*4-. *4-0

17.1

32

3.92

15.3

*47

*4. 1*4

16.1

62

*4.27

1 6 .6

92

3.99

15.5

122

*4.05

1 5.8

152

3.76

1*4.6

182

3.62

1*4. 1

212

3. *41

13.3

2*4-2

3.31

12. 9

362

2.75

10.7

44

T ab l e

6.

Collection
p er i od (min. )

Decrease

in U r i n e R a d i o a c t i v i t y

Av. Time
(min.)

c.p.s.
p e r ml.

P e r c e n t of
first sample

0-16

8

6.89

100

16-46

31

•6.35

46-?6

61

7.01

76-106

91

5.6 7

82.2

106-136

121

4.93

71.5

136-166

151

3.8 1

55.2

166-196

181

2.96

4 2. 9

196-226

211

2.32

33.6

226-256

241

2.10

30 .4

256-376

316

1.49

21.6

37 6- 4 9 6

436

2.20

31.9

92.1

101

^5
The d e c r e a s e

In u r i n e r a d i o a c t i v i t y o c c u r r e d

the r a d i o a c t i v i t y at ^36 m i n u t e
was 32 p e r c e n t
The d a t a

that

suggest

in the k i d n e y
and e x c r e t e d

of

during

at
t ha t

( a v . ) collection period

the 8 - m i n u t e period.
lodinated

(femoral v e i n ) ,

first e x p e r i me n t.

by

fro m the blood

dog was

in jected,

plasma

of l o d l n e - 1 3 1

Twenty-five

tap w a t e r

s t o r e d u n de r r e f r i g e r a t i o n .
the r a d i o a c t i v i t y

the u n d l a l y z e d urine,

is t h e r e f o r e

concluded

for 3 days,

the

of

the d ia lyzed

passes

the same for both the

900 counts p e r minute.

It

th at most or all of the r a d i o a c t i v e
into

lodinated

the u r i n e
casein,

i m m e d i a t e l y f o ll o wi n g

is either

p ro t e i n or a large u n d i a l y z a b l e metabolite.
eliminate f re e

in a ce l lo p ha n e

of the u n d l a l y z e d urine.

d ia l yz e d a n d

the I n j e c t i o n of

The u rine

A r a t e m e t e r was then

of 6 ml.

The r a d i o a c t i v i t y w a s

which

syringe.

one w a s p l a c e d

casing and d i a l y z e d asrainst r u n n i n g

Results.

in the

d o e w a s d r a i n e d by means

to a h y p o d e r m i c

in t o two po r ti o ns ;

urine w i t h 6 ml.

l o d i n a t e d ca s ei n

m i n u t e s a f te r the injection,

of the u n a n e s t h e t i z e d

used to c o m p a r e

intravenously

the m e t h o d a l r e a d y d e s c r i b e d

of a c a t h e t e r a t t a c h e d

material,

concentrated

in U r i n e

w i t h 9 ml.

solution p r e p a r e d

other w a s

case in w a s

the r a p i d c l e a r a n c e

A 19-kllogram male

was d i v i d e d

slowly;

i n t o the u r i n e at a s l ow e r rate.

Radioactive Material

the b l ad d er

more

the u n a l t e r e d
T hi s w o u l d

t h y r o x i n e and d i l o d o t y r o s i n e as possibilities.

DISCUSSION
The f i n d i n g
administered

of R e i n e k e

iddlnated

et al

(19^2)

that p a r e n t a l l y

casein exhibits hormonal activity

led to the q u e s t i o n of its mo de of action;
b eg i nn i ng of th is paper:
exert a b i o l o g i c a l
release

of

the

the

lo d in a t e d p r o t e i n per se

e f f e c t or is it first m e t a b o l i z e d w i t h the

t hy r ox i ne ?

According
herein,

Does

as stated in the

to the r e s u l t s of the e x p e r i m e n t s r e p o r t e d

thyroidal

gent on the m e t a b o l i c

effect

of l o d i n a t e d

casein

is c o n t i n ­

s p l i t t i n g off of thyroxine.

evidence is not p r e s e n t e d
not h o r m o n a l l y effective.
re sults p r e s e n t e d , that

Direct

that l o d i n a t e d p r o t e i n per se is
How ever,

it is clear,

thyroxine-like

iodine

f ro m the

is h y d r o l y z e d

fro m l o d i n a t e d c a s e i n in the liver and re l e a s e d

to the plasma.

That

the p r o t e i n

per

synthetic

t h y r o p r o t e i n acts twice;

s e , and a g a i n as fr e e

once as

thyroxine, w o u l d a p p e a r

to be h i g h ­

ly Im probable.
F ig u re

1 shows the c o n c e n t r a t i o n of l o d i n at e d casein In

v ar i ou s t i s s u e s

of the rat 100 m i n u t e s after its i nt r a­

veno us a d m i n i s t r a t i o n .
in the liver,

wh e re

The h i g h e s t

c o n c e n t r a t i o n w a s found

It was more than 10 times

iodide w h i c h h a d b e e n a d m i n i s t e r e d
tratio ns of l o d i n a t e d

similarly.

that of sodium
Higher c o n c e n ­

c a s e i n than Iodide we r e also found in

k?

the spleen,

kidney,

concentration
tine was

lunsr,

stomach,

of i o d i n a t e d c a s e i n

similar

to iodide.

io dinated c a s e i n

in liver,

w it h a s i m i l a r r e p o r t ,

and

sm all Intestine.

in m u s c l e a n d

The

large i n t e s ­

The hicrh c o n c e n t r a t i o n s of
k idney,

and

by H a u r o w i t z

spl een is in accord

and C r a m p t o n

(1952),

on

lodinated ovalbum i n.
Li ver

c o n c e n t r a t e s a l ar^e p r o p o r t i o n of I n je c t e d i o d i n ­

ated ca s ei n d ue

to its

effective

its large

1] gm.

in a 200

size,

In view of
is k no w n

these

to be an i m p o r t a n t

site of i o d i n a t e d

a c o m p a r i s o n of

casein an d

its

of r e l a t i v e

tu r no v er rates.

turnover,

i o d i n a t e d ca sein,

like iodi ne
c en t ra t i o n
vals.

(Fig.

4),

the ra te

of

and pe r mi t

The r e s u l t s

show

t h yr o xi n e in the

of l o d i n a t e d c a se i n supplied
its

will be m a i n t a i n e d at a c on s t a n t
m in i st e re d

in liver and

In te r dependent.

the q u a n t i t y
the

of lodinated

in d icate

to thyro xine,

of i o d i n a t e d p r o t e i n an d

the liver e q u a l s

the fact that liv er

iodine metabolite

ca s ei n

liver and b l o o d p l a s m a are
As loner a s

192*0.

ca s ei n metabolism.

time I n t e r v a l s wo u l d

of i o d i n a t e d

the l e v e l s

(D onaldson,

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

thyroxine-like

plasma at d i f f e r e n t

that

r at

and

in p r o t e i n m e t a b o l i s m it w a s a s su m ed

Therefore,

e s t i m a ti o ns

vm.

c o n s i d e r a t i o n s as w e l l as

to take p a r t

h yd r ol y si s

c o n c e n t r a t i n g ability,

to

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

level.

Intravenously a d ­

r e p r e s e n t e d by combined

th yroxine

s howed a 99 p e r c e n t d e cr e a s e in c o n­

in the pit. sma b e t w e e n the li^— and 2*+— hou r in t er ­

Since

l i ve r r e a d i l y

c o n c e n t r a t e s i o d i n a t e d casein

48
(Fig.

1),

it

is p r e s u m e d

the blo od p l a s m a
fact that

the

(Fig.

l e v e l of

was s u p p l i e d

t h at
to

and the l i v e r

s ee n

that there w a s a

24-hour interva l s.

the t u r n o v e r rate,

is

percent decrease

in the p l a s m a b e t w e e n

c asein,

decrea s ed .

d ecrease in the p l a s m a
A recent report

the

in i o d i n ­
l£- a nd

li v er c o n ­

the a m o u n t of t h y r o x i n e f r e e d

Th is w a s

f o l l o w e d by an 87 p e r c e n t

l e v e l of f r e e

by

That

shown in f i g u r e 3, w h e r e

As a r e s u l t of the d e c r e a s e d

c e n t r a ti o n of i o d i n a t e d
to the p l a s m a

99

thyroxine

C a m p b e l l et al

(1950)

(Fig.

the a l i m e n t a r y

is a b s o r b e d

tract of r a t s and

zation of t h i s b u t a n o l - i n s o l u b l e
1^-, 1 2 , a n d 48 hours.
tion c o n t a i n s
was a l s o s h o w n

(Figure

t h y r o x i n e — like

i od i ne

studied, w a s

8)

sheep.

of it,

A further characteri­

fraction was
I nd i cated

i odine

that mo re

In the p l a s m a ,

in the free,

that

in t o the b l o o d p l a s m a f r o m

The r e s u l t s

thyroxine-like

5).

indicates

i od i na t ed c a s e i n or som e b u t a n o l - i n s o l u b l e m e t a b o l i t e
as w e l l as t h y r o x i n e ,

inter­

the l o d i n a t e d p r o t e i n

c o ul d n ot be m ai n ta i ne d .

decreased

ated c a s e i n c o n c e n t r a t i o n

24-hour

the 7 2 - h o u r i n t e r ­

the l i v e r d e c r e a s e d b e l o w

in fact,

The

l i ve r w a s

the l£- a n d

the r a t e a t w h i c h

concentration

the supply had,
it can be

constant between

from

the liver.

i o d i n a t e d c a s e i n in the

7) a n d d e c r e a s e d 91 p e r c e n t by

val i n d i c a t e s

of c l e a r a n c e

I n d i c a t e s a r a p i d u p t a k e by

m a i n t a in e d f a i r l y
vals

th at a h i g h r a t e

carried
that

in a c o m b i n e d

out at

this f r a c ­
form.

It

than 50 p e r c e n t of the
at the time

butanol-soluble,

form.

i nt e r v a l s

49
In a
casein,

st u dy

of the d i s t r i b u t i o n

it w a s

many of the

f ou n d

same

N e v e r t h el e ss ,
abi lity for

ti s s u e s

no t i s s u e

slightly

iodinated casein.
spleen,

th at

tha t c o n c e n t r a t e
s h o w e d as

liver,

lodinated

in

casein.

effective a concentrating

i o d i n a t e d c a s e i n as

kidney,

iodinated

this p r o t e i n w a s c o n c e n t r a t e d

Tissues with

s t omach,

of s l i g h t l y

that of l i ve r for

the h i g h a c c u m u l a t i o n s w e r e :
large

intestine,

lung,

and

small intes tine.
In f i g u r e

1 it w a s

s h o w n that the k i d n e y a c c u m u l a t e s

iodinated c a s e i n rapidl y.
the r apid r a t e

of c l e a r a n c e

blood p l a s m a of

the dog

mlnute I n t e r v a l s ,
radioactivity
minute

When

these data are

of l o d i n a t e d c a s e i n fr o m the

(83 p e r c e n t

t able 5) a n d

in the u r i n e

collection periods,

the

b e t w e e n the
slower rate

(32 p e r c e n t b e t w e e n
table 6),

f r o m the bl o o d

the u rine at a slow rate.
case in f r o m
et al

the b l o o d is

lodinated c a s e i n
hours a f t e r

its

the 8- and 436 -

clearance

that

into

of i od i na t ed

in a c c o r d w i t h the r e p o r t
of the r a d i o a c t i v i t y

of C a m p b e l l
of l a be l le d

s t r e a m of sheep 7

I n t r a v e n o u s a d m i n is t ra t io n .
characterization

of the r a d i o a c t i v e m a t e r i a l

25 m i n u t e s a f t e r the i n t r a v e n o u s injection of

iodi ne - 131 l a b e l l e d i o d i n a t e d casein,
undialyzable.

in

in the k i d n e y d u r i n g

is r e m o v e d f r o m the blood

The a u t h o r ' s
in the u r ine,

of change

p l a s m a and e x c r e t e d

The r a p i d

that 90 p e r c e n t

(1950)

1- an d 17-

it is c o n c l u d e d

iodinated c a s e i n Is r a p i d l y a c c u m u l a t e d
its r ap i d c l e a r a n c e

compared with

i n di c at e s

that It is

The r a d i o a c t i v i t y

in the u r i n e

iodinated p r o t e i n or

may r e p r e s e n t

some large me tabolite.

the int act

SUMMARY
1.

Distribution
ated

studies

casein reveal

highest

concentration was

more

lung,

t h a n 10

Slightly

concentrate
casein was

not as

and

same

it

intravenously,

tissues which wer e

casein.

The s l i g h t l y

s t om a ch and

in the
l ar p e

c a s e i n is a d m i n i s t e r e d

in the

The a m o u n t

where

The

s h o w n to
lodinated

li ver bu t more
intestine

than

casein.

thyroxine-like
protein,

intestine.

in the liver,

administered

concentrated,

in the

iodinated

the l i v e r ,

that of s o d i u m i o d i d e w h i c h had

casein,

lodinated

the i o d i n a t e d

found

a n d small

in

iodin­

similarly.

in the

concentrated

When

s t omach,

ti mes

iodinated

concentrated

administered

it is c o n c e n t r a t e d

kidney,

be en a d m i n i s t e r e d

3.

that

sp leen,

w as

2.

of i n t r a v e n o u s l y

iodine

is h y d r o l y z e d

liver,

and r e l e a s e d

of l o d i n a t e d

the p l a s m a l e v e l of

Intravenously,
from the

l od i n a t e d

to the plasms.

casein available

to the liver

t h y r o x i n e - l i k e i o d i n e are

interdependent.
Following

the o r a l a d m i n i s t r a t i o n of i o d i n a t e d

thyroxine-like
f ou n d

iodine,

in a c o m b i n e d form,

in the b l o o d plasma.

protein

or

some

large

T h i s may be the

metabol i te .

casein,

can be
intact

5.

Most of the t h y r o x i n e - l i k e
after

the plasma,

the oral a d m i n i s t r a t i o n of lodinated casein,

in the free,
6.

iodine a pp e ar i ng in

butanol

soluble,

Intravenously administered
accumulated

form.

lodin ated casein is

In the k idney and

is

rapidly

there is a slow excretion

of its metabolite.
7.

The l o d i n a t e d m a te r i a l exc reted
the first 25 m i nutes

into the urine

during

is not dl a ly z ab l e and may r ep r e­

sent the Intact i od i nated p r o t e i n or a large metabolite.

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Appendix I

THE DISTRIBUTION OF IODINATED CASEIN IN THE RAT

Tissue
ilood

Rat
No.

Wet
wt.
(gm. )

Dry
wt.
(gm. )

Percent
solid

1
2

.1037
.0943
.0602
. 1014
.1035
.1023
.0988
.0511
.0351
. 0850
mean

21.59
21.15

7

.4802
.4457
.3535
.5107
.4961
.5070
.4952

8

.3061

9

.2253
.4560

3
4
5

6

10
Plasma*

1
2

19.85

20.86
20.17
19.95
16. 69
15.57
18.64
19.14

68.44
53 . 7 4
31.07
6 2 .62
6 1 . 81
64.59
65 .34
28.88
19.71

69.66
63.89
50.17
29.00
58.46
57.70
6 0 .30

8

.2469
. 2292
. 1818
. 2626
.2551
. 2607
.2546
.1574

9

.1158

10

.2345

18.40
65.03

1
2

. 8600
.5967
. 6263
2 .1342
.4 6 0 4
.7813
.4405
.5857
.4113
.3979

3
4
5

6
7

Glut eal
muscle^

17.02

cps per
cps^ gm wet
tissue

3
4
5

6
7

8
9

10

61.00
26.96

.2120
. 1471
. 1544

50.48
23.57

.5261

90.54

. 1135
.1926
. 1086
. 1444
. 1014

13.01
20.16

.0981

15.00

23.16

14.58
24.31
14. 31

cps per gm tissue
ops per gm p l a s m a

142.52
120.57
87.89

122.61
124.18
127.39
1 31.94
94.34
87.48
152.76

258 .76
218.89
159.51

222.61
226.18
231.30
239.59
1 7 1 .28
158.89
277.31
58.69
39.50
36.97
42. 42
28.25

25.80
33.09
41. 50
34.79
37.69

m^an
standard error

.2268
» 1804
.2317
.1905
.1249
.1115
.1381
.2422
.2189
.1359

.1800
.0154

57
THE DISTRIBUTION OF IODINATED CASEIN IN THE R\T (cont.)
Ti ssue

Rat
No.

Wet
wt.

Iehu.2
Lung

1

.5324

2

.3345
.4703

3
4

5

.4 7 4 0

7

.3280

9

.2 8 2 7
.5123
.5025

6
8

10

L iver

1
2

.3826

. 1141
. 0717

.1008
.0 7 8 4
. 1016
. 0820
. 0703
. 0606
. 1098
.1077

. 228 4

1
2

.7061

.1514

.6 1 8 4

.1326

3

.6856

.1470
.1335

6
7

8
9

10

4

.6226

5
6

.6343
.6907
. 6986
.5391
.6 9 5 4

7

8
9

10

.5601

Percent
solid

cps per
pm. w e t
tissue

cps

73.35
53.01
52. 54
41. 04
55.33
5 0 . 88
65 . 1 9
35 .49
6 9 . 88
92. 78

gm tissue
c p 8 per gm p l a s m a

137.77
15 8 . 4 7
1 1 1 .71
112.19

.5324
.7239
.7003
.5039

116.72

.5160

13 2.9 8
19 8.7 5
125.53
136 .40
184.63

.5749
.8295
.7328
.8584
.6657
.6637
. 0404

mean
s tandard error

.8 7 6 4
.7052
.6335
.5652
. 7847
.4735
.6354
. 8096
.7 5 7 4
.6795

3
4
5

Kidney

.3658

Dry
wt.
(gm. )

.1838
. 1651
.1473
. 2 045
.1234
. 1656
. 2110
.1 9 7 4
.1771

.1360
.1 4 8 1
.1498

.1156
. 1491

.1201

1050.56
691.51
6 7 1 .^6
639.69
847. 4-3
462.92
82 6 . 4 2
1059.33
896.24
1155.18

1198.72
980.58
1060 .07
113 1 . 7 9
1 07 9 . 9 4
977. 65

4.6325
4.4797

6. 645 7
5.0841
4. 7746
а .2267
5.4285
1300.62
7.6393
1308.46
118 3.31
7.4473
б. 130 4
1700.04
m e a n 5 .6488
.3940
standard error

183.31
241.26
222.85
223.35
255.44

261.11
185.26
235.06
232.17

296.42
351 .89
357.93

352.12
369 .82
373.76
343.64

338.02

1.3 541

2.2060
1.6078
1.5568
1.5988
1.5599

2.0063
2.1273
1.4947

41 4 . 5 1
mean 1.7235
standard error .1949

58
THE DISTRIBUTION OF IODINATED CASEIN IN THE RAT (eont.)
Tissue

Rat
No.

Wet
wt.

Dry
wt.

XeeuJ- isaui.
S pleen

.1101

10

.5149
.3 928
.5252
.4415
.42?4
.4321
.4822
.3390
.5299
.3531

1
2

.2154
.4705

.0509
. 1112

.2310

.0 5 4 6
.0434
. 0610
. 0 4 65
. 059 0
.0 7 4 2
. 0828
. 0636

1
2
3
4
5

6
7

8
9

S t omach

4
5

6
7

Small
Intes­
tine

.3 836

.2581
.1967
.2496

8

.31^0

9

.3 5 0 4

10

.2691

1
2

.5863
.3 ^78
.3^13
.4400

3
4
5

6
7

8
9

10

.5108
.3876
.2552
.3223
.^335
.440 9

.0840
. 1123
.0 9 4 4
.0914
.0 9 2 4
. 10 31
.0725
.1133
.0755

. 1266
.0 751
.073?
.0950
.1103
.0 837
.0551
. 0696
.0936
.0952

Percent
solid

cp s

^ cps p e r
gm. w e t
tissue

^57.05
287.38
305.15
427.96
238.17
358.97
316.47

216.58

cps per gm tlsgue
cps p er g m ~ p l a s m a

887.64
731.61

581.01
969.33
557.25
830.75

656.30
638.87

3.^303
3 .3^ 23
3.6424
^ .35 ^3
2 .4637
3.5916
2.7392
3 .72 99

6 86. 39
4.3199
802.46
2 .8937
m e a n 3.4507
standard error . 1974-

363.72
283.35

31.87
^9.3^
40. 80
57. bo
51.89
47.71
41. 24

1^7.95
104.86

5^ .19
45. 01
42 . 4 3
47.43
3^ .39
53 . 3 5
28.46
4 2.22
30 .42
71.17

92.42
129.^1
124.31
107.79
67.32
13 7.6 4
111.52
130.99
70.17
161.41

176.62

.5717
.4790
1.3 072
1 . 404 3

312.63
201.04
.8888
1.
0486
242.55
1 6 5 .22
.6895
58.26
185. *54
1.0832
47.41
135.30
.8515
59.02
219.32
.7908
me an
. 89 14
st andard error
.0879
.3571
.5912
.7793
.48 42
.2976
.5950
.465^
.7647
.4416

.5820

mea n
.5358
standard error .0500

59
THE DISTRIBUTION OF IODINATED CASEIN IN THE RAT (cont.)
Tissue
Large
intes­
tine

Rat
No.

Wet
,wt. .
(gm. )

Dry
Percent
wt.
solid
(^m. )

1
2

.22 80

. 04 24
.0925
. 0726
.0632
. 0883
. 0 834
. 1051
. 0682

7

.4975
.3905
.3399
.4749
.4486
.5653

8

.3668

9

.2797
.2711

3
4
5

6

10

cps per cps per (?m tissue
gm. we t cps per gm plasma
tissue

cps

18.72
35.^9
36.23
28. 86
24.99
2 5 .14
54.06

28.18
1 6 . 04
22.30

.0520
. 0 504

82.10
71.33
92.77
84.90

52.62
56.04
95.63
76 . 82
57.34
82.25

standard
Rat No,

Body wt.
(gm. )

1
2

170

.3172
.3258
.5815
.3813
.2326
.2422
.3991
.4485

.3608
.2965
mean .3585
error .0325

166
180
166

3
4
5

174

6

182

7

158

8

160

9

184
151

10
m e an

*

168

Counts p e r second corrected for background, physical decay
(appendix V I ) , and s e l f -absorption (appendix VI]).
^ Plasma v a l u e s were computed from the blood data,
>d x 0.5143 = gm. wet plasma lappenai
gm. wet blood
(appendix
0.9336
=
pla
s
m
a
cps
(appendix
V I I I ).
blood cps

V)

3 Wet wt. w e r e computed from dry wt.
Percent solid was d e t e r ­
mined in tissues in the iodide distribution experiment.
Both
experi m ents were run on rats from the same original group.
percent

j. x 100

solid

= wet wt.

(gm. )

^ The standard error(S.E.) was computed from the standard devia­
tion (S) as described in Dixon et
p Q ^ l ) ^ a n d Snedecor (1946),
T O
respectively.
jT
N - 1
3 -E - ”

xjH

S

60
A p p e n d i x II
TH E D I S T R I B U T I O N OF IODIDE IN THE RAT

V*

1

2
3
4
5

6
7

8

W ®t
D
r
y
7 cps per
wt.
wt.
solid
cps
gm. w et
IgrcuJ___LgffiiJ_________________ tissue
.1415
.2157
.1536
.1640
.2042
.2617
.1379

.1901

9

.1985

1

.0727
.1109
.0789
.0843

2
3
4
5

6
7

8
9
1

2
3
4
<

6
7

8
9

.0271
.0413
.0294
.0314
.0391

55.95
121.15
55.88
52.36
10.24
97.50
77.07
70 . 02
63.75

.0501
.0 2 6 4
. 0 364
. 0380

34 .64
75.02
34. 60
32.42
76.32

.1050

.3585
.574
.4585
.6935
.5365
.4640
.717 5
.4475
.6495

395.40
561.65
363.80
319.26
603.52
372.56

55 8.88
368.33
321.15

47. 72
43.36
39. 48

476.47
676.46
438.52
384.57
726.85
4 4 8 .92
673.06
4 4 3 .80
387.05

60.38

.1345
.0 709
.0977
. 1020
.0945
.1370
.1063

26.35

23.86
23.18

33.13
55-22
46.75

96 .20
101.96

.1678

24.19
23.05
32.19
22.70
22 . 81
23.52

65.66

94.67

44.36
31.99
57.17
36.09
53.50

82.68

.1237
. 1494
. 1629
. 1021
. 1528

cps per gm. tissue
ops per gm. pl a s m a

92 ..41

68.94
79.67
80 . 64
82.37

mean,
standard e r r o r 4.
1
2
3
4
5

6
7

8
9

. 3780
.209
.3000
.4160

.3260
.2300
.5005
.3570
.6550

. 0902
.0457
. 0 664

.0910
. 0678
.0510

23.86
2 1 .86
22.13
21.87
20.79
22 .17
20.71

.1037
. 0782
21.90
17.64
. 1156
mean 21.43

63.50
30.98
56.99
57.32
48.65
2 2 . 16
63.52
54.15

67.62

167.98
148.2?
189.96
137.78
149.23
96. 34
126.91
1 5 1 .68
103.23

mean
standard error

.1939
. 1422
.2325
.2461
.1137
.1535
.1183
.1817

.2128
.1771
.0159
.3525
.2191
.4331
.3582
.2053
.2146
.1885
.3417
.2667
.2866
.0288

61
DISTRIBUTION OF IODIDE IN THE RAT (cont.)
u•

1

2
3
5

6
7

8
9

1

2
3
4
5

6
7

8
9

""Wet
wt.
Igrc.)

Cry
wt.
(g m .)

J’ct.
solid

.4920
.7645
.6410
.8710
.5560
.8195
.8710
.7320
.7615

. 1256
. 1980
. 1708
.2262
. 1392
.2193
.2339
. I960
.1915
mean

25.52
25.89
26. 64
25.97
25.03
2 6 . 76
26.85
26.77
25.14
26.06

98.56
118.51
118.32
117.29
9 4.04
131.06
145.07
173.20
108.93

200.32
155.01
184.58
134.66
169.13
159.92
166.55
236.61
143.04

. *1-204
.2291
.4209
.3531

.565
.556 0
.5045
.8025
.6420
.6545
.7730
.5740
.6520

. 1140
. 1180
.1077
.1686
.1391
. 1401
. 1658
. 1208
.1433
mean

20.17
21.22
21.34
2 1 .00
21.66
21.40
2 1 .44
22.78
21.97
21.44

103.70
84.62
8 8 . 06
138.76
106.80
93.40
103.00
88.57
97.66

183.53
152.19
174.54
172.90
166.35
142.70
133.24
154.30
149.78

.3851
.2249
.3980
.4495

_ cps per cps per gm. tissue
cps
gm. wet cps per prm. plasma
______ tissue

.3562
.2474
.5331
.3695
mean .3510
standard 1
.0338

2
3
4
5

6
7

8
9

.443
.4220
.484
.5525
.4955
.3570
.6105
. 4285
.4185

.0880
.0794
.0907
.1332
.1115
.0733
.1293
.1005
.0977
mean

19.86
18.81
18.73
24.10
2 2 . 50
20.53
21.17
23.45
23.34
21.38

9 0 . 57
58.98
81.97
187.34
82.58
54.50
1 05.94
107.53
105.51

204.44
139.76
169.35
339.07
166.65
152.66
173.52
250.94
252.11

standard
1

2
3
4
5

6
7

8
9

.143
.2500
.3390
.4935
.6185
. 3720
.3620
.3010
.6410

.0323
.0479
. 0792
. 1240
.1432
.0901
.0895
.0708
.1723
mean

22.58
19.16
23.36
25.12
23.15
24.22
24. 72
23.52
26. 87
23.63

.2288
.3178
.1979
.3476
.3869
mean .3262

standard <
1

.2326

28.11
40.23
61.06
2 1 5 .14
138.96
60.01
57.78
100.22
157.96

. 0298
.4290
.2066

.3861
.8833
.2292
.3400
.2578
.5654
.6513
mean .4387
.0746

.4125
.2378
.4107
.1335
.3091
.3593
.2371
.7502
.63 66
mean .4985
.0980
standard error
196.57
160.92
180.11
435.94
224.67
161.31
159.61
332.95
246.42

62
THE DISTRIBUTION OF IODIDE IN THE RAT (cont.)
Tissue

Rat
No.

Wet
Dry
wt.
wt.
J
p
m
. )
JjS!n.i.L

Pet.
solid

cps per c p s per gm.
c p s 1 gm. wet cps per gm.
tissue

Small
intes­
tine

1
2
3
4
5
6
7
8
9

.0775
.2140
.170
.2780
.4355
.0900
.0975
.1415
.3760

.0150
.0452
.0282
. 0616
. 0837
. 0226
. 0267
. 0273
.0910
mean

19.35
21.12
16.58
22.15
19.21
25.11
27.38
19.29
24.20
21.59

11.35
28.29
20.32
41.38
7 2 . 24
10.59
12.42
27.88
55.00

Large
Intes­
tine

1
2
3
4
5
6
7
8
9

.2590
.1280
.342
.2060
.2835
.1685
.1390
.2840
.2840

.0503
.0238
.0599
. 0459
. 0621
.0319
.0276
.0509
.0596
mean

19.42
18.59
17.51
2 2 .28
21.90
18.93
19.85
17.92
20.98
18.59

45.07
18. 29
52.89
54. 71
48. 90
24.03
14.58
49.94
39.71

Cecal
con­
tents

1
2
3
4
5
6
7
8
9

. 6665
.6675
.7057
.6727
.7293
.6879
.7130
.7352
.5998

.1512
.1395
. 1526
.1340
.1645
.1509
.1429
.1390
.1054
mean

22.68
2 0 . 89
2 1 .62
19.91
22.55
21.93
20.04
18.90
17.57
20.67

85.59
71.06
85.10
76.50
79.27
72.42
73.96
61.77
54.05

1
2
3
4
5
6
7
8
9

Body wt.
180
188
184
202
168
202
196
176
184
mean
186

146.45
132.19
119.52
148.84
165.87
117.66
127.38.
197.03
146.27

.3073
.1954
.2725
.3870
.2282
.2620
.1892
.4439
.3779
mean .2959
standard error
.0300
1 7 4 .01
142.39
154.64
265.58
172.48
1 4 2 .61
104.89
200.96
139.82

.3652
.2112
.3526
.6905
.2372
.3176
.1558
.4528
.3612
mean .3493
standard error
.0523

(gm. )

55.02
45-51
53.28
49. 04
48.38
45.77
45.88
37.86
36.01

.1154
.0672
.1214
.1275
. 0665
.1019
. 0681
.0853
.0929
mean . 0940
standard error
.0074
Hematocrit
.50
.46
.47
.50
.42
.50
.40
.49
.49
mean
.47

63
THE DISTRIBUTION OF IODIDE IN THE RAT (cont.)
1 Co unt s p e r second c o r r e c t e d for background, p h y s i c a l decay
(appendix V I ) , and s e l f - a b s o r p t i o n (appendix VI3).
^ Wet wt. w e r e com p u t e d f r o m dry wt.
Percent solid (19. l M was
d e t e r m i n e d in tis s u e s in the io din a t e d casein d i s t r i b u ­
tion experiment.
B o t h e x p e r i m e n t s were ru n on rats of
the same o r i g i n a l group.
Dry w t
(gm ) x 100 = w et
(gm- )
p e r c e n t solid
3 Pl asm a v a l u e s w e r e c o m p u t e d from the blood data.
gm. w e t b l o o d x 0 . 5 1 ^ 3 = gm. w e t plasma (appendix V
blood cps x 0.6193 = pl a s m a cps (appendix IX)
^ See f o o t n o t e k, a p p e n d i x I.

)

64
A p p e n d i x III

THE DISTRIBUTION CF SLIGHTLY IODINATED CASEIN IN THE RAT
Tissue

Rat
No.

______________ —

Plasma

Gl uteal
muscle

Lung

Wet
wt.

iSHU-l

Dry
Percent
cps per cps per gm tissue
wt.
solid cps
gm. w e t cps per gm p l a s m a
(gm. )___________________tissue_______________________

.2661
.1592
.1212
.1592
.1781
.1 8 0 8
.1330
.1811
.1114
. 1282

.0 2 6 4
9.92
.0 1 4 4
9.04
10.
80
.0131
. 0162
10.17
.0 2 0 4
11.45
.0147
8.13
. 0132
9.92
9.00
.0163
. 0100
8.97
9.75
.0125
mean
9.71

17.88
12.13
7.59
25 . 4 0
15.73
9.65
12. 04
7. 88
8.85
11.89

67.19
76.19
62.62
159.54
88.32
53-37
90.52
43.51
79.44
92.74

1
2
3
4
5
6
7
8
9
10

.7522
. 7402
.6734
.8438
.7315
.7716
. 6908
.8335
.8402
.8765

.1902
.1 8 1 1
.1639
.2098
.1891
.2017
.1632
.2055
.2 1 1 8
.2 2 7 4
mean

25.28
24.46
24.33
24.86
25.85
2 6.14
23.62
24.65
25.20
25.94
25.03

13.90
15.13
9.06
18.00
7.7 6
13.05
13.15
1 0.99
18.19
19.01

18.47
20. 44
13.^5
21.33
1 0 .60
16.91
19.03
1 3.18
21.64
21.68

•1
2
3
4
5
6
7
8
9
10

.5379
.4638
.5889
.6518
.4417
.4582
. 4648
.4880
.3778
.3825

.1233
. 1112
.1357
.1458
.1039
.1023
.0995
.1121
.0766
.0797
mean

22.92
23.97
2 3 . 04
22. 36
23.52
22.32
21.40
22.97
20.27
20.83
22.36

26.97
2 6 .56
29.87
61.64
24.93
19.66
28.29
16.66
18.50
23.54

1
2
3
4
5
6
7
8
9
10

.2748

.2682
.214?
.1336

.1200

.3168
.2102
.3029
.2724
.2337

mean
standard error
50.13
57.26
50.72
94.56
56.44
42. 90
6 0 .86
34.13
48.96
61.5**

mean
standard error

.7460
.7515
.8099
.5927
.6390

.8038
.6723
.7844
.6163
.6635
.7079
.0254

65
THE DISTRIBUTION CF SLIGHTLY IODINATED CASEIN (cont.)
Tissue

Rat
No.

Wet
wt.
Cp;m..)

Dry
Percent
wt.
solid
(cm. )

cps per cps per gm tissue
gm wet
cps per gm p l a s m a
tissue

cps1

Liver

1
2
3
4
5
6
7
8
9
10

.7726
1.0130
.9566
.9392
1.0371
.7939
1.0 1 3 6
.9233
1.1029
.8556

.1991
.2478
.2579
.2528
.2692
.2149
.2835
.2733
.2813
.2234
mean

25.77
24.46
26.96
26.91
25.95
27.06
27.96
29.60
25.50
26.11
26.62

49.05
75.99
72.09
135.78
179.70
43.14
94. 84
45.08
87.47
108.90

Kidney

1 1.0303
2
.8185
.8968
3
4
.6333
.8538
5
. 8400
6
.8568
7
8
.9793
.7769
9
.7352
10

.2274
. 1662
.1961
. 1432
.1902
.1907
.1952
. 2366
.1703
.1607
mean

2 2 .07
20.30
21.86
2 2 . 61
22. 27
2 2 .70
2 2 . 78
24.16
21.92
21.85
22.25

131. 14
1 1 8 .66
158. 71
94. 94
137. 53
147. 15
148. 59
81. 54
89. 96
131. 34

.3856
.4449
.3700
.3248
.2877
.4751

.0900

23.34

.0987
.0877
.0732
. 0667

22.18

.1092

7

.3^61

.0760
.0995

9

.4026
.3670
.4327

22.98
21.95
24.71

70.84
1 11.31
7 7.44
50.27
67.50
92.72
86.87
33.01
58.66
89.40

Spleen

1

2
3
4
5

6

8
10

.0806

23.70
22.53

23.18

21.96

. 0942
21.77
mean 22.83

63.48
75.01
75.36
144.56
173.27
54.33
93.56
48.82
79.30
127.27
mean
standard error
127. 28
144. 97
1 7 6 .97
149..91
1 6 1 .07
175. 17
173.,42
83.,26
115.,79
1 7 8 ..64

.9447
.9845
1.2034
.9061
1.9618
1.0142
1.0335
1.1220
.9982
1.3723
1.1540
.0997
1.8742
1.9027

2.8260

.9396
1.8237

3.2821
1.9158
1.9135
1.4575

1 .9 2 6 2

mean 1 . 9 8 6 1
2060
standard error

.

2.7341
3.2837
3.3422
.9701
2.6563
3.6565
2.7727
1 . 8843
2.0119
2.2277
mean 2.5539
.2542
error

183.71
250.19
209.29
154.77
234.61
195.15
250.90
81.99
159.83
206.60

standard

66
THE DISTRIBUTION OF SLIGHTLY IODINATED CASEIN (cont.)
ftat
Wet""
Tissue
No.
wt.
______________ (gnu )

Dry Percent
wt.
solid
(gm. )_________

Stomach

1
2
3
4
5
6
7
8
9
10

.2693
.3429
.226 7
.2786
.3154
.3189
.3222
.3631
.4461
.1815

.0731
. 0880
.0726
.06 97
.0813
.0845
. 0947
. 1106
. 1100
.0507
mean

27.14
25.66
32.02
25.01
25.77
26.49
30.45
24.65
27.93
23.53
26.86

55.03
84.51
40.52
35.45
36.11
62.12
58.70
41.60
78.07
31.18

Small
intes­
tine

1
2
3
4
5
6
7
8
9
10

. 1606
.2868
.1989
.136?
.3162
.2653
.2366
.5052
.3782
.3543

. 0378
.0653
. 0441
. 0298
. 0740
.0582
. 0490
.1085
.0841
. 0768
mean

23.53
22.76
22.17
21.79
2 3.40
21.93
20.71
21.47
22.23
21.67
22.16

11.18
12.44
7.60
4.52
21.15
14.32
8.60
18.09
13.08
11.04

Large
i ntes­
tine

1
2
3
4
5
6
7
8
9
10

.3048
.2519
.2961
. 1614
.2119
.2612
.3165
.2885
.2294
.2592

.0861
. 0644
.0859
.0397
.0557
.0589
.0688
.0709
.0539
.0531
mean

28.24
25.56
29. 01
24.59
26.28
22.54
21.73
24.57
23.49
20.48
24. 64

16.14
15.78
15.92
4.69
22.09
19.81
24.20
14.61
12.83
15.20

ops per cps per gm tissue
cps
srra wet
cps per srm plasma
_______ t Issue
204.34
246.45
178.73
127.24
114.48
194.79
182.18
114.56
175.00
171.79
mean
standard error

3.0412
3.2346
2.8541
.7975
1.2961
3.6498
2.0125
2.6329
2.2029
1.8523
2.3573
.2826

69.61
1.0360
43.37
.5692
38.21
.6101
33.06
.2072
66.88
.7572
53.97
1.0112
36.34
.4014
35.80
.8227
34.58
.4325
31.16
.3359
mean
.6183
standard error
.0895
52.95
62.64
53.76
29.05
104.24
75.84
76.46
50.64
55.92
53.64
mean
standard error

.7880
.8221
.8585
1.8208
1.1802
1.4210
.8446
1.1638
.7039
.6323
1.0235
.1177

67
THE DISTRIBUTION OF SLIGHTLY IODINATED CASEIN (cont.)
Tissue

RatWet
No.
wt.
(gm. )

Cecal
1
.8640
contents 2
.8011
.7473
3
4
.8598
5
.8059
6
.8074
.9326
7
8 1.0045
. 8860
9
10
.7503

Dry
wt.

Percent
solid

.158?
.1374
.1291
. 1686
.1 4 9 4
.1508
.1680
.1743
. 1861
. 1362
mean

18.36
17.15
1 7 .2 ?
19.60
18.53
18.67
18.01
17.35
21.00
18.15
18.40

cps per cps per gm tissue
cps
gm wet
cps per gm plasma
_______ tissue
24.28
21. 70
22. 27
25.32
18. 80
19.94
31.24
17.04
23.02
17.70

28.10
2 7 . 08
29.80
29.44
23.32
24.69
33.49
16.96
25.98
23.59

mean
standard error

.4182
.3554
.4758
.1845
.2640
.4 626
.3699
.3897
.3270
.2543
.3501
.0296

Body wt.
(gm. )
268
244
258
226
239
259
240
336
235
228

Rat No.
1
2
3
4
5
6
7
8
9
10
mean

253

1 «
Counts p e r second corrected for background, p h y s i c a l decay
(appendix VI) f and s e l f - a b s o r p t i o n (appendix VII).
^ See f o o t n o t e 4, a p p e n d i x

I.

Appendix IV
THE METABOLISM OF IODINATED CASEIN, INTRAVENOUS ADMINISTRATION

___________________
1
1
2
Plasma
Liver
Liver
1 ml
0.92 gm
1.11 gm

Time (hr.)
Rat No.
Tissue
»t. or vol.

12
2
Plasma
1 ml

3
Liver
1.56 gm

13.37
4.26
172.12
38.87

11.97
5.15
31.33
5.73

3
Plasma
1 ml

%
Liver
1.48 gm

4
Plasma
1 ml

cps per i_ml.
butanol
a3

B
HA
HB

27.59
7.43
125.72
18.28

11.20

2.62
224.33
50.95

22.29
5.35
108.55
17.72

5.75
-2.22
4.08
0,62

13.78
4.84
41.62
7.40

11.30
2.86
8.97
1.13

cps per gra or
ml of tissue
A

248.5
66.9
HA 1359.0
HB 197.5

B

89.6
20.9
1794.6

107.6

242.2
58.1
1415.8
231.0

106.9
34.0
1376.9
310.9

76.7
33.0
240.9
44. 0

46. 0
17.7

32.6
4.96

93.1
32.7
337.4
57.0

90.4
22.8
71.7
9.04

THE METABOLISM OF IODINATED CASEIN, INTRAVENOUS ADMINISTRATION (cont.)

. ......J 2

24

Time (hr.)
Rat No.
Tissue
Wt. or vol.

5
Liver
1.82 gm

5
Plasma
1 ml

6
Liver
1.69 gm

I
Plasma
1 ml

7
Liver
1.12 gm

7
Plasma
1 ml.

8
Liver
1.59 gm

'8
Plasma
1 ml

cps per iLml.
butanol
a3

B
HA
HB

32.32
9.04
202.33
45.51

12.90
3.82
7.73
0.46

23.40
6.59
129.79

29.12

8.00
1.59
5.74
0.47

3.73
1.45
20.87
2.36

0.61
0.33
1.48
0.10

4.43
1.89
30.95
2.92

0.61
0.40
1.76
0.10

cps per gm or
ml of tissue
A
177.5
B
49.6
HA 1334.0
HB 300.0

103.2
30.5

61.8
3.68

138.4
38.9
921.5
206.7

64.0
12.7
45.9
3.76

33.3

12.9
223.5
25.2

4.88
2.64
11.8
0.80

27.8
11.8
233.5
22.0

4.88
3.20
14.0
0.80

Rats 3 and 4 were injected with a different preparation of lodinated casein than had
been administered to the other rats in this experiment.
The preparation also had a
different radioactivity.
2

■i

Counts per second corrected for background and physical decay (Appendix VI).

3 A unwashed butanol extract
B alkaline washed butanol extract
HA unwashed butanol extract of hydrolysate
HB alkaline washed butanol extract of hydrolysate

Appendix IV (cont.)
THE METABOLISM OF IODINATED CASEIN, ORAL ADMINISTRATION

Time (hr.)
Rat No.
Tissue
Wt. or vol.

______________ l£_____
“T
1 ~ 2
Liver
Plasma
Liver
2,21 gm
1 ml
1.93 gm

2
Plasma
1 ml

3
Liver
1.13 gm

3
Plasma
1 ml

“5
Liver
1.50 gm

37.75

6.90

19.07

1.80

1.12

23.48

4.56
1.87

3.02
9.61

5.03
0.70
3.^6
0.17

V

Plasma
1 ml

cps per £ ml
butanol1
A2
B
HA
HB

M l

6.60

0.11

0.29

2.09
0.19

5 M

A
B
HA
HB

18.1

52.8

0.49
11.3
1.03

2.32
43.8
1.84

0.23

16.55
0.77
9.40
0,48

0.69

0.45

16.61
2.21
7.80
0.41

cps per gm or
ml of tissue

302.0

61.0

3.88

14.4

56.9
2.90

187.8

9.91
48.4

83.5

5.52

19.8

152.5
24.1

76.8
3.60

3.35
4.66

27.6
1.36

132.8
17.6
62.4
3.28

•Nj
O

THE METABOLISM OF IODINATED CASEIN, ORAL ADMINISTRATION (cont.)

48

Time (hr.)
Rat No.
Tissue
Wt. or ml.
cps per £ ml
butanol1

5
Liver
2.00 gm

«
AT

5
Plasma
1 ml

6
Liver
2.01 gm

6
Plasma
1 ml

B
HA
HB

1.59
0.30
1.15
0.12

2 .2 6

2.47
0.07
1.07

0.12

0.05

3.74
0.26
2.7^
0.14

A
B
HA
HB

7.95
1.50
6.90
0.72

14.6
3.60
18.0
0.9 6

12.2
0.34
6.36
0.29

29.9
2.08
21.9
1.12

1.83
0.45

cps per gm or
ml of tissue

1 „

Counts per second corrected for background and physical
decay (appendix VI).

p

A unwashed butanol extract
B alkaline washed butanol extract
HA unwashed butanol extract of hydrolysate
HB alkaline washed butanol extract of hydrolysate

72
Appendix V
S t an d a r d R a t B l o o d V a l u e s and C o m p u tat ion of Correcti on Factors

Values
Sp ec i f i c gra v i t y of blood ( f e m a l e s ) 1
^
Sp ec i f i c gravity of pla sma (mixed sex)
P e r c e n t solid in blood, by wt.

1.05^
1.023
19.1^

Percent plasma in blood, by

53.

vol.^

Computations
I o d l n a t e d Casein D i s t r i b u t i o n Experim ent
1 gm. w e t b l o o d / 1 . 05 ^ * 0.9^87 ml. blood
0. 9^8 ? ml. blood x 0.53 = 0 . 5 0 2 8 ml. plasma
0.5028 ml. p l a s m a x 1.023 = 0.51^3 wm. w et plasma
therefore, gm. w et blood x 0.51^3 = gm . w e t plasma
Iodide D i s t r i b u t i o n Expe ri m e n t
1 gm. dry b l o o d / 6 . 191^ = 5 - 2 2 k gm. we t blood
5 . 2 2 k gm. wet b l o o d / 1 . 05 ^ = ^.956 ml. blood
^■.956 ml. blood x 0.53 = 2 . 6 2 6 ml. plasma
2.626 ml. p l a s m a x 1.023 = 2.686 gm. wet plasma
therefore, gm. dry blood x 2.686 = gm. wet plasma

1 A l b r i t ton, 1952
2 Iodlnated casein distrib uti on experiment, this paper.
3 Iodide d i s t r i b u t i o n experiment, this paper.
1.00 - he mat ocr it

73
A p p e n d i x VI
C O R R E C T I O N FOR P H Y S I C A L D E C A Y

ib =

a n t l l °g

A

present activity

A0

original activity

t

time elapsed

T

half-life

For the p u r p o s e

case of The M e t a b o l i s m
where

of isotope

(1-131,

192 hr.)

of c o r r e c t i n g for p h y s i c a l decay,

24 -hour day w a s d i v i d e d

Administration,

since A

Into 4 - h o u r
of Iodlnated

periods,
Casein,

except

in the

Int r a v e n o u s

8-hour p e r i o d s w e r e used.

the

A p p e n d i x VII
C o r r e c t i o n for S e l f - a b s o r p t i o n
Due

to the v a r y i n g th icknesses

of the tissue

w h i c h r a d i o a c t i v i t y d e t e r m i n a t i o n s w e r e made,
necessary

to co rre ct

samples on

it w a s deemed

the observed cps for self-absorption

due to tissue.
Iodine-131

io dlnated casein w a s a d m i n i s t e r e d intra­

ve nou sly to a large rat.

Cne h u n d r e d minutes a f t e r the in jec ­

tion the rat wo.s sa cri fic ed and a po r t i o n of the liver was
removed.

This w as f i n e l y divided and mixed w e l l w i t h the aid

of a single edge razor

blade and p i n c h forceps.

am oun ts of the mi xture w ere
cm.

di ame ter

evenly distri but ed

steel planchets.

The

hours at 9 0 ° C.
a desiccator,

The tissue

in an air oven,

and w e i g h e d

on tared 1.9

liver on the planchets

varied from a m o u n t s barely covering
filling the planchet.

Varying

the surface to completely

samples were dried for 14

cooled to room temperature

on a G-ramatlc balance.

in

Radioactiv­

ity d e t e r m i n a t i o n s were made by the same method described in
the iodlnated casein and iodide distri but ion experiment.
planchet w a s pla ced in the same p o s i t i o n in the

shield

The

(middle

shelf) so that the geo m e t r y was reproduced.
The log of the r a d i o ac tiv ity per tissue v e i g h t
m g . ) was found to vary as the tissue w eig ht
basic data r a d i o a c t i v i t y per mg., w a s
b e twe en 0 and

(mg.)

(cps per
From the

computed for samples

550 mg. from the r e g r e s s i o n equation

75

log

Y

= log

Y

c o r r e c t i o n fa c t o r s w e r e

+

(X - X)

then co mputed from the equation,
A

c o r r e c t i o n factor

The p r o d u c t

-

of the a p p r o p r i a t e

Y at zero mg.
“ 5\
Y at x mg.

correction factor and the

e x p e ri men tal ly d e t e r m i n e d r a d i o a c t i v i t y of a sample equals the
radioactivity

cor rec ted for self-absorption.

Appendix VII

(cont.)

SELF-ABSORPTION CORRECTION FACTORS FOR IODINE IN TISSUE

Tissue dry
wt. (mg.)

cps*

Y

X
22.83
35.21
45.40
72.46
82.70
85 .OO
99.68

19.3
32.1
42.9
81.4
102.7
1C1.1
139.5
Sum
Mean

Deviations
from mean

cps per
mg.

y

X

.1056
.0712
.0549
- .0 1 6 9

-54.8
-42.0
- 31.2
7.3
28.6
27.0
65.4

log Y
.0718
.0374
.0211
-.0507
-.0942
-.0758
-.1463

1.18
1.09
1.05
.890
.805
.840
.714

519.0
74.14

-.0604
-.0420
-.1125

-.236?
-.0338

log

A
Y

= log Y

log

r

i -. 0 3 3 8 .

?

= antllog ( .0989 - .00179 X)

4

^ A

Square of
deviations

x2

Product of
deviations

xy

3003.04
1764.00
973.44
53.29
817.96
729.00
4277.16

-5.78
-2.99
-1.71
-0.12
-1.72
-1.13
-7.35

11617.89

-20.80

(X-X )

( x-74.14)

* Counts per second corrected for background and physical decay (appendixVI )

SELF-ABSORPTION CORRECTION FACTORS FOR IODINE IN TISSUE (cont.)
Tissue wt.
(gm)#

.000
.010
.020
.030
.040

.050
.060
.070
.080
.090
.100
.110
.120

.130
.140
.150

.160
.170
.180
.190
.200
.210
.220

.230
.240

.250
.260
2?0

Tissue wt.
(gm)

cps per
mg.

Correction
factor

cps per
. mg, .

Correction
factor

1.25

1.0 0

.280

.396

3.15

1.20

1,04

.290

.380

3 .28

1.15

1.08
1.12

.300

1.17

.320

.364
.350
.335

1.22

.330
.3^0
.350
.360
.370
.380
.390
.400
.410
.420
.430
.440
.4 50
.460
.470
.480
.490

1.11
1.06
1.-02
.980
.9^1
.903
.866
.830
.798
.765
.73^
.705
.676
.649
.623
.598
.550
.528
: 8
.467
.448
.430
.412
_J$6.

1.27
1.32
1.38
1.44
1.50
1.56
1.63
1.70
1.77
1.84,
1.92
2.00
2.09
2.18
2.27
2.36
2.46
2.57
2.67
2.79
2.90
.2aJ92™.... .

.310

.322

.500
.510

.520
.530
.540
^55P„

____

.309
.296
.284
.273
.262
.251
.241
.231
.222
.213
.204
.196
.188
.181
.173
.166
.160
.153
.147
.141
.135
.130

3.^3
3.57
3.73
3.88
4.04
4.22
4.40
^.57
^.77
4,98
5.18
5.^1
5.63
5.86
6.12
6.37
6.64
6.90
7.22
'
7.53
7.81
8.16
8.50
8.86
9.25
_____5.61 . ...

78
A p p e n d i x VIII
The D i s t r i b u t i o n of R a d i o a c t i v i t y in Blood 100 Minutes
After

the In t r a v e n o u s A d m i n i s t r a t i o n of

Iodine-131

Labelled Iodlnated Casein

The d i s t r i b u t i o n of r a d i o activity

between the plasma and

red cell f r a c t i o n s of blood was determined 100 minutes after
the i n t r a v e n o u s a d m i n i s t r a t i o n of iodine-131 labelled iodlnated
casein.

This inf o r m a t i o n was used

to compute

the error involved

when all the r a d i o a c t i v i t y is assumed to be in the plasma.
The i o d l n a t e d casein was p r e p a r e d by the same method
de s c ribed In the iodlnated
with

casein

the m o d i f i c a t i o n s outlined

lism experiment.
vein, and

in the iodlnated

casein m e t a b o ­

The a d m i n i s t r a t i o n was by way of the femoral

the blood

abdominal aorta.

distribution experiment,

sample

One ml.

planc h e t and the r e m a i n d e r
crit and to separate

(3.^ nil. ) was removed from the
of blood was pipetted

onto a nickel

centrifuged to determine the h e m a t o ­

the plasma.

One ml.

of the plasma was

p i p etted onto a nickel planchet and both blood and plasma
samples w e re dried at room temperature.
ments were made with a gamma
The results

Radioactivity m e a s u r e ­

tube in conjunction w i t h a ratemeter.

indicate that 93.36 percent of the radioactivity

per gram of blood is in the plasma fraction.

Therefore the

r a d i o a c t i v i t y per gram of blood multip l i e d by 0.9336 equals
the r a d i o a c t i v i t y in the pl a s m a fraction.
tations follow:

The data and c o m p u ­

79

Data
Sample

Counts per mi n u t e '

pla sma

34?0

"blood

1970

percent plasma2

53

Computations
I

cpm per ml. plasma) (ml. plasma per gm. wet blood)
cpm per ml. blood) (ml. blood per gm. wet blood)
percent of cpm per gm. of wet blood,

cpm per ml. plasma
cpm per ml. blood
o
ml. plasma per gm. wet blood
ml. blood per gm. wet blood^
percent of cpm per gm. of wet blood,

in theplasma

x 100 =
In the plasma
3470
1970
.502 8
.948?
93.36

1 corrected for background
^(1.00 - hematocrit) x 100
^JA,
^ appendix V

80
A p p e n d i x IX
The D i s t r i b u t i o n of R a d i o i odi de
B e t w e e n P l a s m a and R ed Cells
The d i s t r i b u t i o n of r a d i o a c t i v i t y between the pl asm a and
red cell f r a c t i o n s of blood was d e t e r m i n e d

100 min u t e s a fter

the i n t r a v e n o u s a d m i n i s t r a t i o n of r a d i o i o d i d e
I n f o r m a t i o n w a s used to compute

(I-I 3 1 ).

This

the error involved w h e n al l

the r a d i o a c t i v i t y Is a s s u m e d to be in the plasma.
The e x p e r i m e n t w a s

c o n d u c t e d on 3 large rats.

was used as the a n e s t h e t i c and In jec tio ns were made
femoral vein.

Blood

samples wer e

aorta w i t h a h e p a r l n i z e d
centrifuge

into the

taken from the a b d o m ina l

s y r i n p e , transferred to 12 to 15 ml.

tubes and c e n t r i f u g e d for 30 minutes at 2000 rpm.

The plasma was
each were

Ether

s e p a ra ted

from the red cells and

samples of

taken and w e i g h e d wet In tared p l a n c h e t s

gramatlc balance.

The

samples w e r e

on a

dried in an air oven at

90 -95 ° C. and w e i g h e d dry.
Computations
cps
cps
gm.
prn.

per gm. red cells, w et vit.
per gm. plasma, w e t wt.
p las ma per prm. blood, viet w t . #
red cells p er gm. blood, viet w t . * *

0.65

1 .00

0.514
0.486

0 65 x 0.486 = 0 . 3 1 5 9 cps in the red cells of 1 erm. of the
viet blood
1 00 x 0.^14 = 0.5140 cps in the pl a s m a of 1 gm. of the
w e t blood
therefore, 6 1 . 9 pe r c e n t of the cps per erm. of w e t blood is in
the plasma

# A p p e n d i x V.
** 1.000 - 0 .5 1 4

THE DISTRIBUTION OF RADIOIODIDE BETWEEN FLASMA AND RED CELLS(cont.)
Percent
Solid

Tissue

Wet
Wt.

Dry
Wt.

Plasma

1

.3168

8.36
8.91
8.97

45.02

.1259

.0265
.0126
.0113

19.53
mean

142.10
152.75
155.12
149.99

2

.2378
.1993

.0203
.0177

8.53
8.88

59.01
51.48
mean

248.14
258.30
253.22

3

.3331
.2950

.0270
.0242

8.10
8. 20

86.07
71.98
mean

258.39
244.00
251.19

l

.3784

.1276

33.72

36.95

p

.3710
.3925

.1312
.1371

35.36
34.92

.3810
.2906

.1281
.0938

Red
cells

3

33.62
32.27

cps*

cps per pm red cells
cps per gm plasma

cps per gm.
vet tissue

Rat
No.

21.60

'

97.64

.650

54.70
61.27
mean

147.*3
156.10
151.76

.598

69.05
49.17
mean

181.23
169.20
175.21
mean

Rat no.
1
2
3

Rat wt.
435
232
2^5

.697
.648

(gm.)

# counts per second corrected for background, physical decay (Appendix VI)
ana self absorption (Appendix VII).

82
Appen d i x X
U- Kilo

Y e l l o w corn meal

( T h o m a n ) .............................. 1400 gm.

Ground w h o l e wheat
W hole milk pow d e r
L i n seed oil meal

"Hoppert" Stock Ration

(Thoman)

1000 gm.

( B o r d e n ) ..........................

800 gm.

( T h o m a n ) ............................... 400 gm.

A l f a l f a leaf meal ( T h o m a n ) ............................ 2k0 gm.
B r e wer's yeast
Table

salt

(Strain G) (A. Busch)

(iodized)

.

120 gm.
^0 gm.