A Study of the Absorption Characteristics of Iodine-131 Labeled Iodinated Casein in Some Mammals

234 15 4MB

English Pages 105

Report DMCA / Copyright

DOWNLOAD FILE

A Study of the Absorption Characteristics of Iodine-131 Labeled Iodinated Casein in Some Mammals

Citation preview

P U R D U E U N IV E R S IT Y

THIS IS TO CERTIFY THAT THE THESIS PREPARED UNDER MY SUPERVISION

BY_______

Donald Jam es C am pbell

________________________

ENTITLED

A STUDY OF THE ABSORPTION. GHARACTERISTIGS_ OF_______

IODINE-131 LABELED IQDINATSD CASEIN IN S(ME MAMMALS________

COMPLIES WITH THE UNIVERSITY REGULATIONS ON GRADUATION THESES

AND IS APPROVED BY ME AS FULFILLING THIS PART OF THE REQUIREMENTS

FOR THE DEGREE OF

__________DactQr--Qr P h ilo so p h y __________________________________

_____________P r o f e s s o r l y C hahoe o f T h e s is

> V-/7

T

^

____________ H e a p o f S c h o o l o r D e p a r t m e n t

(T December 20

is 49

TO THE LIBRARIAN;----



THIS THESIS IS NOT TO BE REGARDED AS CONFIDENTIAL

P B ozsssoR

GSAI>. SCBOOIi FOBM 0— 3 -4 9 — 131

n r oha sob

A STUDY OF THE ABSORPTION CHÂRACTEBISTICS OF IODINE-131 LABELED lODINATED CASEIN IN SOME m m A LS

A T h e s is S u b m itte d t o t h e F a c u lt y of P urdue U n i v e r s i ty by Donald Jam es C am pbell 3ii P a r t i a l F u l f i l l m e n t o f t h e R e q u irem en ts f o r t h e D egree of D o cto r o f P h ilo s o p h y

F e b ru a ry , 1950

ProQuest Number: 27712215

All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is d e p e n d e n t upon the quality of the copy subm itted. In the unlikely e v e n t that the a u thor did not send a c o m p le te m anuscript and there are missing pages, these will be noted. Also, if m aterial had to be rem oved, a n o te will ind ica te the deletion.

uest ProQuest 27712215 Published by ProQuest LLO (2019). C opyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C o d e M icroform Edition © ProQuest LLO. ProQuest LLO. 789 East Eisenhower Parkway P.Q. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346

ACKKaŒEDGMEKT The w r i t e r w ish e s t o acknow ledge h i s d e b t o f g r a t i t u d e t o t h e c o - d i r e c t o r s o f t h i s r e s e a r c h . P r o f e s s o r Jo h n E . C h r i s t i a n o f t h e P urdue S c h o o l o f Pharm acy and P r o f e s s o r F . N. Andrews o f t h e D e p art­ m ent o f A nim al H usbandry i n t h e P urdue S c h o o l o f A g r i c u l t u r e .

The

s p i r i t o f c o o p e r a tio n w hich p r e v a i l e d , i n c o n ju n c tio n w ith t h e u n s t i n t e d a id and a d v ic e w hich was g r a n te d t h e w r i t e r , p ro v ed t o b e a m ost n e c e s s a r y a d ju n c t t o t h e s u c c e s s f u l c o m p le tio n o f t h i s p ro b lem . M ention sh o u ld a l s o b e nm.de o f th e h e l p f u l a s s i s t a n c e o f Mr* W illia m M adison, g ra d u a te s t u d e n t i n th e S c h o o l o f Pharm acy, and Mr. E . J . Turman, g ra d u a te s t u d e n t i n t h e D epartm ent o f A nim al H u sb an d ry .

TABLE OF CONTENTS Page INTRODUCTION

....................................................... *................

LI1ERATURE

..........................................................................

EXPERIMENTAL

1

................................................................................................. 22

The p r e p a r a t i o n o f io d o c a s e in . . . . . . . . . . . . . . . . . . . . . . . . .

22

A n a ly s is o f io d o c a s e in

23

......... .

.

..................... ..

1.

C o n c e n tr a tio n o f s o l u t i o n . . . . . . . . . . . . . . . . . . . . . .

2.

I o d in e c o n te n t

3.

T h y ro x in e c o n te n t

............

. . . . . . . . .

...........

23 24 26

The i s o l a t i o n o f th y r o x in e from io d o c a s e in . . . . . . . . . . . . . . . 27 The a b s o r p tio n sp e ctru m o f io d in e -1 3 1 i n aluminum

30

The p r e p a r a t i o n o f io d o c a s e in la b e le d w ith io d in e -1 3 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33

îh e p r e p a r a t i o n o f th y r o x in e la b e le d w ith io d in e - 1 3 1

34

The a n a l y s i s o f io d o c a s e in f o r th y r o x in e c o n te n t by t h e i s o t o p e d i l u t i o n m ethod . . . . . . . . . . . . . . . . . . . . 35 The a b s o r p tio n o f l a b e le d io d o c a s e in fro m t h e a lim e n ta r y t r a c t o f t h e r a t . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 The d i s t r i b u t i o n o f io d in e - 1 3 1 i n t h e p lasm a o f t h e r a t f o llo w in g o r a l a d m i n i s t r a t io n o f l a b e le d io d o c a s e in ..........

39

The in tr a v e n o u s i n j e c t i o n o f la b e le d io d o c a s e in i n t o sh eep . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 The a b s o r p tio n o f la b e le d io d o c a s e in from th e a lim e n ta r y t r a c t o f t h e sh e e p

45

1.

O r a l and sm a ll i n t e s t i n e a d m i n i s t r a t i o n . . . . . . . . . . 46

2.

O r a l, v e n t r a l rum en s a c , s m a ll I n t e s t i n e and cecum a d m i n i s t r a t io n . . . . . . . . . . . . . . . . .

46

TABLE OF CONTENTS (C o n t.) Page The d i s t r i b u t i o n o f io d in e - 1 3 1 i n t h e p lasm a o f sh e e p fo llo w in g t h e o r a l a d m i n i s t r a t io n o f .................. l a b e le d i o d o c a s e in

47

The a d m i n i s t r a t io n o f la b e le d i o d o c a s e in t o l a c t a t i n g ewes .............

49

The d i s t r i b u t i o n o f io d in e -1 3 1 i n t h e m ilk o f l a c t a t i n g ewes f o llo w in g o r a l a d m i n i s t r a t io n o f l a b e le d io d o c a s e in

51

The a d m i n i s t r a t io n o f l a b e le d io d o c a s e in t o b e e f cows ................. ..... ...

. .. . . . . . .

52

P ro c e d u re s ta k e n f o r t h e c o n t r o l o f c o n ta m in a tio n from r a d i o a c t i v e s u b s ta n c e s . . . . . . . . . . . . . . . . . . . . . . .

53

DISCUSSION ..........................................

55

SUMMARY AND CONCLUSIONS

59

BIBLIOGRAPHY

........................

........................

62

In tr o d u c tio n R e se a rc h on t h e i o d i n a t i o n o f p r o t e i n s d a te s b a ck t o t h e l a t t e r p a r t o f t h e n i n e t e e n t h c e n tu r y fo llo w in g Baumann* s d is c o v e r y (1 ) o f io d in e i n o rg a n ic c o m b in a tio n i n t h e t h y r o i d g la n d .

In a d d itio n to

g iv in g much im p e tu s t o i n v e s t i g a t i o n s on t h e n a t u r a l t h y r o i d horm one, th e s e e a r l y d i s c o v e r i e s l e d t o a number o f a tte m p ts t o d u p l i c a t e t h e a c t i v e s u b s ta n c e a r t i f i c i a l l y by t h e i o d i n a t i o n o f p r o te in s #

I n 1915,

K e n d a ll (2 2 ) i n a s e r i e s o f c l a s s i c a l e x p e rim e n ts su c c e e d e d i n i s o l a t i n g th y r o x in e from t h e t h y r o i d and to d a y m ost w o rk e rs f e e l t h a t t h i s sub­ s ta n c e i s t h e c i r c u l a t i n g t h y r o id horm one. P a r a l l e l e x p e rim e n ts on a r t i f i c i a l l y i o d in a t e d p r o t e i n s f i n a l l y r e s u l t e d i n t h e I s o l a t i o n o f c r y s t a l l i n e th y r o x in e b y Ludwig and von M utzenbecher ( 2 5 ) .

S u b seq u en t r e s e a r c h h a s b e e n d i r e c t e d p r i n c i p a l l y

to w ard t h e im provem ent o f t h e i o d i n a t i o n m ethod t o form p r o d u c ts w ith a h ig h e r th y r o x in e c o n te n t, and d e te i? n in a tio n o f t h e e f f e c t s o f a c t i v e io d in a te d p r o t e i n s on v a r io u s p h y s i o l o g ic a l p r o c e s s e s . R e c e n tly , E ein ek e and T u rn e r (2 ? ) h a v e r e p o r te d t h e s y n t h e s i s o f io d in a t e d c a s e i n w hich shows a h ig h d e g re e o f t h y r o id a c t i v i t y .

They

h a v e c a r r i e d o u t a number o f i n v e s t i g a t i o n s t o d e te rm in e t h e p o s s i b i l i t y o f i n f lu e n c i n g v a r io u s p r o d u c tiv e p r o c e s s e s i n d o m estic a n im a ls by t h e a d m i n i s t r a t io n o f t h i s s u b s ta n c e .

E x p e rim e n ts o f t h i s ty p e w ere b a se d

on t h e c o n c e p t t h a t th ro u g h i t s in f lu e n c e on g e n e r a l m e ta b o lism , and p o s s ib l y on e n d o c rin e sy ste m s i n t e r r e l a t e d w ith t h e t h y r o i d , t h e a d m in is­ t r a t i o n o f s m a ll am ounts o f t h y r o i d a l s u b s ta n c e w i l l f a v o r a b ly in f lu e n c e c e r t a i n p h y s i o l o g ic a l f u n c t i o n s such a s m ilk p r o d u c tio n , egg p ro d u c tio n o r g ro w th .

2 P a r t o f t h i s work (5 8 ) d e a l t w ith c o m p ariso n s o f t h e amount o f t h y r o i d a l l y a c t i v e i o d i n a t e d p r o t e i n r e q u i r e d t o p ro d u c e a l a c t a t i o n r e s p o n s e i n d a i r y c a t t l e w ith t h e d o sa g e o f th y r o x in e t o p ro d u ce th e same e f f e c t when i n j e c t e d .

The r e s u l t s i n d i c a t e d t h a t i o d i n a t e d p r o t e i n

i s u t i l i z e d q i i t e i n e f f i c i e n t l y i n ru m in a n ts .

As ju d g e d b y t h e d o sa g e

o f i o d i n a t e d p r o t e i n r e q u i r e d t o c a u se a s ta n d a r d w e ig h t r e d u c t io n i n t h e s h e e p , o n ly 5% a s much i o d in a t e d p r o t e i n was r e q u ir e d by s u b c u ta n e o u s i n j e c t i o n a s when g iv e n o r a l l y .

Mhen t h e io d in a t e d p r o t e i n was p la c e d

d i r e c t l y i n t h e abomasum th ro u g h a perm anent c a n n u la i n o r d e r t o b y p a ss th e rum en, no i n c r e a s e i n t h e u t i l i z a t i o n was o b s e rv e d .

Thus t h e rumen

was r u l e d o u t a s a p o s s i b l e s i t e o f i n a c t i v a t i o n o f t h e a c t i v e p r i n c i p l e . The o r a l u t i l i z a t i o n was in c r e a s e d som evhat by p r e lim in a r y h y d r o l y s i s o f t h e i o d in a t e d p r o t e i n w ith a c i d , i n d i c a t i n g t h a t p o o r u t i l i z a t i o n o b ta in e d w ith t h e w hole p r o t e i n may b e p a r t l y due t o i n c o s ^ l e t e d i g e s t i o n . However, i t was f e l t t h a t f u r t h e r l i ^ t c o u ld be th ro w n on t h e s u b j e c t o f a b s o r p tio n o f i o d in a t e d c a s e i n from th e a lim e n ta r y t r a c t o f ru mi na n ts i f th e m a t e r i a l f o r a d m i n i s t r a t i o n was l a b e l e d w ith one o f th e r a d i o a c t i v e i s o t o p e s o f i o d i n e , nam ely io d in e - 1 3 1 .

By d o in g s o , t h e

b lo o d l e v e l o f i o d in a t e d c a s e i n , o r i t s m e t a b o l i te s , may be d e te rm in e d q u i te s i n g l y b y c a r r y in g o u t r a d i o a c t i v e a s s a y s on t h e b lo o d a t p e r i o d i c i n t e z v a l s fo llo w in g t h e a d m i n i s t r a t io n o f t h e i o d i n a t e d p ro te in * The te c h n iq u e o f i s o t o p e d i l u t i o n w hich h a s become i n c r e a s i n g l y p o p u la r w ith w o rk e rs a tte m p tin g t o i d e n t i f y s u b s ta n c e s p r e s e n t i n q u a n t i t i e s to o sm a ll t o be d e te rm in e d b y g r a v im e tr ic m eth o d s, was a ls o enqployed h e r e f o r t h e p a r t i a l i d e n t i f i c a t i o n o f t h e m a t e r i a l a b so rb e d from th e a lim e n ta r y t r a c t and a p p e a rin g i n t h e b lo o d s tre a m .

A lth o u g h t h e sh e e p was t h e c h i e f an im al o f i n t e r e s t i n t h i s w ork, p r e lim in a r y s t u d i e s w ere a l s o c a r r i e d o u t on t h e r a t and t h e cow a s a f u r t h e r a id t o t h e e l u c i d a t i o n o f t h e p ro b le m .

L ite ra tu re Baumann (1 ) d is c o v e r s i o d in e i n th e t h y r o i d g la n d i n o rg a n ic c o m b in a tio n . H u tc h iso n (2 ) r e p o r t s t h a t t h e g r e a t e r p a r t o f t h e io d in e o f t h e th y r o id o c c u rs i n c o m b in a tio n w ith th y r o g lo b u lin , th u s e s t a b l i s h i n g t h e f a c t t h a t t h e a c t i v e s u b s ta n c e i s a c t u a l l y i o d o p r o te in . H arin g to n (3 ) s t a t e s t h a t th e io d in e i n th e th y r o id g la n d e x i s t s i n t h e form o f two w ell-know n e n t i t i e s , nam ely d i io d o t y r o s i n e and th y ­ r o x i n e , a c c o u n tin g f o r p r a c t i c a l l y a l l t h e o rg a n ic io d in e i n t h e g la n d . T hese two compounds a r e n o t p r e s e n t i n t h e f r e e form b u t a r e com bined w ith o t h e r am ino a c id s t o form t h e c h a r a c t e r i s t i c t h y r o id p r o t e i n , th y r o g lo b u l i n . H a rin g to n (4 ) re v ie w s t h e e v id e n c e i n f a v o r o f t h e h y p o th e s is t h a t d i io d o t y r o s i n e i s t h e p r e c u r s o r o f th y ro x in e i n t h e th y r o id g la n d . l i e b r i c h t ($ ) com bines io d in e w ith c a s e in i n a b a s ic s o l u t i o n and o b ta in s a p ro d u c t s i m i l a r t o t h e i o d o th y r i n i s o l a t e d by Baumann from t h e th y r o id g la n d . Boehm and Berg (6 ) p r e s e n t r e s u l t s t o show t h a t p r o t e i n s w i l l com­ b in e w ith io d in e i n o rg a n ic c o m b in a tio n .

I n t e r e s t i n th e s e r e a c t i o n s

was i n t e n s i f i e d when l a t e r i t was r e p o r te d t h a t io d in e was s i m i l a r l y bound t o p r o t e i n s i n t h e t h y r o i d . Morse (7 ) i s f i r s t t o c la im t h a t a r t i f i c i a l l y io d in a t e d p r o t e i n s p o s s e s s p h y s i o l o g ic a l a c t i v i t y . Lerman and S a l t e r (8 ) f i n d t h a t i o d in a t e d p r o t e i n s and t h e i r de­ g r a d a tio n p r o d u c ts a r e e f f e c t i v e i n t h e r e l i e f o f myxadema i n man* E ein ek e and T u rn er (9 ) p r e s e n t d a t a t o show t h a t io d in a te d p r o t e i n s

p r e p a r e d u n d e r o p tim a l c o n d itio n s have s e v e r a l tim e s th e t h y r o i d a l a c t i v i t y o f s ta n d a r d t h y r o id p o w ie r Ü .S .P . by b i o l o g i c a l a s s a y , o r by t h e c h e m ic a l c o n te n t o f th y r o x in e . R ein eke and T u rn e r (1 0 ) r e p o r t t h a t t h e pH o f t h e s o l u t i o n a f f e c t s b o th th e t h y r o i d a l a c t i v i t y o f t h e i o d in a t e d p r o t e i n and i t s th y r o x in e c o n te n t. R ein ek e (1 1 )

s t a te s t h a t th e th y r o id a l a c t i v i t y o f io d in a te d

p r o t e i n s may b e in c r e a s e d b y c o n t r o l l i n g t h e d e g re e o f i o d i n a t i o n . R e in e k e , W illia m s o n and T u rn e r (12) i n v e s t i g a t e d t h e e f f e c t o f tem ­ p e r a t u r e on t h e t h y r o i d a l p o te n c y o f i o d in a t e d c a s e i n and soybean p r o t e i n . H o fm e is te r ( 1 3 ) , Blum (1 4 ) and Oswald ( 1 5 ) , ( l 6 ) , ( 1 7 ) , (1 8 ) d e s c r ib e i n d e t a i l th e c h e m ic a l ch an g es t a k i n g p la c e d u rin g t h e i o d i n a t i o n o f v a r io u s p r o t e i n s . K u ra je e f (1 9 ) u s in g t h e n o w -e s ta b lis h e d m ethods f o r i o d i n a t i o n o f p r o t e i n s shows t h a t t y r o s i n e and try p to p h a n a r e t h e amino a c id s c h i e f l y a lte r e d by io d in a tio n . P a u ly (2 0 ) shows t h a t i o d in e a l s o s u b s t i t u t e s i n th e im id a z o le r i n g o f h i s t i d i n e a s w e l l a s o x id iz in g th e SH g ro u p o f c y s t i n e . S p ie g e l—A dolph, H am ilto n and Henny (2 1 ) fo llo w in g a s tu d y o f t h e X -ra y d i f f r a c t i o n p > atterns o f t y r o s i n e , d iio d o t y r o s i n e and th y r o x in e c o n c lu d e t h a t i o d i n a t i o n c a u s e s some s t r u c t u r a l re a rra n g e m e n t o f t h e amino a c id s i n th e p r o t e i n s . K e n d a ll (2 2 ) i s o l a t e s c r y s t a l l i n e th y r o x in e from t h e t h y r o id g lan d o f hogs by h y d r o l y s i s . H a rin g to n (2 3 ) d ed u ces th e c h e m ic a l c o n f i g u r a ti o n o f th y r o x in e and l a t e r (2 4 ) p r e s e n t s a m ethod o f s y n t h e s i s o f d , 1 - th y r o x in e .

Ludvdg and von M utzen becher ( 2 5 ) , (2 6 ) r e p o r t t h e i s o l a t i o n o f c r y s t a l l i n e th y ro x in e fro m a r t i f i c i a l l y i o d in a t e d p r o te in s # R ein ek e and T u rn e r (2 7 ) r e p o r t a much h ig h e r y i e l d o f c r y s t a l l i n e th y r o x in e fro m i o d in a t e d c a s e i n th a n h a s b e ^ o b ta in e d b e f o r e . H a rin g to n and S a l t e r (2 8 ) o b t a i n a m in u te amount o f 1 - th y r o x in e b y t h e h y d r o ly s is o f t h y r o i d w ith p r o t e o l y t i c enzym es. R ein ek e and T u rn e r (2 9 ) f i n a l l y su c ce ed i n i s o l a t i n g 1 - th y r o x in e from io d o c a s e in .

They c la im t h a t i t p o s s e s s e s tw ic e t h e t h y r o i d a l

p o te n c y o f t h e d , l m ix tu r e . B lo ck (3 0 ) von M utzenbecher ( 3 1 ) , Jo h n so n and Tew kesbury (3 2 ), B a r k d o ll and Ross (3 3 ) and H a rin g to n (3 4 ) p r e s e n t m ethods o f p r e p a r in g th y r o x in e from d iio d o ty r o s in e *

R ein ek e and T u rn e r (3 5 ) m en tio n t h a t p r e ­

p a r a t i o n b y t h i s method in v o lv e s th e same c o n d itio n s a s th o s e n e c e s s a r y f o r t h e p r e p a r a t i o n o f t h y r o i d a l l y a c t i v e io d in a te d p r o t e i n s .

Jo h n so n

and Tevdcesbury (32) a l s o o u t l i n e a p l a u s i b l e r e a c t i o n m echanism f o r t h i s c o n v e r s io n , in v o lv in g t h e o x i d a t i v e c o u p lin g o f two m o le c u le s o f d iio d o ­ t y r o s i n e w ith Idle e li m in a ti o n o f one s i d e c h a i n . H a rin g to n and P i t t - R i v e r s (3 6 ) c o n c lu d e t h a t th e above m echanism a l s o a p p l i e s i n th e fo r m a tio n o f th y r o x in e d u rin g t h e i o d i n a t i o n o f p ro te in s . R ein ek e and T u rn e r (3 7 ) s t a t e t h a t t h e th y ro x in e c o n te n t o f i o d in a t e d c a s e in i s i n c r e a s e d when p re p a re d i n t h e p re s e n c e o f m anganese compounds. Ray and D eysach (3 8 ) s t a t e t h a t th e th y r o id h a s a s p e c i a l c a p a c i ty t o s t o r e m anganese.

T h is m anganese may a c t i n v iv o a s w e l l a s i n v i t r o

i n p ro m o tin g t h e o x i d a t i v e f o rm a tio n o f t h y r o x in e . H a m ilto n , Power and A lb e r t (3 9 ) c la im t h a t t h e th y r o x in e c o n te n t o f io d o c a s e in i s in c r e a s e d b y t h e u s e o f a m anganese c a t a l y s t p re p a re d b y th e

7 m ethod o f F riedem aim and K e n d a ll ( 4 0 ) . K o h le r (4 1 ) d e s c r ib e s an open a s h in g m ethod f o r d e te r m in in g t h e i o d in e c o n te n t o f io d in a te d c a s e i n .

I t i s c la im e d t o g iv e r e p r o d u c ib le

and a c c u r a te r e s u l t s on m a t e r i a l s c o n ta in in g 0 .1 t o 10^ io d in e # S hahrokh ( 4 2 ) o u t l i n e s a m ethod f o r d e te rm in in g i o d in e i n a r t i f i c ­ i a l i o d o p r o te in s W iich , i t i s c la im e d , i s s a t i s f a c t o r y f o r i o d o p r o te in s c o n ta in in g a s much a s 13^ i o d i n e . H arvey (4 3 ) p r e s e n t s a c o m p le te su rv e y and c r i t i q u e o f m ethods f o r d e te rm in in g i o d i n e i n b i o l o g i c a l m a t e r i a l s . Roche and M ich el (4 4 ) r e p o r t a n e f f i c i e n t m ethod f o r t h e a n a l y s i s o f th y ro x in e b a s e d upon t h e d i s t r i b u t i o n o f t h a t s u b s ta n c e i n n - b u t a n o l . Le%#is (4 5 ) g iv e s a c o m p reh en siv e r e p o r t on t h e a n a l y t i c a l c h a r a c t e r ­ i s t i c s o f i o d i n a t e d p r o t e i n s and d ry th y r o id pow der.

I t i s s t a t e d i^ ia t

i s o - e l e c t r i c p r e c i p i t a t i o n i s s u f f i c i e n t t o rem ove in o r g a n ic i o d in e . M ention i s a l s o made o f a new c h e m ic a l a s s a y m ethod f o r th y r o x in e . Lugg ( 4 6) g iv e s a d e t a i l e d r e p o r t on t h e d e te r m in a tio n o f t y r o s i n e i n p r o t e i n s and s t a t e s t h a t t h o s e w ith a h ig h t y r o s i n e c< xitent a r e th o s e w hich a r e m ost s u i t a b l e f o r t h e p r e p a r a tio n o f t h y r o a c ti v e p r o t e i n s by io d in a tio n . S a l t e r e t a l ( 4 7 ) , ( 4 8 ) , (4 9 ) u s e s t h e c o r r e c t i o n o f myxadema and e l e v a t i o n o f t h e m e ta b o lic r a t e i n man t o m easu re t h e p o te n c y o f i o d in ­ a te d serum p r o t e i n s . G u d e rsn a tc h ( 5 0 ) , (5 1 ) i s f i r s t t o r e p o r t t h e d ra m a tic m etam orphosis o f am phibian l a r v a e v*ien f e d a sm a ll amount o f th y r o id s u b s ta n c e . Wokes ( 52) shows t h a t th e p e rc e n ta g e d e c r e a s e i n body l e n g t h o f a ta d p o le g iv e n t h y r o id s u b s ta n c e , b e a r s a s t r a i g h t l i n e r e l a t i o n s h i p t o th e lo g o f t h e d o s a g e .

s R ein ek e and T u rn e r (53) a p p ly in g t h e ta d p o le m ethod t o th e q u a n t­ i t a t i v e a ssa y o f th e th y r o id a l a c t i v i t y o f io d in a te d p r o te in s , fin d t h a t th e ta d p o le w i l l re s p o n d when i t i s i n j e c t e d i n t o t h e body c a v ity #

T h is

m a t e r i a l d i f f e r s from t h y r o id p o v d e r o r th y r o x in e i n t h a t i t i s n o t a b so rb e d when p la c e d i n s o l u t i o n around t h e ta d p o le # W h ite , M cGinty, A nderson and W h ite ( 5 4 ) , D r e s s ie r and R o llin g (5 5 ) s u c c e s s f u l l y employ t h e e l e v a t i o n o f m e ta b o lic r a t e and d e c r e a s e i n body w e ig h t o f t h e g u in e a p ig a s a m ethod o f a s s a y o f t h y r o i d a l l y a c t i v e s u b s ta n c e s # MLxner, R eineke and T u rn e r (5 6 ) r e p o r t a more c o n v e n ie n t a s s a y m ethod b a se d om t h e a b i l i t y o f t h y r o i d a l l y a c t i v e m a t e r i a l t o re d u c e t h e e n la rg e d th y r o id s o f t h i o u r a c i l t r e a t e d c h ic k s and r a t s #

The p e rc e n ta g e r e d u c tio n

i n t h y r o i d w e l ^ t o f t h e t h i o u r a c i l t r e a t e d a n im a ls g iv e n g ra d e d am ounts o f th y r o x in e shows good p r o p o r t i o n a l i t y w ith d o sa g e , and a g re e s w e ll w ith t h e r e s u l t s o f m etab o lism m easurem ents, i t i s claim ed# J a n v i e r (5 7 ) f i n d s t h a t i o d in a te d p r o t e i n s have a l a c t a t i o n s tim ­ u l a t i n g a c t i o n when g iv e n t o p re g n a n t women. R ein e k e and T u rn e r (5 8 ) d e m o n s tra te t h a t a r t i f i c i a l t h y r o p r o te in s w i l l s t i m u l a t e m ilk p r o d u c tio n when f e d f o r s h o r t p e r io d s t o g o a ts i n d e c l in i n g l a c t a t i o n .

I n c r e a s e s w ere n o te d i n th e m ilk y i e l d o f l a c t a t i n g

cows a s w e ll# Graham ( 5 9 ) , (6 0 ) r e p o r t s t h a t t h e a d m i n i s t r a t io n o f t h y r o i d sub­ s ta n c e o r th y r o x in e t o n o rm al cows w H l c a u s e a r a p i d i n c r e a s e i n b o th m ilk y i e l d and p e rc e n ta g e o f f a t i n m ilk# T u rn e r (6 1 ) c a l l s a t t e n t i o n t o t h e p o s s i b i l i t y o f s t i m u l a ti n g m ilk p r o d u c tio n i n d a i r y c a t t l e b y th e u s e o f t h y r o a c t i v e i o d in a t e d p r o t e i n s . R e in e k e and T u rn e r (6 2 ) p r e s e n t d a ta on t h e a c t u a l s t i m u l a ti o n o f

l a c t a t i o n o f cows b y f e e d in g i o d i n a t e d p r o t e i n s . B l a x t e r (6 3 ) w orking i n E n g la n d , r e p o r t s f a v o r a b le r e s u l t s fromi f e e d in g i o d in a t e d p r o t e i n s t o l a c t a t i n g cow s. R ein ek e and T u rn e r ( 6 4 ) c la im t h a t fo llo w in g o r a l a d m i n i s t r a t io n o f i o d i n a t e d c a s e i n t o d a i r y c a t t l e , no t h y r o id hormone p a s s e s i n t o th e m ilk .

T h is c o n c lu s io n was re a c h e d fo llo w in g an e x p e rim e n t w h e re in 100 m l.

o f m ilk from cows r e c e i v i n g a h ig h d osage o f i o d in a t e d p r o t e i n was i n t u r n f e d t o g u in e a p i g s . n o te d .

No m e ta b o lic ch an g es i n t h e g u in e a p ig s was

S i m ila r r e s u l t s a r e o b ta in e d i n t r i a l s w ith th y ro id e c to m iz e d g o a ts .

E ein ek e and T u rn e r (6 5 ) fro m co m p ariso n s o f t h e amount o f th y r o id ­ a l l y a c t i v e i o d i n a t e d p r o t e i n r e q u ir e d to p ro d u ce a l a c t a t i o n re s p o n s e i n d a i r y c a t t l e w ith t h e d o sag e o f th y r o x in e to p ro d u ce th e same e f f e c t when i n j e c t e d , i n d i c a t e t h a t i o d in a t e d p r o t e i n i s u t i l i z e d q u i t e i n e f f i c i e n t l y i n ru m in a n ts .

When t h e i o d in a t e d p r o t e i n was p la c e d d i r e c t l y i n t o t h e

abomasum o f t h e sh e ep i n o r d e r t o by—p a s s t h e rum en, no i n c r e a s e i n u t i l i z a t i o n was n o te d .

Thus t h e rumen i s r u l e d o u t a s a p o s s i b l e s i t e

o f in a c tiv a tio n o f th e a c tiv e p r in c ip le .

The o r a l u t i l i z a t i o n was s l l ^ t l y

in c r e a s e d b y th e p r e lim in a r y h y d r o ly s is o f t h e i o d in a t e d p r o t e i n w ith a c i d , i n d i c a t i n g t h a t t h e p o o r u t i l i z a t i o n o b ta in e d w ith t h e w hole p r o t e i n may b e p a r t l y due t o i n c o n p le te d i g e s t i o n . K raus and Mouvre (6 6 ) s t a t e t h a t m ilk pro d u ced b y a n im a ls f e d on i o d i n e - r i c h fo o d s c o n ta in s o n ly a t r a c e o f t h e i o d in e f e d . S c h a r r e r (6 7 ) c la im s t h a t t h e i o d in e i n m ilk i s m o s tly i n o rg a n ic c o m b in a tio n , o n ly a s m a ll f r a c t i o n b e in g i n th e in o r g a n ic form*

The serum

o f m ilk c o n ta in s m ost o f t h e i o d i n e . R ein ek e and T u rn e r (6 8 ) show t h a t t h e o r a l a d m i n i s t r a t i o n o f th y r o ­ a c t i v e p r o t e i n t o young th y ro id e c to m iz e d g o a ts a r r e s t e d t h e symptoms o f

10 c r e t i n i s m and s tim u la te d grow th a p p ro a c h in g t h e n o rm a l. K oger and T u rn e r ( 6 9 ) fo llo w in g o r a l a d m i n i s t r a t io n o f io d o c a s e in o v e r b ro a d ra n g e s o f d o s a g e , r e p o r t th e a c c e l a r a t i o n o f t h e grow th r a t e o f r a t s , r a b b i t s o r g u in e a p i g s .

H ow ever, m ice showed a d e f i n i t e i n c r e a s e .

P a r k e r ( 7 0 ) , I r w i n , R ein ek e and T u rn e r ( 7 1 ) , (7 2 ) p r e s e n t e v id e n c e t o show t h a t when th e p h y s i o l o g ic a l d o s e s o f io d o c a s e in a r e g iv e n o r a l l y t o grow ing c h ic k s , t h e r e was a s i g n i f i c a n t s t i m u l a ti o n o f f e a t h e r g ro w th . Grew (7 3 ) r e p o r t s th e r e j u v ^ i a t i o n o f aged fo w ls by t h e a d m in is tr a ­ tio n o f d e s ic c a te d th y r o id .

T here was a ls o an i n c r e a s e i n egg p r o d u c tio n .

T u rn e r, K em pster, H a l l and E ein ek e (7 4 ) s t a t e t h a t t h e egg p ro d u c t­ io n o f h e n s was m a in ta in e d th ro u g h o u t th e h o t w e a th e r o f summer a t t h e c u sto m a ry w in te r l e v e l , fo llo w in g t h e o r a l a d m i n i s t r a t io n o f io d o c a s e in . T h is i s t h e p e rio d when t h e r a t e o f t h y r o id hormone s e c r e t i o n i s n o rm a lly d e c re a se d . B ran d s (7 5 ) s t a t e s t h a t t h e grow th o f p ig s i s s tim u la te d b y t h e o r a l a d m i n i s t r a t io n o f io d o c a s e in . l iv in g o o d and S ea b o rg (7 6 ) d is c u s s t h e p r e p a r a t i o n o f io d in e -1 3 1 b y t h e d e u te r o n bombardment o f t e l l e r i u m i n th e c y c l o t r o n . A b e rso ld (7 7 ) a ls o r e p o r t s t h e p r e p a r a t i o n o f io d in e -1 3 1 from t h e u ran iu m c h a in —r e a c t i n g p i l e , b o th by n e u tr o n bombardment o f t e l l e r i u m and a s a f i s s i o n p ro d u c t o f Ü-235* P e rlm a n , M orton and C h a ik o ff (7 8 ) o u t l i n e a p ro c e d u re f o r o b ta in in g r a d io a c tiv e io d in e , c a r r i e r - f r e e . Downing, D eutsch and R o b e rts (7 9 ) p r e s e n t a d i s c u s s i o n o f th e d ecay scheme o f io d in e —131# M a rs h a ll ( ^ ) g iv e s t h e v a lu e h e d e te rm in e d f o r t h e h a l f - v a l u e a b s o r p tio n o f t h e b e t a r a y o f io d in e —131 i n aluminum).

11 F e a th e r (8 1 ) p r e s e n t s a n e m p ir ic a l e q u a tio n r e l a t i n g t h e maximum e n e rg y o f a b e t a r a y , and i t s ra n g e i n th ic k n e s s o f aluminum#

The equa­

t i o n i s n o t a p p li c a b l e t o b e t a r a y s h a v in g an e n e rg y maximum g r e a t e r th a n 0 .6 ME7. l i b b y (8 2 ) d is c u s s e s th e te c h n iq u e s in v o lv e d i n t h e c o u n tin g o f i s o t o p e s , such a s io d in e - 1 3 1 , w hich have low e n e rg y d i s i n t e g r a t i o n schem es « B e rlo v ic h (8 3 ) p r e s e n t s some f a u l t s o f e x i s t i n g t h e o r i e s o f m is s e s i n r e c o r d s w ith Gr-M c o u n te r s .

A new fo rm u la i s d e riv e d f o r com puting

c o r r e c t i o n s and m is s e s . Schoup and O dishaw (8 4 ) re v ie w p r o g r e s s i n n u c le a r r e a c t i o n s . H evesey (8 5 ) re v ie w s p a p e rs p u b lis h e d p r i o r t o 1939 d e a lin g w ith t h e u s e o f a l l a v a i l a b l e r a d i o a c t i v e i s o t o p e s a s p h y s i o l o g ic a l t r a c e r e le m e n ts . H evesey (8 6 ) p r e s e t s a le n g th y d is c u s s io n o f t h e a p p li c a t io n o f r a d i o a c t i v e i n d i c a t o r s i n b io c h e m is tr y , a n im a l p h y s io lo g y and p a th o lo g y . P a r t i c u l a r l y u s e f u l i s th e s e c t i o n d e v o te d t o sh o rtc o m in g s o f r a d i o ­ a c tiv e in d ic a to r s . H evesey (8 ? )

d i s c u s s e s t h e u s e o f la b e le d atom s i n b i o l o g i c a l

re se a rc h . H evesey (8 8 ) d is c u s s e s t h e a p p l i c a t i o n o f r a d i o a c t i v e is o t o p e s i n b i o l o g i c a l r e s e a r c h , s t a t i n g t h a t t h e m ost im p o r ta n t a p p l i c a t i o n s a r e t o be found i n t h e d e te r m in a tio n o f r a t e , p la c e and seq u en ce o f fo r m a tio n o f t h e o rg a n ic c o n s t i t u e n t s o f t h e b o d y . L isc o (8 9 ) p r e s e n t s a n i n t e r e s t i n g d is c u s s io n on t h e p ro p e r d o sa g e l e v e l s f o r a d m i n i s t r a t i o n o f r a d i o a c t i v e i s o t o p e s t o a n im a ls and man. S to n e (9 0 ) d is c u s s e s a p p l i c a t i o n s o f n u c le o n ic s t o b i o l o g i c a l r e s e a r c h and t o d ia g n o s is and th e r a p y i n m e d ic in e .

12 B a le ( 9 1) p r e s e n t s a d i s c u s s i o n i n d i c a t i n g t h e o r d e r a p p ro x im a tin g th e e a s e id iic h e le m e n ts may b e u s e d a s r a d i o a c t i v e i n d i c a t o r s i n b i o ­ lo g ic a l re se a rc h . Rawson and M cArthur (9 2 ) re v ie w th e c l i n i c a l and t h e r a p e u t i c a p p l i ­ c a tio n s o f ra d io a c tiv e io d in e . H a m ilto n and Law rence (9 3 ) g iv e f i g u r e s on t h e d o sa g e o f io d in e - 1 3 1 which w i l l by i n j e c t i o n , b r in g ab o u t a lm o st conç>lete d e s t r u c t i o n o f t h e t h y r o id i n dogs and r a b b i t s . F in d le y and L eblond (94) s t a t e t h a t t h e y w ere a b le t o b r i n g a b o u t p a r t i a l d e s t r u c t i o n o f t h e r a t th y r o id u s in g l a r g e d o s e s o f io d in e - 1 3 1 . Mann and L eblond (9 5 ) r e p o r t on t h e c h e m ic a l tr a n s f o r m a tio n o f io d in e f i x e d b y th e t h y r o i d g la n d . A r i e l l and h i s a s s o c i a t e s (96) d e m o n s tra te w ith r a d i o a c t i v e i o d i n e , t h a t th o u g h th e t h y r o id c o l l e c t s by f a r t h e h i p e s t p e r c e n ta g e o f t h e a d m in is te r e d io d in e , lu n g and k id n e y t i s s u e c o l l e c t s m ore r e l a t i v e l y th a n do th e o t h e r t i s s u e s o f t h e bo d y . P e rlm a n , M orton and C h a ik o ff (9 7 ) r e p o r t t h a t fo llo w in g t h e o r a l a d m i n i s t r a t io n o f io d in e - 1 3 1 , 50^ o f t h e a d m in is te r e d d o se a p p e a re d i n t h e t h y r o id 5 h o u rs a f t e r a d m i n i s t r a t io n and a s much a s 65^ i s found t h e r e 25 and 30 h o u rs l a t e r . Mann, L eblond and W arren (98) d i s c u s s th e d i s t r i b u t i o n o f r a d i o ­ a c t i v e i o d i n a t e d compounds i n th e t h y r o i d , fo llo w in g t h e i n j e c t i o n o f io d in e -1 3 1 i n t o d o g s . B a rry (9 9 ) d e s c r i b e s a c o n v o i e n t m ethod f o r m ea su rin g io d in e -1 3 1 i n t h e n o rm al r a t t h y r o i d .

I n s te a d o f th e u s u a l m ethod, w hereby a l l t h e

i o d in e i s c o n v e rte d i n t o sodium i o d id e w ith 10^ sodium h y d ro x id e , a compound o f p a lla d iu m i s added w hich form s a p r e c i p i t a t e o f p a lla d iu m

13

io d id e w ith n e a r l y q u a n t i t a t i v e r e c o v e ry o f t h e io d in e - 1 3 1 p r e s e n t . P e rlm a n , M orton and C h a ik o ff (1 0 0 ) c o n s id e r in g t h e q u e s tio n o f exchange o f r a d i o a c t i v e io d in e w ith t h e o r g a n i c a l l y bound i o d in e o f t h e t h y r o i d , f i n d t h a t Wien io d in e - 1 3 1 was added t o d e s i c c a t e d t h y r o i d t i s s u e and c a r r i e d th ro u g h t h e r e g u l a r a n a l y s t i c a l p ro c e d u re , t h e r a d i o ­ i o d in e was r e c o v e re d a lm o st q u a n t i t a t i v e l y i n t h e in o r g a n ic f r a c t i o n . L eb lo n d , P e a c o c k , G ro ss and Evans (101) r e p o r t on t h e d i s t r i b u t i o n o f i o d in a t e d compounds i n t h e r a t th y r o id fo llo w in g t h e i n j e c t i o n o f io d in e - 1 3 1 . L eb lo n d , G ro s s , P eacock and E vans (1 02) f i n d t h a t io d in e a f t e r e n te r in g t h e t h y r o id g la n d a s in o r g a n ic i o d i n e , was r a p i d l y t r a n s f e r r e d i n t o d iio d o t y r o s i n e and d e p o s ite d a s su ch i n th e c o l l o i d o f t h e th y r o id fo llic le . H e r tz , R o b e rts and E vans (1 0 3 ) f o llo w in g t h e in tr a v e n o u s i n j e c t i o n o f io d in e - 1 3 1 i n r a t s , f i n d t h a t t h e u p ta k e i n t h e t h y r o id r e a c h e s a maximum i n 10 m in u te s . E lm er ( 1 0 4 ), H am ilton and S o le y (105) r e p o r t t h a t l a r g e q u a n t i t i e s o f i o d i n e a r e e x c r e te d i n t h e u r i n e fo llo w in g th e in tr a v e n o u s i n j e c t i o n o f io d in e - 1 3 1 . L eblond (106) s t a t e s t h a t th e r o u t e o f a d m i n i s t r a t io n o f i o d i n e 131 d o es n o t a f f e c t i t s b e h a v io u r i n th e b o d y .

S i m i la r r e s u l t s w ere

o b ta in e d b y g iv in g t h e same d o se t o g u in e a p ig s e i t h e r by gavage o r by in tr a v e n o u s i n j e c t i o n . L eblond and Mann (1 0 ?) o b se rv e t h a t r a t s on a n i o d in e d e f i c i e n t d i e t su p p lem en ted b y so y a b ean s c o l l e c t more io d in e - 1 3 1 i n t h e i r t h y r o i d s t h a n do r a t s sim p ly on an io d in e d e f i c i e n t d i e t .

14 D voskin (108) a s s e r t s t h a t e le m e n ta ry i o d in e when g iv e n o r a l l y o r s u b c u ta n e o u s ly to t h e r a t o r c h ic k h a s a th y r o x in e —li k e a c t i o n . I o d id e d o es n o t p o s s e s s t h i s p ro p e rty * M o rto n , P erlm an , A nderson and C h a ik o ff (109) f i n d t h a t 24 h o u rs a f t e r t h e in tr a v e n o u s i n j e c t i o n o f i o d i n e , 80^ o f t h e i o d in e i n t h e p la sm a i s fo u n d i n th e th y r o x in e f r a c t i o n .

T h is c l o s e l y p a r a l l e l s th e c o n c e n t­

r a t i o n o f p ro te in -b o u n d i o d in e i n th e plasm a# S i l v e r (H O ) i n d i c a t e s t h a t a l l t h e io d in e n o rm a lly o c c u r r in g i n t h e b lo o d i s i n a form w hich d o es n o t p a s s t h r o u ^ a c e llo p h a n e membrane Wien t h e b lo o d i s d i a l y s e d a g a i n s t w a te r . B ru g e r and Member (1 1 1 ) show t h a t a l l th e io d in e o f p lasm a i s q u a n t i t a t i v e l y p r e c i p i t a t e d when z in c h y d ro x id e i s added a s t h e p r e c i p i t a ­ t i n g a g e n t.

They a ls o show t h a t when c r y s t a l l i n e th y r o x in e i s added t o

plasm a i t to o i s q u a n t i t a t i v e l y p r e c i p i t a t e d w ith t h i s r e a g e n t . S i l v e r (1 1 2 ) s t a t e s t h a t a l t h o u ^ m ost o f t h e i o d in e i n th e t h y r o id e x i s t s i n c o m b in a tio n w ith g l o b u l i n , i . e . t h y r o g lo b u l i n , t h i s i s n o t t h e c a s e i n th e p e r i p h e r a l b lo o d .

I n s t e a d , b o th th e alb u m in and g l o b u li n

f r a c t i o n s c o n ta in i o d i n e , t h e amount v a r y in g w ith t h e am ount o f p r o t e i n i n each f r a c t i o n .

U n p u b lish e d s t u d i e s show t h a t b o th alb u m in and g lo b u li n

have t h e a b i l i t y t o f i x d i io d o t y r o s i n e and th y r o x in e i n v i t r o . T revorrow and Lerman (1 1 3 ), (1 1 4 ) p r e s e n t p ro o f t o show t h a t plasm a io d in e i s n o t i n t h e fo rm o f th y r o g lo b u l i n . S a l t e r (1 15) f i n d s t h a t t h e i o d i n e i n plasm a i s a tt a c h e d t o s m a lle r p r o t e i n m o le c u le s . S a l t e r , B a s s e t and S a p p in g to n ( H 6 ) , L o w e n ste in , B ru g e r and H in to n (117) d i s c u s s t h e d e te r m in a tio n o f p ro te in -b o u n d i o d in e i n p lasm a a s a

15

m ethod o f d ia g n o s is f o r t h y r o id d is e a s e s * W in k le r, R iggs and Thompson ( 1 1 8 ) , T a lb o t, B u t l e r , S altm an and R o d rig u ez (119) r e p o r t t h e u s e o f t h e p ro te in -b o u n d f r a c t i o n o f p la s m a i o d in e a s an i n d ic a t o r * R ig g s, L a v ie te s and Man (1 2 0 ) r e p o r t i n g on t h e n a t u r e o f b lo o d io d in e s t a t e t h a t t h e b lo o d d o e s n o t c o n ta in f r e e i o d i n e , b u t t h a t i t i s in c o r p o r a te d i n t o t h e p lasm a p r o t e i n j u s t a s a n y o t h e r amino a c id may be* B a s s e t t , Coons and S a l t e r (121) d i s c u s s th e p ro te in -b o u n d i o d i n e i n b lo o d , from t h e s ta n d p o in t o f t h e n a t u r a l l y o c c u r r in g io d in e f r a c t i o n s and t h e i r c h e m ic a l b e h a v io r* M eans, L em an and S a l t e r ( 1 2 2 ), H a rin g to n (1 2 3 ) and S a l t e r (1 2 4 ) c o n c lu d e d t h a t th y r o x in e i s n o t t h e f o m o f th e c i r c u l a t i n g t h y r o id h o r ­ mone a s t h e b i o l o g i c a l a c t i v i t y o f t h e t h y r o g lo b u l i n c a n n o t b e c o r r e l a t e d w ith i t s th y r o x in e c o n te n t# T h o n ^ o n , B r a i l e y , Cohen and Thompson (125) r e a c h th e same c o n c lu s io n b a se d on t h e i r d is c o v e r y t h a t a n im a ls i n j e c t e d w ith th y r o x in e show a d e la y e d re sp o n se * H a rin g to n (1 2 6 ) fo llo w in g t h e s e r e s u l t s , p o s t u l a t e s i n 1935 t h a t t h e c i r c u l a t i n g hormone i s a p e p t i d e , c o n ta in in g b o th th y r o x in e and d iio d o ty r o s in e * H a rin g to n (1 2 7 ) how ever, r e c e n t l y r e e v a l u a ti n g t h i s o l d e r e v id e n c e h a s a r r i v e d a t t h e c o n c lu s io n t h a t t h e p e p tid e i s an u n n e c e s s a ry com­ p l i c a t i o n and t h a t th y r o x in e i t s e l f i s p ro b a b ly t h e hormone* W illia m s an d W h itte n b e rg e r (1 2 8 ) fo llo w in g a s tu d y d e a lin g w ith th e e f f e c t s o f t h y r o x in e , t h y r o g lo b u lin and t h y r o t r o p i n on t i s s u e r e s p i r a t i o n .

16

a r e l e d t o t h e c o n c lu s io n t h a t t h e a c t i v e fo rm o f th e t h y r o id horm one i s p r o b a b ly a th y r o x in e p e p tid e *

T h e ir m ain e v id e n c e i s t h a t th y r o x in e

h a s no c a l o r i g e n i c e f f e c t on l i v e r s l i c e s , v diereas th y r o g lo b u l i n d id * T h is l a t t e r r e s u l t was n o t c o n firm e d by W illiam s-A shm an (129)# C h a ik o ff , T aurog and R e in h a rd t (130) r e p o r t a m ethod f o r o b ta in in g t h e l a b e le d t h y r o id hormone i n i t s n o rm a l p h y s i o l o g ic a l s t a t e # f r e e io d in e -1 3 1 i s i n j e c t e d i n tr a v e n o u s ly i n t o r a t s .

C a rrie r-

The i s o t o p e i s

r a p i d l y ta k e n u p by t h e t h y r o id g la n d , c o n v e rte d t h e r e t o o rg a n ic io d in e , and t h e n r e l e a s e d i n t o th e p la s m a .

W ith in 24 h o u r s , a b o u t 90^ o f t h e

r a d i o a c t i v e io d in e i n th e p lasm a i s p r o te in - b o u n d , and n e a r l y a l l t h i s p e r c ^ t a g e i s p resu m ab ly th e t h y r o id hormone* Z i l v e r s m i t , Entenman and F i s h i e r (1 3 1 ) g iv e a fo rm u la f o r f i n d in g t h e r e l a t i o n betw een t h e r a t e o f d e c r e a s e o f t h e r a d i o a c t i v i t y i n t h e plasm a and t h e r a t e a t vAiich t h e new ly form ed p r o te in -b o u n d i o d in e a p p e a rs. T aurog, C h a ik o ff and Entenm an (1 32) i n a p a p e r c o n c e rn e d w ith t h e r a t e o f t u r n o v e r o f p r o te in - b o u n d io d in e i n t h e b lo o d , r e p o r t t h a t w ith in 7 h o u r s , th e t h y r o id o f t h e dog s e c r e t e s a n am ount o f hormone e q u a l t o t h e p ro te in -b o u n d i o d in e o f t h e e n t i r e c i r c u l a t i n g v o lu m e. S c h u ltz e and T u rn e r (133) e s tim a te t h e r a t e o f t h y r o id s e c r e t i o n o f t h e fo w l, g o a t and cow by f i r s t t r e a t i n g t h e a n im al w ith t h i o u r a c i l i n o r d e r t o i n h i b i t t h e fo rm a tio n o f t h y r o id horm one.

T h y ro x in e was th e n

g iv e n i n g rad e d d o se s u n t i l an i n c r e a s e i n t h y r o id s i z e was n o t e d .

T h is

i n t u r n r e p r e s e n t s th e norm al o u tp u t o f t h e th y r o id * S a l t e r e t a l (134) u s in g r a d i o a c t i v e io d in e - 1 3 1 was a b le t o c a lc u ­ l a t e t h e tu r n o v e r r a t e o f th y r o x in e i n t h e r a t .

A new m ethod o f c o u n tin g

17 sam p les i s g iv e n , vdiich e li m in a te s many o f th e in d e te r m in a te e r r o r s w hich p la g u e r e s e a r c h w o rk e rs m aking s t u d i e s o f r a d i o a c t i v i t y i n b i o l o g i c a l sy s te m s. H evesey (1 3 5 ) a s earT y a s 1924 p o i n ts o u t t h a t a s im u lta n e o u s s tu d y o f r a d i o a c t i v e an d s t a b l e is o t o p e s i n a b i o l o g i c a l sy ste m c o u ld be u se d t o d e te rm in e th e tu r n o v e r r a t e .

T h is o f c o u r s e a p p l i e s o n ly to sy stem s

w here t h e t o t a l c o n c e n tr a ti o n re m a in s c o n s t a n t , S a l t e r and M c A llis te r (1 3 6 ) p r e s e n t a n e x c e l l e n t d is c u s s io n on t r a c i n g t h e th y r o id hormone i n p e r i p h e a l t i s s u e s .

They f i n d t h a t th y ­

ro x in e added t o t h e b lo o d does n o t f o llo w t h e r e s p o n s e o f t h e o r g a n i c a l l y bound i o d in e t o c e r t a i n c h e m ic a l a g e n ts . Taurog and C h a ik o ff (1 3 7 ) s t a t e t h a t 90^ o f t h e io d in e i n p lasm a may b e removed b y b u t y l a lc o h o l e x t r a c t i o n . i n t h e th y r o x in e f r a c t i o n .

Of t h i s am ount, 80^ s t a y s

They s tr e n g th e n t h e i r c la im t h a t th y r o x in e

i s t h e c i r c u l a t i n g th y r o id hormone by a l s o show ing t h a t th e t h y r o id hormone o f p la sm a , l a b e le d by means o f io d in e - 1 3 1 , b e h av e s e x a c t l y t h e same a s added th y r o x in e c a r r i e r , a s ju d g ed by t h e l a t t e r * s r e c r y s t a l l ­ i z a t i o n t o c o n s ta n t s p e c i f i c a c t i v i t y and by i t s d i s t r i b u t i o n betw een two im m isc ib le s o l v e n t s .

I n t h e t h y r o id g la n d h ow ever, d iio d o t y r o s i n e

r e p r e s e n t s a t l e a s t 60^ o f t h e t o t a l i o d in e , a c c o rd in g to t h e s e a u th o r s ( 138) .

Thus, t h ^ s t a t e t h a t t h e r e m ust b e a p r e f e r e n t i a l r e l e a s e o f

th y r o x in e b y t h e g la n d i n t o th e c i r c u l a t i o n ; o n ly a sm a ll f r a c t i o n o f th e g la n d ’s io d in e le a v e s th e g la n d a s d i i o d o t y r o s i n e . T rev o rrow ( 1 3 9 ) , E lm er ( I 4 0 ) and W illia m s (1 4 1 ) a l l r e p o r t th e u s e o f b u t y l a lc o h o l f o r t h e f r a c t i o n a t i o n o f b lo o d i o d i n e , b u t t h e i r e f f o r t s le a d to no ag re em e n t a s t o th e n a tu r e o f t h e c i r c u l a t i n g t h y r o id horm one.

18 Taurog and C h a ik o ff (142) c la im t h a t t h e s e d i f f i c u l t i e s h a v e b e e n due t o e r r o r s made i n m e a su rin g t h e s m a ll q u a n t i t i e s o f i o d in e in v o lv e d . (5 gamma p e r 100 m l. o f p la s m a ).

They p r e s e n t a s e n s i t i v e and r e l i a b l e

m ethod f o r t h e d e te r m in a tio n o f m ic ro am ounts o f i o d i n e . L elan d and F o s t e r (143) r e p o r t t h a t n - b u ta n o l w i l l e x t r a c t a l l o f th e th y r o x in e from a th y r o id g la n d t h a t h a s been s u b je c te d t o s t r o n g h y d r o ly s is w ith 2 N sodium h y d ro x id e , L eblond (1 44) s e p a r a t e s t h e th y r o x in e i n t h e th y r o id g la n d u s i n g n b u ta n o l i n th e above f a s h i o n . B lau (1 45) c la im s t h e d is c o v e r y o f a r e a g e n t w hich w i l l e x t r a c t d i io d o t y r o s i n e and in o rg a n ic i o d i n e , b u t n o t th y r o x in e , from b u t y l a l c o h o l. M orton and C h a ik o ff (146) p ro d u c e r a d i o a c t i v e th y r o x in e and d iio d o ­ t y r o s i n e b y p la c in g t h y r o id t i s s u e s from s h e e p , dogs an d r a t s i n a R in g e r ’s s o l u t i o n c o n ta in in g io d in e - 1 3 1 . H oreau and Sue (147) h a v e p r e p a r e d r a d i o a c t i v e th y r o x in e fro m 3 , 5 d iio d o th y r o x in e u s in g am m oniacal io d in e c o n ta in in g io d in e -1 3 1 a s t h e i o d i n a t i o n a g e n t.

However, t h e r e i s some u n c e r t a i n t y a s t o t h e n a t u r e

o f t h e i o d in e atom s i n th e two a ro m a tic r i n g s . M i l le r (148) g iv e s t h e s y n t h e s i s o f l a b e le d d i i o d o t y r o s i n e , employ­ in g a sim p le exchange r e a c t i o n .

I t i s c la im e d t h a t a t a te m p e r a tu re o f

50^C. and pH 5*0 t h e io d in e - 1 3 1 exchanged a lm o st c o m p le te ly w ith io d in e o f d i io d o t y r o s i n e i n 90 m in u te s , F r ie d e n , l i p s e t t and W in z le r (1 49) p r e s e n t a number o f m ethods f o r l a b e l i n g th y r o x in e w ith io d in e -1 3 1 and th y ro x in e #

How ever, t h e q u e s tio n

o f w h ich i o d in e atom s a r e in v o lv e d i n t h i s exchange i s d i f f i c u l t t o

19 so lv e *

A more c o n v e n ie n t m ethod o f p r e p a r in g th y r o x in e w ith io d in e -1 3 1

o f id e n t i c a l s p e c ific a c t i v i t y i n a l l fo u r p o s itio n s i s by th e i n v i t r o i o d i n a t i o n o f p r o t e i n s su c h a s c a s e i n w hich a r e h ig ^ i n t y r o s i n e c o n t e n t , L eb lo n d , Sue and Chamorro ( 1 5 0 ), J o l i o t , C o u r r i e r , H oreau and Sue ( 151) , and J o l i o t (152) com pare t h e u p ta k e and e x c r e t i o n o f l a b e l e d th y r o x in e i n j e c t e d i n t r a v e n o u s ly i n t o r a b b i t s w ith th e b e h a v io u r o f i n ­ j e c t e d i o d i n e - 131*

Up t o f i f t y tim e s a s much l a b e l e d th y r o x in e a s

la b e le d i o d id e i s found t o b e e x c r e te d th ro u g h th e b i l e .

I t was a l s o

found t h a t t h e t h y r o id g la n d ta k e s up a p p r e c ia b ly more io d in e a f t e r t h e a d m i n i s t r a t io n o f r a d i o - i o d i d e th a n a f t e r t h e a d m i n i s t r a t io n o f r a d i o th y ro x in e * G ro ss and L eblond (1 53) f i n d t h a t f o llo w in g t h e i n j e c t i o n o f r a d i o ­ t h y r o x in e , o n ly 2% o f t h e a d m in is te r e d d o se i s l e f t i n t h e b lo o d two h o u rs f o llo w in g a d m in is tr a tio n *

A t t h e b e g in n in g , t h e r e i s l i t t l e

r a d i o a c t i v i t y fo u n d i n t h e t h y r o i d , b u t l a t e r , p resu m ab ly b e c a u s e th y ­ r o x in e i s b ro k e n down t o i o d i d e , more r a d i o a c t i v i t y a c c u m u la te s i n t h i s gland*

A lso 24 h o u rs a f t e r i n j e c t i o n o f a l a r g e d o se o f r a d i o - t h y r o x in e ,

a b o u t 75^ o f i t may b e fo u n d i n th e fe c e s *

Thus t h e a u th o rs c o n c lu d e

t h a t th e l i v e r and g a s t r o i n t e s t i n a l sy ste m p l a y a n im p o rta n t r o l e i n t h e m etab o lism o f th y ro x in e * M i l le r and F e lle n b e r g (154) p r e s e n t r e s u l t s to show t h a t th y r o x in e l o s t none o f i t s a c t i v i t y when added t o b lo o d and in c u b a te d i n v i t r o f o r 48 h o u r s , w h ile i t v a n is h e d f a i r l y r a p i d l y from t h e b lo o d i n e x p e r i­ m ents c a r r i e d o u t i n v iv o *

The a u th o r s assum e t h a t t h e b lo o d i t s e l f

d o es n o t c a u s e th e d is a p p e a ra n c e o f t h y r o x in e , b u t t h a t some o t h e r o rg an w ith d raw s t h i s s u b s ta n c e from t h e c i r c u l a t i o n *

20

K e n d a ll (155)> K ra y e r (156) and A sim o ff, E s t r i n and M ile tz k a ja (1 5 7 ) f i n d t h a t fo llo w in g l a r g e d o s e s o f th y r o x in e , t h e l i v e r t a k e s up th y ­ r o x in e from t h e b lo o d and r e l e a s e s i t i n t o t h e b i le * K ellaw ay e t a l (158) n o te s t h a t l a r g e d o se s o f th y r o x in e p ro d u c e a g r e a t e r m e ta b o lic re s p o n s e i n h e p a te c to m iz e d th a n i n no rm al a n im als*

It

i s s u g g e s te d t h a t some i n a c t i v a t i o n o f t h e l a r g e r d o se s o f th y r o x in e t a k e s p la c e i n t h e l i v e r * M orton e t a l (159) a f t e r a d m i n i s t r a t io n o f io d in e - 1 3 1 t o t h y r o i d e c t om ized r a t s , d e m o n s tra te s t h e p re s e n c e o f b o th r a d io - th y r o x in e and r a d i o — d i io d o t y r o s i n e i n t h e i r t is s u e s * Oswald ( l 6 0 ) , F o s t e r and Gutman ( l 6 l ) i n v e s t i g a t i n g th e m e ta b o lism o f d ü o d o t y r o s i n e , o b se rv e t h a t p a r t o f t h e dose i s d e s tr o y e d , w h ile a l a r g e amount was e x c r e te d i n t h e u rin e * L eblond and Sue ( l6 2 ) r e p o r t t h a t d iio d o ty r o s i n e i s r a p i d l y b ro k e n down i n t o io d id e i n t h e blood* Blum and G ru tz n e r (163) s t a t e t h a t when t h y r o g lo b u l i n i s i n j e c t e d i n t o th e b lo o d , i t i s r a p i d l y b ro k en down i n t o io d id e * A b e lin and S c h e in f in k e l ( I 6 4 ) , V e il and S t u m (165) and Zawadowsky and A s in o f f (166) s t a t e f u r t h e r t h a t a f t e r l a r g e d o se s o f a t h y r o id p r e p a r a t i o n , t h e b lo o d io d in e r e tu r n e d t o a norm al l e v e l i n 24 h o u r s . A s in o ff and E s t r i n ( I 67) a l s o fo u n d t h i s t o be t r u e f o r th y r o x in e . I t i s s t a t e d t h a t i t i s a p p a re n t t h a t n e i t h e r th y r o g lo b u lin , th y r o x in e n o r th e io d id e d e r iv e d from t h e i r breakdown rem ain s i n th e b lo o d f o r a lo n g tim e* W oolley ( 168) s u g g e s ts t h a t a n a lo g u e s o f th y ro x in e m i ^ t b e s y n th e s iz e d iféiich would i n h i b i t th e p e r i p h e r a l f u n c tio n o f th e horm one.

21 w ith o u t a f f e c t i n g th e g la n d i t s e l f .

Thus a new ty p e o f a n t i - t h y r o i d

d ru g would became a v a i l a b l e . Niemann (1 69) f u r t h e r p o s t u l a t e s t h a t an a n t i - t h y r o i d hormone d ru g would h av e t o i n t e r f e r e w ith th e q u in o id re s o n a n c e i n t h e th y r o x in e m o le c u le vdiich i s p ro b a b ly c o n c e rn e d w ith i t s p a r t i c i p a t i o n i n o x i d a t i v e phenomena i n t h e body, F r a n k l in , C h a ik o ff and L e m e r (170) o b s e rv e t h a t t h e p re s e n c e o f t h i o u r a c i l i n s u r v iv in g t h y r o id s l i c e s a f f e c t s t h e c o n v e r s io n o f i o d id e t o o r g a n ic a lly -b o u n d i o d i n e , b u t does n o t a f f e c t th e u p ta k e o f i o d i n e . S c h a ch n e r (171) a l s o n o te s t h e same r e s u l t w ith p -am in o b en zo ic a c id ,

© lis d e m o n s tra te s t h a t t h e c o n c e n tr a ti o n o f i o d in e by t h e t h y r o id

i s an e n t i r e l y d i f f e r e n t m echanism th a n i s in v o lv e d i n t h e f o im a tio n o f d iio d o t y r o s i n e and th y r o x in e i n t h e g la n d , t h e a u th o r s t a t e s . Astwood ( 172) and McGinty (173) s u b s e q u e n tly f i n d u s in g i n t a c t a n im a ls , t h a t t h i o u r a c i l tr e a tm e n t does n o t a b o li s h th e i o d i n e - c o n c e n tr a ­ t i n g c a p a c i ty o f th e g la n d , even th o u g h i t b lo c k s t h e p r o d u c tio n o f th e th y r o id horm one. V an d erlan n and B is s e l l (1 7 4 ) show t h a t u n d e r t h e s e c o n d it i o n s , t h e io d in e does n o t rem ain i n t h e g la n d f o r lo n g ; th e maximum c o n c e n tr a tio n i s re a c h e d w i t h in th e f i r s t h a l f h o u r f o llo w in g i n j e c t i o n . V a n d e rla a n and V a n d e rla a n (175) i n m ore r e c e n t o b s e r v a tio n s p r e s e n t th e f o llo w in g c o n c lu s io n s c o n c e rn in g t h e u p ta k e o f io d in e b y th e t h y r o id s o f a n im a ls made g o i tr o u s by t h e fe e d in g o f t h i o u r a c i l , 1.

I t rem a in s i n t h e form o f in o r g a n ic i o d i n e ,

2.

I t d o es n o t p r e c i p i t a t e w ith p r o t e i n s .

3.

I t i s c o m p le te ly d i a l y s a b l e .

22 EXPERIMENTAI, THE PREPARATION OF IQDOGASEIN

P r i o r t o th e p r e p a r a t i o n o f io d o c a s e in u s in g io d in e - 1 3 1 , some sam p les w ere p re p a re d u s in g s t a b l e io d in e - 1 2 ? a s th e i o d i n a t i n g a g e n t.

V a rio u s

m ethods o f s y n t h e s i s w ere t r i e d and th e f o llo w in g b a s e d upon t h e m ethod o f R ein ek e and T u rn e r (9 )

p ro v ed t o b e t h e b e s t fro m th e s ta n d p o in t o f

e a s e o f h a n d lin g and th y r o x in e - io d i n e c o n te n t. Twenty gram s o f ( v i ta m i n - f r e e ) c a s e in c o n ta in in g 4 .9 ^ t y r o s i n e was p la c e d i n 700 m l, o f d i s t i l l e d w a te r i n a 2 l i t e r 3 -n ec k ed ro u n d b o tto m ed f l a s k f i t t e d w ith a H irs c h b e rg s t i r r e r and a r e f l u x c o n d e n s e r. m illig r a m s o f trim a n g a n ic t e t r a o x i d e was added a s c a t a l y s t .

Twenty

The c a t a l y s t

was p re p a re d by h e a tin g manganous c a rb o n a te f o r 18 h o u rs i n an e l e c t r i c m u ffle s e t a t SOO^C,

The f l a s k was s e t i n a G la s c o l h e a tin g m a n tle and

t h e v a r i a c was a d ju s te d so a s t o m a in ta in t h e te m p e ra tu re a t 4 0 % ,

A

t o t a l o f 4*8 g* o f powdered i o d in e d is s o lv e d i n m eth an o l was s lo w ly

add­

ed o v e r a 4 h o u r p e r i o d , t h e s o l u t i o n b e in g a g i t a t e d a t a r a t e o f 600 r ,p ,m .

A t t h e c o n c lu s io n o f t h e 4 h o u r i n c u b a tio n p e r io d , t h e v a r i a c was

a d ju s te d t o b r in g t h e r e a c t i o n te m p e ra tu re t o 7 0 % ., v h e re i t was k e p t f o r 20 h o u r s ,

A pH o f 8 ,5 was m a in ta in e d th ro u g h o u t t h e r e a c t i o n by t h e

a d d it i o n o f sodium b i c a r b o n a t e ,

A N a tio n a l T e c h n ic a l L a b o r a to r ie s Beckman

m odel AC o p e ra te d g l a s s e le c t r o d e pH m e te r was u s e d f o r t h i s c o n tin u o u s ty p e o f o p e r a t io n . The io d o c a s e in was f r e e d o f in o r g a n ic i o d in e b y i s o —e l e c t r i c p r e ­ c ip ita tio n ,.

When d i l u t e h y d r o c h lo r ic a c id was added t o b r i n g th e pH

t o 3 * 8 , th e io d o c a s e in was p r e c i p i t a t e d .

The io d o c a s e in was th e n r e ­

d i s s o l v e d i n 4 l i t e r s o f d i s t i l l e d w a te r b y b r in g in g i t t o a pH o f 8 ,5

23 w ith d i l u t e sodium h y d ro x id e .

I t was th e n r e - p r e c i p i t a t e d b y th e add­

i t i o n o f a c i d , and a f t e r s e t t l i n g , t h e s u p e r n a ta n t l i q u i d was p o u red o f f . T h is p ro c e d u re was r e p e a te d s e v e r a l tim e s u n t i l th e s o l u t i o n no lo n g e r c o n ta in e d a n y l o o s e l y com bined o r in o r g a n ic i o d i d e .

The p re s e n c e o f

i o d id e was c o n v e n ie n tly d e te c t e d by a d d in g some o f t h e io d o c a s e in s o l u t i o n t o an a c id s o l u t i o n o f p o ta s s iu m perm anganate o r h y d ro g en p e r o x id e .

The

p u r i f i e d io d o c a s e in was th e n d is s o lv e d i n minimum amount o f d i l u t e sodium h y d ro x id e by h e a tin g t o 7 0 % ,

I t was found t h a t f o r a d m i n i s t r a t ­

io n t o a n im a ls , t h e io d o c a s e in was more e a s i l y h a n d le d when i n s o l u t i o n , th a n a s a d r i e d pow der.

How ever, t h e d r i e d powder may b e o b ta in e d by

b r in g in g th e io d o c a s e in t o i t s i s o - e l e c t r i c p o i n t , and f i l t e r i n g i t o f f th ro u g h a p ie c e o f c h e e s e c l o t h ,

A B uchner f u n n e l was found t o b e

u n s u i t a b l e f o r t h i s p u rp o s e , due t o c lo g g in g o f t h e f i l t e r p a p e r .

The

w et m ass o f p r o t e i n was p la c e d on a m e ta l s h e e t and a llo w e d t o d r y i n a i r f o r two d a y s .

The h a r d , b r i t t l e mass form ed on d r y in g was th e n ground

t o a pow der, ANALYSIS OF IODOCASEIN 1,

C o n c e n tr a tio n o f s o l u t i o n : As t h e io d o c a s e in was n o t i s o l a t e d i n a d r y c o n d i t i o n , i t was

n e c e s s a r y t o f i n d a m ethod o f d e te rm in in g t h e w e ig h t o f m a t e r i a l i n a g iv e n volume o f s o l u t i o n , p r i o r t o t h e a d m i n i s t r a t i o n t o a n im a ls .

The

m ost c o n v e n ie n t m ethod o f w e ig h t a n a l y s i s would b e t o remove th e w a te r by vacuum d ry in g and t o w eigh th e d r i e d r e s i d u e re m a in in g .

T his p ro c e d u re

was i n v e s t i g a t e d i n t h e f o llo w in g m anner, A w eighed amount o f d r y , pow dered io d o c a s e in was d is s o lv e d i n a m easu red amount o f sodium h y d ro x id e s o l u t i o n w ith a pH o f 8 ,0 t o g iv e a

24

s o l u t i o n h a v in g 0 ,1 g , o f io d o c a s e in p e r m i l l i l e t e r .

T h is a p p ro x im a te s

t h e c o n c e n tr a ti o n s t h a t w i l l b e d e a l t w ith i n t h i s w ork,

A 0 ,2 m l,

p o r t io n o f t h i s s o l u t i o n was p la c e d i n a vacuum d e s i c c a t o r c o n ta in in g anhydrous p h o sp h o ru s p e n to x id e as d ry in g a g e n t , m a in ta in e d f o r 8 h o u r s .

A vacuum o f 0 ,5 mm, was

Upon w e ig h in g , th e d r i e d r e s i d u e ch eck ed w ith in

1 mg, o f t h e c a l c u l a t e d w e ig h t p e r m l, o f s o l u t i o n ,

T h is r e l a t i o n s h i p

was found t o h o ld f o r saniples o f s o l u t io n up t o 0 ,5 m l, volum e, 2,

I o d in e c o n te n t: The p ro b lem o f io d in e a n a l y s i s o f o rg a n ic m a t e r i a l h a s alw ay s be«a

d i f f i c u l t , c h i e f l y due t o t h e f a c t t h a t d u r in g t h e a s h in g s t e p , some io d in e i s l o s t by v o l a t i l i z a t i o n .

The fo llo w in g m ethod, b a se d on th e

c l a s s i c a l m ethod o f K e n d a ll (2 2 ) and m o d ifie d by K o h le r (4 1 ) surm ounted t h i s d i f f i c u l t y by a v e ry c a r e f u l c o n tr o l o f t h e h e a t i n g .

The p ro c e d u re

i s a s f o llo w s : 8y m eans o f a sc o o p , 5 g , o f anhydrous A ,E , sodium c a rb o n a te was added t o a 100 m l, n i c k e l c r u c i b l e and f i f t y m illig ra m s o f io d o c a s e in l a y e r e d on t h e sodium c a r b o n a te .

The m ix tu re was dam paied th o ro u g h ly

w ith ID m l, o f e th a n o l and f i v e m l, o f s a t u r a t e d sodium h y d ro x id e was th e n ad d ed , fo llo w e d

b y a n o th e r 5 g . o f sodium c a r b o n a te .

p la c e d on a h o t

p l a t e u n t i l a l l th e e th a n o l was rem oved. G r a d u a lly , t h e

te m p e ra tu re was

i n c r e a s e d u n t i l th e w a te r and e th a n o l had b een rem oved

an d th e sam ple d r i e d .

The c r u c i b l e was

D uring th e p re c e e d in g o p e r a tio n , c a r e was tak en ;

t h a t th e c o n te n ts d id n o t s p l a t t e r o u t o f t h e c r u c ib le »

The c r u c i b l e

and c o n te n ts w ere c o v e re d and p la c e d i n a m u ffle fu rn a c e e q u ip p e d w ith a u to m a tic te m p e ra tu re c o n t r o l s and h e ld a t 5 0 0 % , f o r 15 m in u te s , a llo w in g

25 f o r t h e tim e r e q u ir e d f o r t h e fu r n a c e t o come b ack t o te m p e r a tu re a f t e r t h e c o ld sam p les w ere a d d e d .

A f t e r c o o lin g , 25 m l, o f w a te r was ad d ed ,

t h e c r u c i b l e c o v e re d w ith a w atch g l a s s and b o i le d g e n t l y f o r 10 m in u te s . The s o l u t i o n was p o u red th ro u g h a f u n n e l i n t o a 200 m l, v o lu m e tric f l a s k , a n o th e r 25 m l, o f w a te r added t o t h e c r u c i b l e and b o ile d f o r a s h o r t tim e t o rem ove a l l o f t h e w h ite s a l t and t h i s added t o th e s o l u t i o n i n t h e v o lu m e tric f l a s k .

The c o n te n ts w ere c o o le d t o 2 5 % , and b ro u g h t up t o

volum e w ith d i s t i l l e d w a te r . moved by c e n t r i f u g a t i o n .

The sm a ll am ount o f c a rb o n p r e s e n t was r e ­

One h u n d red and f i f t y n d l l i l e t e r s o f t h e c l e a r

c e n t r i f u g a t e was c a r e f u l l y p i p e t t e d i n t o a 500 m l, w ide-m outhed E rle m e y e r fla s k .

A f t e r c o o lin g t o 2 0 % , , a few g l a s s b e a d s w ere added and t h e

s o l u t i o n was n e u t r a l i z e d w ith 85^ p h o sp h o ric a c i d , 0,5% m e th y l o ra n g e i n e th a n o l b e in g u se d a s i n d i c a t o r .

F o llo w in g t h i s , 1 m l, o f a c id was added

i n e x c e s s a lo n g w ith 25 m l, o f s a t u r a t e d brom ine w a te r . brom ine was a c h ie v e d b y g e n t l e b o i l i n g .

Removal o f t h e

Ten d ro p s o f f r e s h l y p re p a re d

5% sodium s a l i c y l a t e was added t o t h e s o l u t i o n w hich had b e en c o o le d t o 20% ,

One m i l l i l e t e r o f 85% p h o sp h o ric a c id and 5 m l, o f a c o o l, f r e s h l y

p re p a re d p o ta s s iu m i o d id e s o l u t i o n was added and t h e l i b e r a t e d i o d in e was t i t r a t e d w ith 0 ,0 0 5 N sodium t h i o s u l f a t e ,

A s o l u t i o n o f 0,5% s o lu b le

s t a r c h s o l u t i o n (M erck) was added a s i n d i c a t o r n e a r th e end o f t h e t i t r a ­ tio n ,

A s u i t a b l e e n d p o in t was one which was s t a b l e f o r a t l e a s t 1 m in u te .

The c a l c u l a t i o n was done a s f o llo w s : % io d in e i n a 50 mg, sam ple =

Volume sodium t h i o s u l f a t e X N o rm a lity o f sodium t h i o s u l f a t e X 56,4*

A p p ly in g t h i s m ethod o f a n a l y s i s t o one o f t h e l o t s o f io d o c a s e in p re p a re d b y t h e m ethod o u t l i n e d e a r l i e r , i t w as found t o h av e an io d in e

26 c o n te n t o f 7# 15%,

T h is c h e c k s c l o s e l y w ith t h e r e s u l t s o b ta in e d by o t h e r

w o rk ers ( 9 ) , ( 4 1 ) , ( 1 0 ) , (1 1 ) u s in g s i m i l a r m ethods o f p r e p a r in g io d o ­ c a s e in , 3,

T h y ro x ine c o n te n t - G ra v im e tric a n a l y s i s : As t h e th y r o x in e c o n te n t o f io d o c a s e in was l a t e r t o be d e te rm in e d

by t h e i s o t o p e d i l u t i o n m ethod u s in g th y ro x in e la b e le d w ith io d in e - 1 3 1 , i t was f e l t a d v is a b le t o f i r s t d e te rm in e t h i s v a lu e b y a c h e m ic a l m ethod (4 4 ).

The p ro c e d u re f o llo w s : One gram o f io d o c a s e in , 32 g , o f h y d r a te d b ariu m h y d ro x id e and

6 ,4 ml# o f d i s t i l l e d w a te r was p la c e d i n a l a r g e t e s t tu b e .

An a i r -

c o o le d r e f l u x c o n d e n se r was a t t c h e d , and t h e t e s t tu b e was p la c e d i n a v ig o r o u s ly b o i l i n g w a te r b a t h .

When th e b a riu m h y d ro x id e had d is s o l v e d ,

th e c o n te n ts o f t h e tu b e w ere s t i r r e d w e ll, c a r e b e in g ta k e n t o work an y m a t e r i a l a d h e rin g t o t h e w a lls down i n t o t h e s o l u t i o n .

The p r e p a r a t i o n

was th e n r e p la c e d i n th e b o i l i n g w a te r b a th and t h e h e a tin g c o n tin u e d f o r 20 h o u r s . The h y dro l y s a t e was m ixed w ith 2$ m l, o f d i s t i l l e d w a te r , and s e t a s i d e f o r a few m in u te s to a llo w th e c o a r s e p r e c i p i t a t e o f b a riu m s a l t s to s e t t l e . fla s k .

The s u p e r n a ta n t f l u i d was d e c a n te d i n t o a 100 m l, v o lu m e tric

The p r e c i p i t a t e o f b a riu m s a l t s re m a in in g i n t h e t e s t tu b e was

decomposed by a d d in g 2 m l, o f n - b u ta n o l and e x a c tl y 5 m l, o f 1 N h y d ro c h lo ­ r i c a c i d ; sh a k in g and warming s l i g h t l y i n a w a te r b a th t o e f f e c t c o m p le te s o lu tio n o f th e p r e c i p it a te .

The d is s o lv e d s u b s ta n c e was t r a n s f e r r e d

q u a n t i t a t i v e l y t o th e v o lu m e tric f l a s k and d i s t i l l e d w a te r was added t o b r in g t h e volume up to 100 m l, A 20 m l, a l i q u o t o f w e ll-m ix ed h y d r o ly s a te was p u t i n a s e p a r a t o r y

27 f u n n e l and 5 d ro p s o f 0,5% b ro m c re so l g re e n i n d i c a t o r was a d d ed .

The

s o l u t i o n was t i t r a t e d w ith 1 N h y d r o c h lo r ic a c id t o a d e f i n i t e y e llo w c o lo r. The th y r o x in e was e x tr a c t e d from t h e h y d r o ly s a te by sh a k in g w ith 20 m l, o f n - b u t a n o l .

The b u ta n o l e x t r a c t i n t u r n was p u r i f i e d b y sh a k in g

w ith an e q u a l volum e, and th e n w ith h a l f t h e volume o f 4 N sodium h y d ro x ­ i d e s o l u t i o n , c o n ta in in g 5% sodium c a r b o n a te .

T h is i s B laus* r e a g e n t

(1 4 5 ) w hich t h e a u th o r c la im s w i l l e x t r a c t in o r g a n ic i o d in e and d iio d o ­ t y r o s i n e frcxn a b u ta n o l s o l u t i o n , b u t n o t th y r o x in e .

A f te r each e x t r a c t i o n ,

t h e s o l u t i o n was l e f t f o r an h o u r t o i n s u r e good s e p a r a t i o n b e f o r e t h e aqueous l a y e r was draw n o f f . The p u r i f i e d n - b u ta n o l e x t r a c t was f i l t e r e d d i r e c t l y i n t o a 100 m l. n i c k e l c r u c i b l e th ro u g h a s o f t l y packed p lu g o f g l a s s w ool t h a t was p la c e d i n t h e ste m o f t h e s e p a r a t o r y f u n n e l.

F i n a l l y , t h e s e p a r a to r y

f u n n e l was r i n s e d w ith 5 m l, o f n - b u ta n o l, w hich was d r a in e d th ro u g h t h e g l a s s wool i n t o t h e c r u c i b l e .

The n - b u ta n o l was m ost c o n v e n ie n tly r e ­

moved b y e v a p o r a tio n on a steam b a th a t a tm o sp h e ric p r e s s u r e . The Io d in e c o n te n t o f th e r e s i d u e may be d e te rm in e d by t h e m ethod o f K o h le r (4 1 ) p r e v i o u s ly o u t li n e d f o r t h e d e te r m in a tio n o f i o d in e i n io d o ­ c a s e in ,

The r e s u l t i n g v a lu e , o b ta in e d from sam ples ru n i n d u p l i c a t e ,

was m u l t i p l i e d by t h e f a c t o r 1 ,5 2 9 t o o b ta in t h e th y r o x in e v a lu e . The r e s u l t s o b ta in e d on a l o t o f io d o c a s e in p re p a re d by t h e m ethod u se d th ro u g h o u t t h i s work was 3,1% th y r o x in e .

T h is com pares f a v o r a b ly

w ith r e s u l t s r e p o r t e d by R ein ek e and T u rn e r ( 9 ) , ( 1 0 ) , ( 1 1 ) , ( 1 2 ) , ( 2 7 ) . THE ISOLATION OF THYROXINE FROM IODOCASEIN P r i o r t o t h e p r e p a r a t i o n o f th y r o x in e la b e le d w ith io d in e - 1 3 1 , i t

28 was f e l t n e c e s s a r y t o work o u t a m ethod f o r th e i s o l a t i o n o f n o n - r a d io a c t i v e th y r o x in e .

The fo llo w in g a p p ro a c h , b a se d on t h e method o f R e in e k e

and T u rn e r (27) p ro v e d t o be m ost s u i t a b l e from t h e s ta n d p o in t o f l a t e r o b ta in in g r a d i o t h y r o x in e . The io d o c a s e in u se d f o r t h e h y d r o l y s i s and re c o v e r y o f th y r o x in e was ta k e n from a b a tc h p re p a re d u n d e r th e p ro c e d u re s d e s c r ib e d p r e v i o u s ly f o r o b ta in in g optimum th y r o x in e c o n t e n t . One h u n d red grams o f t h i s m a t e r i a l was d i s s o lv e d i n d i l u t e sodium h y d ro x id e , pH 8 ,0 , an d t o t h i s s o l u t i o n was added 320 g , o f h y d ra te d b ariu m h y d ro x id e , fo llo w e d by g e n t l e h e a t in g u n d e r r e f l u x f o r 20 h o u r s . The p r e c i p i t a t e o f b a riu m s a l t s was removed b y ru n n in g t h e h o t s o l u t i o n th ro u g h a s u c tio n f i l t e r .

The p r e c i p i t a t e (A) and f i l t r a t e (B) w ere t h e n

t r e a t e d s e p a r a t e l y a s f o llo w s :

P r e c i p i t a t e (A) was t r e a t e d w ith d i l u t e

h y d r o c h lo r ic a c id w ith v ig o u ro u s s t i r r i n g u n t i l t h e r e a c t i o n becam e s l i g h t l y a c id t o Congo r e d p a p e r, f i l t e r e d w ith s u c t io n and washed w ith d ilu te a c id .

F i l t r a t e (B) was c o o le d and t h e c r y s t a l l i n e barium h y d ro x id e

was rem oved by f i l t r a t i o n , r e d is s o lv e d and c r y s t a l l i z e d a g a in , and t h e second f i l t r a t e was com bined w ith t h e f i r s t . The combined f i l t r a t e was a c i d i f i e d w ith d i l u t e h y d r o c h lo r ic a c id t o pH 4 * 5 , w hereupon a l i g h t - c o l o r e d p r e c i p i t a t e s e t t l e d o u t .

The p r e c i p i t a t e

was re c o v e re d by c e n t r i f u g i n g , washed s e v e r a l tim e s w ith d i l u t e a c e t i c a c i d , d r i e d , and added t o p r e c i p i t a t e (A ),

A f t e r b e in g d r i e d , t h e combin­

ed p r e c i p i t a t e was e x t r a c t e d w ith t h r e e ch an g es o f e t h e r , and th e n d i s s o lv e d i n 0 .1 N sodium h y d ro x id e c o n ta in in g a s m a ll amount o f sodium: s u l f a t e f o r t h e rem o v al o f t h e b a riu m , and b o i l e d .

The barium , s u l f a t e

form ed was removed by c e n t r i f u g i n g , t h i s p ro c e s s b e in g r e p e a te d u n t i l a

29 c l e a r a m b e r-c o lo re d s o l u t i o n was o b ta in e d .

The s o l u t i o n was h e a te d t o

90% * and c a u t i o u s l y a c i d i f i e d w ith d i l u t e s u l f u r i c a c id u n t i l a g r a n u l a r p r e c i p i t a t e a p p e a re d (pH 5 . 0 ) ,

The p r e c i p i t a t e was r e c o v e re d b y f i l t r a t ­

i o n , w ashed w ith d i l u t e a c e t i c a c i d , and d r i e d i n vacu o o v e r D r i e r i t e * The y i e l d o f a c i d - i n s o l u b l e s u b s ta n c e from 100 g . o f io d o c a s e in was u s u a l l y aro u n d 3 grams* The a c i d - i n s o l u b l e s u b s ta n c e was d i s s o lv e d i n t h e minimum amount o f 0 , 1 N sodium c a r b o n a te s o l u t i o n , c e n t r if u g e d to rem ove t r a c e s o f un­ d i s s o l v e d , d a r k - c o lo r e d s u b s ta n c e , and th e n c h i l l e d t o 5 % . A h eav y w h ite p r e c i p i t a t e o f t h e monosodium s a l t o f th y r o x in e formed*

The p r e ­

c i p i t a t e was c o l l e c t e d by c e n t r i f u g i n g , and p u r i f i e d b y c r y s t a l i i z in g s e v e r a l tim e s from 0*1 N sodium c a r b o n a te s o l u t i o n .

A d d itio n a l am ounts

o f t h e monosodium s & lt w ere o b ta in e d by s u c c e s s iv e ly c o n c e n tr a tin g t h e m o th er l i q u o r s by b o i l i n g and p u r i f y in g t h e w h ite p r e c i p i t a t e t h a t form ed upon c o o lin g . The monosodium s a l t was ta k e n up i n 70% a l k a l i n e e th a n o l, h e a te d t o b o i l i n g , and a c i d i f i e d w ith a c e t i c a c i d .

T h y ro x in e s e p a r a te d im m e d ia te ly

i n c h a r a c t e r i s t i c b u n d le s o f w h i t e , m ic ro sc o p ic n e e d l e s .

The m e ltin g

p o i n t was 2 3 0 -2 3 3 % ,, vfoich ch eck ed c l o s e l y w ith t h e v a lu e r e p o r t e d i n th e l i t e r a t u r e . The u s u a l y i e l d o f th y r o x in e from t h i s p ro c e d u re was b etw een 200 and 300 mg.

As shown e a r l i e r , t h e th y r o x in e c o n te n t o f io d o c a s e in i s

a p p ro x im a te ly 3%> so t h i s m ethod i s f a r from b e in g a q u a n t i t a t i v e ap p ro ach t o t h e pro blem o f o b ta in in g th y r o x in e .

However, i t do es p ro v id e a conven­

i e n t m ethod f o r o b ta in in g th y r o x in e la b e le d w ith io d in e - 1 3 1 ,

30 THE ABSORPTION SPECTRUM OF IODINE-131 IN ALÜMIMUM B e fo re b e g in n in g t h e p a r t o f t h i s r e s e a r c h i n w hich r a d i o a c t i v e i o d in e was em ployed, i t was f e l t t h a t t h e ty p e o f r a d i a n t e n e rg y b e in g m easured sh o u ld be d e te rm in e d .

F o r t h i s w ork, a n Eck and K rebs ty p e

th in - w a l le d b e t a c o u n te r was u se d i n c o n ju n c tio n w ith a C y c lo tro n S p e c i a l i t i e s G-M s c a l e - o f - 8 a m p lify in g c i r c u i t .

The in ç su lse r e g i s t e r

was m a n u fa c tu re d by t h e C y c lo tro n S p e c ia lta e s Congiany and a c c u r a t e l y r e g i s t e r s u p t o 60 im p u lse s p e r se c o n d .

An e x t e r n a l N e h er-H arp e r ty p e

c i r c u i t f o r e x tin g u is h in g t h e c o u n te r tu b e and a m p lify in g t h e p u ls e s was a l s o employed* I t was found t h a t t h i s assem b ly would s a t i s f a c t o r i l y re p ro d u c e c o u n ts from sam ples c o u n tin g a s h ig h a s 3000 c o u n ts p e r m in u te ( c .p .m .) * I n a l l t h e work r e p o r te d h e r e , c o n s ta n t g e o m e try , background and d ecay tim e c o r r e c t i o n s have b een made, w here n e c e ssa ry * As o p e r a tin g v o lta g e and quenching c i r c u i t s e t t i n g a r e th e m ain f a c t o r s c o n t r o l l i n g th e ty p e o f r a d i a n t e n erg y m easu red , t h e e x p erim en t was c a r r i e d o u t a t t h e u s u a l s e t t i n g s employed d u rin g a t y p i c a l e x p e r i­ m ent w ith io d in e - 1 3 1 la b e le d - c a s e in * A sam ple o f io d in e - 1 3 1 was p u t i n a g l a s s cup and c o u n te d .

F o llo w ^

in g t h i s , v a r io u s th ic k n e s s e s o f aluminum ra n g in g from 1 m g ./s q .c m . t o 280 m g ./s q .c m . w ere i n te r p o s e d betw een t h e sam ple and t h e c o u n te r tu b e . The lo g b a se t e n o f t h e c .p .m , o b ta in e d w ere p l o t t e d a g a i n s t t h e t h i c k ­ n e s s , a s shown on Graph 1 . , t h e g rap h b e in g p r e p a r e d on a s e m i-lo g b a s e t e n g rap h p a p e r, o f t h e tw o c y c le v a r i e t y .

From t h i s g ra p h , a c u rv e was

o b ta in e d w hich c o u ld be d e s c r ib e d b y th e e q u a tio n :

GRAPH I,

10 0 0 4 -

900--

The absorption spentror.ent

20

-

Absorber thickness in œ g./sq.cji. o f alu^lnum # t

31

—dS dt

— uS

S t u -KiS dt

= c.p#m# o b ta in e d w ith z e ro a b s o r b e r B t h ic k n e s s o f alm ninum a b s o r b e r » a b s o r p tio n c o e f f i c i e n t = r a t e a t udiich t h e c .p .m . d e c r e a s e w ith i n c r e a s e i n a b s o r b e r t h ic k n e s s t .

w here

To p u t t h i s e q u a tio n i n a form s u i t a b l e f o r g ra p h in g on lo g b a s e 10 g rap h p a p e r, i t was i n t e g r a t e d .

S

=

udt

( - lo g g S )g = i i t ♦

c o n s ta n t

lO gg S - lO ggSg = -U t

-u t

s

S e

So w here

now r e p r e s e n t s t h e c .p .m » o b ta in e d w ith z e ro a b s o r b e r , and

S r e p r e s e n ts t h e c.p#m* o b ta in e d a f t e r p a s s a g e th ro u g h t h ic k n e s s t o f a b so rb e r# T h is may b e changed t o b a s e 10 lo g form i n t h e f o llo w in g m anner: - lo g ^S

=

- log]_()S

= ut 2 .3 0 3

Then

ti ^

=

ut

Q>693 u

c o n s ta n t

when S i s h a l f t h e o r i g i n a l v a lu e o f

and

ti

32 i s known a s t h e " h a l f - v a l u e a b s o r p t i o n ." When t h e v a lu e o f S q aiKi S a r e ta k e n from t h e g ra p h and s u b s t i t u t e d i n t h e e q u a tio n t

2 0 .3 6 m g ./s q .c m .

T h is v a lu e c h e c k s c l o s e l y w ith t h e v a lu e found i n t h e l i t e r a t u r e (8 0 ) f o r t h e h a l f - v a l u e a b s o r p tio n o f i o d i n e -1 3 1 i n alum inum . From th e g ra p h , a d e p a r tu r e from l i n e a r i t y is n o te d , b e g in n in g a t 100 m g ./s q .c m . o f alum inum .

T h is i s due t o t h e two s o f t gamma com ponents

a s s o c i a t e d w ith io d in e - 1 3 1 . When t h i s l a t t e r p a r t o f t h e c u rv e i s e x t r a p o la t e d b a c k , and su b ­ t r a c t e d from t h e t o t a l a b s o r p tio n c u r v e , th e t r u e b e t a r a y a b s o r p tio n c u rv e i s o b ta in e d .

Xt i s th e n s e e n , t h a t t h e maximum ra n g e o f th e b e t a

r a y i n aluminum i s 159 m g ./s q .c m . T h is c h ec k s c l o s e l y w ith t h e v a lu e o b ta in e d from t h e e m p ir ic a l e q u a tio n o f F e a th e r ( 8 1 ) . R m 0 .5 4 3 Emax.

- O .I 6O

w here R

=

Maximum ra n g e o f th e b e ta r a y i n aûLtiminum, e x p re s s e d a s g ./ s q .c m .

and Emax. *

Maximum e n e rg y v a lu e o f t h e b e t a r a y , w hich f o r io d in e - 1 3 1 i s 0 .5 9 5 MEV.

Thus

«

0 .5 4 3 (0 .5 9 5 ) - O .I6O

»

0 .1 6 3 g ./ s q .c m .

=

163 m g ./s q .c m .

R

Thus i t i s a p p a re n t t h a t ^ e n t h e above o p e r a t in g c o n d it i o n s w ere u se d i n t h i s r e s e a r c h , b o th t h e b e ta and gamma r a y s from t h e io d in e - 1 3 1 w ere b e in g c o u n te d .

33 THE PREPARATION OF lODOCASEIM lAEEIED WITH IODIME-131 The p ro c e d u re h e r e i s s i m i l a r t o t h a t em ployed i n t h e p r e p a r a t i o n o f o r d i n a r y io d o c a s e in , w ith a few c h a n g e s made n e c e s s a r y by th e u s e o f io d in e - 1 3 1 .

F r ie d e n , L i p s e t t and W in z le r (149) a ls o u s e d t h i s m ethod

t o o b t a i n th y r o x in e l a b e le d w ith io d in e -1 3 1 * Ten gram s o f c a s e i n ( v i ta m i n - f r e e ) was u se d i n s t e a d o f t h e u s u a l 20 g ram s.

T his g iv e s an e n d -p ro d u c t o f h ig h e r s p e c i f i c a c t i v i t y .

Io d in e -

131 i n t h e fo rm o f sodium i o d id e i n sodium b i s u l f i t e s o l u t i o n was o b ta in e d from t h e Oak R idge N a tio n a l L a b o ra to ry o p e ra te d by t h e C a rb id e and C arbon C hem ical C o rp o ra tio n f o r th e Atomic E nergy Com m ission, Oak R id g e, T e n n ., i n c a r r i e r - f r e e c o n d it i o n .

I t i s produced i n l a r g e am ounts

by th e u ran iu m p i l e r e a c t o r ( 7 7 ) , and h a s a n e g a tiv e b e t a p a r t i c l e w ith a n e n e rg y maximum o f 0 .5 9 5 MEV.

Each b e t a p a r t i c l e i s a s s o c i a t e d w ith

two gamma r a y s i n c a s c a d e , w ith e n e rg y v a lu e s o f 0 .3 6 7 and 0*080 MEV (7 9 )# The i o d in e —131 was added t o t h e m eth a n o l s o l u t i o n o f io d in e -1 2 7 w hich s e rv e d a s c a r r i e r .

I n t h i s form i t was added t o th e r e a c t i o n m ix tu re

and t r e a t e d f o r t h e p r e p a r a t i o n o f io d o c a s e in i n t h e m anner p r e v io u s ly d e s c rib e d .

The r e a c t i o n f l a s k was su rro u n d e d by a l e a d s h i e l d , t o c u t

down on t h e r a d i a t i o n b e in g a b so rb e d by t h e p e rs o n n e l i n t h e l a b o r a t o r y . As b e f o r e , t h e io d o c a s e in was n o t i s o l a t e d a s a powder f o llo w in g t h e p u r i f i c a t i o n , b u t was l e f t i n s o l u t i o n a t pH 8 .0 .

T h is was d o n e , a s t h i s

i s a m ore c o n v e n ie n t fo rm f o r a s s a y , and f o r su b se q u e n t a d m i n i s t r a t io n t o a n im a ls . The r a d i o a c t i v e p r o t e i n was c o u n te d by m ounting 0 .5 m l. o f t h e s o l u t i o n , d i l u t e d one h u n d red f o l d , on a f i l t e r s t r i p , 2 j " b y 2 " .

T h is

i n t u r n , was p la c e d on a p ie c e o f b a c k in g p a p e r , 3" by 2 j " , made from

34 o r d in a r y mimeo p a p e r .

The f i l t e r s t r i p was s e a le d t o t h e b a c k in g p a p e r

w ith a p a n e o f Dupont c e llo p h a n e , 3 ” by 2 g " , s e t down w ith s c o tc h t a p e . I n t h i s f a s h i o n t h e s a n g le i s c o m p le te ly s e a l e d i n , th u s a v o id in g con­ ta m in a tio n d i f f i c u l t i e s .

I n f r a - r e d l i g h t was u se d t o d r y t h e sam ples

on t h e f i l t e r s t r i p , p r i o r t o m o u n tin g . The m ounted f i l t e r s t r i p s w ere f a s te n e d d i r e c t l y aro u n d t h e c o u n te r tu b e , t o i n s u r e c o n s ta n t g eom etry and a h ig h d e g re e o f m e a su ra b le a c tiv ity . I n some c a s e s , t h e l a b e le d i o d o p r o te in was a s s a y e d u s in g aluminum c u p s , s e t u n d e r an end-w indow c o u n te r tu b e .

T h is p ro c e d u re i s d e s c r ib e d

i n th e s e c t i o n d e a lin g w ith t h e p r e p a r a t i o n o f la b e le d t h y r o x in e .

As

i n t h e f i l t e r s t r i p m ethod, 0 .5 m l. o f t h e s o l u t i o n , d i l u t e d one h u n d red f o l d , was u s e d . THE PREPARATION OF THYROXINE lABELSD WITH IODINE-131 E s s e n t i a l l y , th e m ethod u s e d i s s i m i l a r t o t h a t o u t l i n e d e a r l i e r f o r t h e p r e p a r a t i o n o f o r d in a r y th y r o x in e . io d in e - 1 3 1 was p re p a re d a s a b o v e .

The io d o c a s e in l a b e le d w ith

I t was l e f t i n s o l u t i o n t o a v o id

t h e c o n ta m in a tio n problem s and c o u n tin g d i f f i c u l t i e s t h a t would p r e s e n t th e m se lv e s i f a d r y sam ple w ere u se d a s s t a r t i n g m a t e r i a l .

The h y d ro ly ­

s i s was c a r r i e d o u t on a 10 g . sam ple o f io d o c a s e in t h a t had b een p re p a re d from 10 g . o f c a s e i n and 10 m i l l i c u r i e s o f io d in e - 1 3 1 . grams o f o r d in a r y io d o c a s e in was added a s c a r r i e r .

T h ir t y

T h is s t e p a s s u r e s

a m ea su ra b le y i e l d o f r a d i o t h y r o x in e , a lth o u g h t h e s p e c i f i c a c t i v i t y i s lo w e re d .

The p u r i f i c a t i o n s t e p was c a r r i e d o u t u s in g s i n t e r e d g l a s s

f u n n e ls f o r s e p a r a t io n o f th e r a d io th y r o x in e , a s th e y p ro v ed t o b e more

35 c o n v e n ie n t t h a n t h e c e n t r i f u g e tu b e s u se d e a r l i e r a s w e ll a s im p ro v in g th e y ie ld . The y i e l d o f r a d io th y r o x in e was fo u n d t o b e 220 mg. h a v in g a s p e c i f i c a c t i v i t y o f 1 0 ,4 0 0 c .p .m ./m g . o f t h y r o x in e .

T h is i s s i m i l a r

t o t h e y i e l d and a c t i v i t y r e p o r t e d b y F r ie d e n , L i p s e t t and W in z le r (1 4 9 ) who u se d a s i m i l a r m ethod o f p r e p a r a t i o n . The r a d i o a c t i v e a s s a y s w ere c a r r i e d o u t u s in g a n end window m ica c o u n te r a sse m b ly h a v in g a window th ic k n e s s o f 2 .7 m g ./s q .c m .

The c o u n te r

was u se d i n c o n ju n c tio n w ith a C y c lo tro n S p e c i a l t i e s Company s c a l e - o f - 8 G-M a m p lify in g c i r c u i t , ty p e A.

The sam ples w ere mounted i n aluminum

sam ple p a n s made e s p e c i a l l y t o f i t d i r e c t l y u n d e r t h e end window tu b e . As t h e aluminum i s o f low atcanic num ber, back s c a t t e r i n g was n e g l i b l e . The sam ple pans m easured 1^" i n d ia m e te r w ith one s i x t h o f a n in c h s i d e ­ w a lls .

The th y r o x in e sam ples w ere d i s s o lv e d i n 0 .0 1 N p o ta s siu m c a r b o n a te

s o lu tio n .

One m i l l i l e t e r sam ples o f t h i s w ere ta k e n f o r c o u n tin g , and

d r i e d on t h e aluminum cups u n d e r i n f r a - r e d l i g h t . THE ANALYSIS CF IODOCASEIN FOR THYROXINE CONTENT BY THE ISOTOPE DILUTION METHOD.

The a n a l y s i s was c a r r i e d o u t on th e same l o t o f io d o c a s e in t h a t had b e en u sed f o r d e te r m in a tio n o f th y r o x in e by c o n v e n t! a l g r a v im e tr ic m eans. I n t h i s way, a c o m p a riso n o f t h e tw o m ethods was made p o s s i b l e .

The

p ro c e d u re was a s f o llo w s : One h u n d red gram s o f io d o c a s e in was h y d ro ly se d f o r 20 h o u rs w ith 40^ b a riu m h y d ro x id e u n d e r r e f l u x c o n d it i o n s .

One h u n d red m illig r a m s o f

r a d io th y r o x in e , h a v in g a s p e c i f i c a c t i v i t y o f 6500 c .p .m ./m g . o f th y r o x in e was added t o t h e m ix tu re o f b ariu m s a l t s of t h e amino a c id s t h a t had b e en form ed b y h y d r o l y s i s .

The i s o l a t i o n o f th y r o x in e was c a r r i e d o u t a s

d e s c r ib e d e a r l i e r , and fo llo w in g t h e p u r i f i c a t i o n , a y i e l d o f 212 mg. o f

36 th y r o x in e was o b ta in e d .

I t p o s s e s s e d a s p e c i f i c a c t i v i t y o f 208 c .p .m ./in g ,

o f t h y r o x i n e , a s d e te rm in e d w ith t h e sanç)le m ounted on a n alum inum cup and u s in g a b e l l - s h a p e d , t h i n window c o u n te r .

The c a l c u l a t i o n s a r e a s

f o llo w s : X^mg, o f th y r o x in e l a b e le d w ith io d in e -1 3 1 c o u n tin g X c .p .m . was added t o t h e h y d r o ly s a te Xgimg. o f u n la b e le d th y r o x in e c o n ta in e d i n th e io d o c a s e in S p e c if i c a c t i v i t y b e f o r e m ixing

=

X %

S p e c if i c a c t i v i t y a f t e r m ixiug T h e r e f o r e , X2

=

( S p e c i f i c a c t i v i t y b e f o r e m ix in g - l ) X^ ( s p e c i f i c a c t i v i t y a f t e r m ix in g )

S u b s t i t u t i n g a c t u a l v a l u e s , t h e f o llo w in g r e s u l t i s o b ta in e d . Xq =

100 mg. o f r a d io th y r o x in e

X c .p .m . *

6 5 0 ,0 0 0 c .p .m .

T h e r e fo r e , t h e s p e c i f i c a c t i v i t y b e f o r e m ixing = =

6 5 0 .0 0 0

=

X ^1

6500 c .p .m ./m g . o f th y r o x in e

100

The s p e c i f i c a c t i v i t y a f t e r m ix in g = X2

»

( 6500 208

208 c .p .m ./m g . o f th y r o x in e

- 1 ) 100

=

3063 mg. o f th y r o x in e

As t h i s i s t h e th y r o x in e c o n te n t o f 100 g . o f io d o c a s e in , t h i s r e p r e s e n ts a v a lu e o f 3*06^ th y r o x in e .

T h is c h e c k s c l o s e l y w ith t h e

v a lu e o b ta in e d by th e g r a v im e tr ic m ethod.

37 THE ABSORPTION OF lABlSLSD IODOCASEIN FROM THE AUMENTARY TRACT OF THE RAT T h is s tu d y was c a r r i e d o u t a s a p r e lim in a r y o p e r a t io n p r i o r t o l a t e r s i m i l a r e x p e rim e n ts w ith l a r g e r a n im a ls .

S e c o n d ly , th e u p ta k e o f

r a d i o a c t i v i t y b y t h e th y r o id was o f p a r t i c u l a r i n t e r e s t a s i t w ould p ro ­ v id e an i n d i c a t i o n o f t h e amount o f in o r g a n ic i o d i d e t h a t was a s s o c i a t e d w ith t h e io d o c a s e in due t o a b s o r p tio n o r c o - p r e c i p i t a t i o n phenomena o c c u r r in g d u r in g i t s p r e p a r a t i o n .

I h i r d l y , any in o r g a n ic i o d id e t h a t may

form a s a m e ta b o lite i n th e a b s o r p tio n o f th e io d o c a s e in w i l l be sw ept i n t o t h e t h y r o i d , due t o i t s p r o p e n s ity f o r c o n c e n tr a tin g t h i s s u b s ta n c e . F o u rte e n l a r g e m ale vAiite r a t s w eig h in g i n t h e neig h b o rh o o d o f 300 g . i n th e p o s t - a b s o r p t i v e s t a t e , w ere each g iv e n 5 m l. o f r a d i o a c t i v e io d o c a s e in o r a l l y by stom ach t u b e .

T h is was m ost s u c c e s s f u l l y done by

s l i d i n g a 3" hypoderm ic n e e d le , s l i g h t l y b e n t and c o v e re d w ith a c o r r e s ­ po n d in g le n g th o f F re n c h No. 12 c a t h e t e r tu b in g , down t h e e so p h a g e a l g ro o v e u n t i l t h e p i t o f t h e r a t ' s stom ach was f e l t .

The m a t e r i a l was th e n

a d m in is te r e d by a t t a c h i n g a 5 m l. g l a s s s y r in g e t o t h e n e e d le .

As t h e

n e e d le end was c o v e re d by t h e c a t h e t e r v h ic h i n t u r n was l i b e r a l l y c o a te d w ith g l y c e r o l , t h e r a t s o f f e r e d l i t t l e r e s i s t a n c e and a d m i n i s t r a t io n was a c c o m p lish e d w ith o u t s t r a p p in g t h e r a t t o a b o a rd o r u s in g an a n e s t h e t i c . The r a t s w ere t h e n p u t i n 4 l i t e r b e a k e rs c o n ta in in g a w ir e g au ze p la tf o r m and c o v e re d o v e r w ith a n o th e r p i e c e o f w ire g a u z e h e ld i n p la c e b y a le a d w e ig h t. These w ere fo und t o be m ost c o n v e n ie n t f o r s t o r i n g a n im a ls t h a t have b e e n g iv e n r a d i o a c t i v e m a t e r i a l s , a s t h e y w ere e a s i l y c le a n e d and t h e r e was l e s s d a n g e r o f c o n ta m in a tio n .

38 The io d o c a s e in s o l u t i o n a d m in is te r e d , c o n ta in e d 0*036 g* o f io d o c a s e in p e r ml* o f s o l u t i o n , and c o u n te d 1 1 5 ,0 0 0 c.p * m ./m l*

At

v a r io u s tim e i n t e r v a l s f o llo w in g a d m i n i s t r a t i o n , each r a t w as a n e s t h e t i z ­ ed by th e i n t r a p e r i t o n e a l i n j e c t i o n o f n e m b u ta l, and i t s b lo o d rem oved by h e a r t p u n c tu re u s in g a 3 /4 " No* 22 gauge n e e d le a t t a c h e d t o a 10 ml* s y rin g e .

As much b lo o d a s p o s s ib l e was rem oved, so t h a t t h e th y r o id

g la n d , w hich was rem oved im m e d ia te ly a f te r w a r d s , was c o u t l e t e l y ex­ s a n g u in a te d *

One t e n t h ml* sam ples o f t h e b lo o d w ere p i p e t t e d o n to a

f i l t e r s t r i p and d r i e d u n d e r i n f r a - r e d l i g h t f o r c o u n tin g .

The d im e n sio n s

o f t h e s a n p le s t r i p w ere a s f o llo w s : F ilte r s trip

2g" by

C e llo p h a n e c o v e rin g -

3" by 3 /4 "

B acking p a p e r -

3" by 2 j "

As b e f o r e , t h e sam ple was p u t t o g e t h e r w ith s c o tc h t a p e . I n t h i s e x p e rim e n t, t h e l a b e le d io d o c a s e in u se d a s a s ta n d a r d was a s s a y e d f o r r a d i o a c t i v i t y i n t h e same f a s h io n a s t h e b lo o d , u s in g a 0*1 ml* sample* Upon re m o v a l, t h e t h y r o id s w ere w ashed th o ro u g h ly w ith d i s t i l l e d w a te r and th e n p u t i n 4 dram v i a l s c o n ta in in g 1 ml* o f 10^ sodium h y d ro x ­ id e .

T w e n ty -fo u r h o u rs l a t e r , a t w hich tim e th e t h y r o id i s c o m p le te ly

d is s o l v e d , a 0 .1 m l. sam ple o f t h e sodium h y d ro x id e sam ple was ta k e n and m ounted on a f i l t e r s t r i p f o r c o u n tin g . Upon c csn p le tio n o f t h e r a d i o a c t i v e a s s a y s o f t h e b lo o d and th e t h y r o i d s , o v e r a 50 h o u r p e r i o d , a c o n s id e r a b le l e v e l was fo u n d i n t h e b lo o d , b u t o n ly a v e ry s m a ll p e rc e n ta g e th e th y r o id s .

T hese r e s u l t s a r e shown on Graph 2 .

of r a d io a c tiv ity was found i n

GRAPH Z

I BOO

o f la b e le d lo lo r a s e in « iiliiaentary tr a c t of tne ra t

1600

Cu

1200

Ô00

£

(200 (1)

400.

200

Hours- after adicialBtr&tton

39 THE DISTRIBUTION OF IODINE-131 P i m S PIA Sm OF THE RAT FOLLOmiGr QRAX ADMINXSTMTIQN OF lABElED IODOCASEIN A lth o u g h th y r o x in e h a s b een i s o l a t e d from t h e t h y r o id g la n d , u n t i l r e c e n t l y (1 3 7 ), t h e r e h a s b e en no i n d i c a t i o n o f i t s p re s e n c e i n t h e blood* L a rg e ly t h i s h a s b een due t o t h e d i f f i c u l t i e s in v o lv e d i n m e a su rin g th e s m a ll q u a n t i t i e s o f io d in e found t h e r e (5 gamma p e r 100 ml* o f p lasm a)* Thus t h e r e h a s b e e n a good d e a l o f c o n je c tu r e c o n c e rn in g t h e n a t u r e o f th e c ir c u la tin g

t h y r o id hormone (1 3 7 ) , (1 3 6 ), (1 2 6 ) , (1 2 4 ), ( 125) , ( 123) ,

( 1 2 7 ) , (1 2 2 ), (128)*

Most o f t h i s worfc h a s in v o lv e d s t u d i e s d e a l in g w ith

t h e e f f e c t s o f v a r io u s s u b s ta n c e s on t i s s u e r e s p i r a t i o n , c o n c lu s io n s r e s t i n g on t h e c a l o r i g e n i c re s p o n s e r e s u l ti n g *

R e c e n tly how ever, Taurog

and C h a ik o ff (1 3 7 ), two p a r t i c u l a r l y a c t i v e w o rk e rs i n t h i s f i e l d , em ploying r a d i o a c t i v e a s s a y s c o u p le d w ith t h e c a r r i e r te c h n iq u e , h av e p r e s e n te d c o n v in c in g r e s u l t s t o show t h a t t h e th y r o id h o m o n e i s , i n f a c t , th y ro x in e * W ith t h i s work i n m ind, i t was d e c id e d t o a tte m p t t h e c l a s s i f i c a t i o n o f t h e i o d in e -1 3 1 l a b e le d compounds a p p e a rin g i n t h e b lo o d o f r a t s , f o llo w in g t h e o r a l a d m i n i s t r a t io n o f la b e le d io d o c a s e in *

As t h e p rep o n ­

d e ra n c e o f e v id e n c e i s i n f a v o r o f th e th e o r y t h a t t h e c i r c u l a t i n g t h y r o id hormone i s th y r o x in e , t h i s work h a s b een c h i e f l y c o n c e rn e d w ith t h e d i s t r i b u t i o n o f t h i s s u b s ta n c e i n t h e plasm a* Ten m l. o f r a d i o a c t i v e io d o c a s e in , c o u n tin g 2 ,3 7 2 ,0 0 0 c.p*m */m l* and c o n ta in in g 0*02? g* o f io d o c a s e in p e r ml* was g iv e n t o f o u r l a r g e r a t s (400 g * ) o r a l l y by stom ach tu b e a s d e s c r ib e d p re v io u s ly *

Ten h o u rs

l a t e r , a t w hich tim e t h e b lo o d l e v e l o f r a d i o a c t i v i t y i s a t i t s p e a k , t h e b lo o d was rem oved from t h e c a r o t i d a r t e r y b y c a n n u la tio n *

C lo ttin g

40

was p re v e n te d by t h e i n t r a p e r i t o n e a l i n j e c t i o n o f 0 .2 5 m l. o f p r i o r to t h e o p e r a t io n .

h e p a r in

The b lo o d from th e f o u r r a t s was p o o le d and

c e n t r if u g e d t o o b t a i n t h e p la s m a , a s n e a r l y a l l t h e i o d in e i n t h e b lo o d a p p e a rs t o b e a s s o c i a t e d w ith t h i s f r a c t i o n (1 2 0 ) . F iv e m i l l i l e t e r s o f t h e h e p a r in iz e d p lasm a was p u t i n a n a rro w ­ n eck ed 50 m l. c e n t r i f u g e tu b e c o n ta in in g 15 m l. o f n - b u t a n o l . was s to p p e re d and sh ak en v ig o r o u s ly *

The tu b e

The b u t y l a lc o h o l l a y e r o b ta in e d

fo llo w in g 10 m in u te s o f c e n t r i f u g a t i o n was p u t i n a 125 m l* s e p a r a t o r y fu n n e l.

The r e s i d u e i n th e c e n t r i f u g e tu b e was once m ore e x t r a c t e d w ith

15 m l. o f b u ta n o l and c e n t r i f u g e d . added t o t h e s e p a r a t o r y fu n n e l*

T h is l a y e r o f b u ta n o l was a ls o

As b u ta n o l i s r e p o r t e d t o d i s s o l v e

th y r o x in e , d i io d o t y r o s i n e and in o r g a n ic io d in e ( 1 4 3 ) , ( 1 3 9 ) , ( 1 4 0 ) , ( l 4 l ) , ( 1 4 4 ) , any o f t h e s e t h r e e s u b s ta n c e s a p p e a rin g i n th e b lo o d fo llo w in g t h e I n g e s t io n o f io d o c a s e in s h o u ld b e p r e s e n t I n t h i s e x t r a c t . The b u ta n o l e x t r a c t i n t h e s e p a r a t o r y f u n n e l was p u t i n a 50 m l. v o lu m e tric f l a s k and b ro u g h t u p t o t h e m ark w ith b u t a n o l .

A 0 .5 m l.

sam ple was ta k e n and p i p e t t e d i n t o an aluminum c o u n tin g cup •

I t was

d r i e d u n d e r i n f r a - r e d , and c o u n te d w ith t h e th in -w in d o w b e l l —sh ap ed c o u n te r d e s c r ib e d p r e v i o u s l y .

I n t h i s f a s h i o n , i t was p o s s i b l e t o d e t e r ­

m ine t h e amount o f r a d i o a c t i v i t y t h a t had b e e n e x t r a c t e d from t h e 5 m l. o f p la sm a . N e x t, t h e b u ta n o l e x t r a c t was sh ak en u p i n a s e p a r a t o r y f u n n e l w ith 50 m l. o f B laus* r e a g e n t w hich w i l l rem ove any in o r g a n ic io d in e o r d i io d o t y r o s i n e p r e s e n t , a c c o rd in g t o B la u ( 1 4 5 ) .

T h is l a t t e r r e a g e n t

c o n s i s t s o f a s o l u t i o n o f 4 N sodium h y d ro x id e c o n ta in in g 5^ sodium c a r b o n a te .

A f te r t h e m ix tu re was a llo w e d t o s ta n d f o r s e v e r a l h o u r s .

41 t h e lo w e r aqueous l a y e r was r u n o u t and t h e b u t y l a lc o h o l f r a c t i o n e x t r a c t ­ ed a seco n d tim e w ith 30 m l. o f th e r e a g e n t .

T h is tim e t h e s e p a r a t i o n

o f t h e two l a y e r s was a llo w e d t o p ro c e e d f o r 15 h o u r s , a t t h e end o f which tim e th e b u t y l a lc o h o l showed o n ly a s l i g h t t u r b i d i t y .

Sam ples w ere

a g a in ta k e n f o r r a d i o a c t i v e a s s a y o f b o th t h e b u t y l a lc o h o l f r a c t i o n and t h e B laus* r e a g e n t f r a c t i o n . The p a t t e r n o f d i s t r i b u t i o n o f io d in e - 1 3 1 i n t h e p lasm a d i io d o t y r o ­ s i n e , in o r g a n ic i o d in e and th y r o x in e f r a c t i o n s i s shown b e lo w . T o ta l A c t i v i t y

5 m l. o f p lasm a B u ty l a lc o h o l e x t r a c t o f plasm a . . . . . . . T hyroxine f r a c t i o n o f p l a s m a D iio d o ty r o s in e - in o r g a n ic io d in e f r a c t i o n o f p lasm a ...........

A c t i v i t y e x p re s s e d a s % o f p lasm a __________________ a c t i v i t y ___________ 8 l6 0 c .p .m . 100^ 3520 c .p .m . 43^ 500 c«p.m . 6% 3010 c .p .m .

3^%

From t h e s e f i g u r e s , i t w i l l b e s e e n t h a t a b o u t 60% o f t h e io d in e - 1 3 1 i n t h e plasm a o f t h e r a t i s n o t e x t r a c t a b l e w ith b u t y l a lc o h o l u n d e r t h e c o n d itio n s f o r e x t r a c t i o n d e s c r i b e d .

An a tte m p t was made t o d e te rm in e

th e amount o f r a d i o a c t i v i t y l e f t i n t h e p lasm a fo llo w in g

b u t y l a lc o h o l

e x t r a c t i o n , b u t t h i s f a i l e d , due t o t h e n a tu r e o f t h e r e s i d u e . As t h e r e was a s l i g h t p o s s i b i l i t y t h a t t h e f r a c t i o n n o t e x t r a c t a b l e w ith b u t y l a lc o h o l was io d o c a s e in , i t was d e c id e d t o ch eck t h e s o l u b i l i t y o f io d o c a s e in i n t h i s s o l v e n t.

I t was found t h a t when; 25 mg. o f io d o ­

c a s e in was added t o plasm a and an e x t r a c t i o n w ith b u t y l a lc o h o l made on t h e p la sm a , no io d o c a s e in a p p e a re d i n th e d r i e d r e s i d u e o f th e e x t r a c t , \dien th e b u t y l a lc o h o l was ro a o v e d b y e v a p o r a tio n . As t h e th y r o x in e f r a c t i o n , a lth o u g h o n ly r e p r e s e n t i n g 6% o f th e

42 t o t a l io d in e -1 3 1 i n th e plasm a^ was o f p a r t i c u l a r i n t e r e s t , i t was de­ c id e d t o p o s i t i v e l y i d e n t i f y i t s p re s e n c e t h e r e a s s u c h .

T h is was m ost

e a s i l y done b y em ploying t h e c a r r i e r te c h n iq u e i n t h e f o llo w in g f a s h i o n , b a se d upon th e m ethod o f Taurog and C h a ik o ff (1 3 7 ). F i f t y mg. o f r e c r y s t a l l i z e d th y r o x in e was added a s c a r r i e r , and t h e b u t y l a lc o h o l e x t r a c t was c o n c e n tr a te d t o d ry n e s s on a b o i l i n g w a te r b a th u n d e r re d u c e d p r e s s u r e .

The th y r o x in e i n th e r e s i d u e was d is s o lv e d

i n h o t 0 ,1 N p o ta s s iu m c a rb o n a te and s e p a r a te d from a u n d is s o lv e d m a t e r i a l b y c e n t r i f u g a t i o n .

sm a.ll

amount o f

The th y r o x in e was th e n r e p e a t ­

e d ly c r y s t a l l i z e d a s f o llo w s : 1.

The f i r s t r e c r y s t a l l i z a t i o n was e f f e c t e d b y th e a d d i t i o n o f g l a c i a l

a c e t i c a c id to t h e h o t p o ta s siu m c a rb o n a te s o l u t i o n , th e n p r e c i p i t a t e d b y c o o lin g t o 0®C. 2.

T h is to o k a t l e a s t 24 h o u r s .

The c r y s t a l s o b ta in e d i n t h i s way w ere washed w ith w a te r , d i s s o lv e d

i n a minimum amount o f h o t p o ta ssiu m c a r b o n a te s o l u t i o n , and th e n p r e c i p ­ i t a t e d b y th e a d d i t i o n o f g l a c i a l a c e t i c a c i d , fo llo w e d b y r a p i d c o o lin g . 3.

The c r y s t a l s o b ta in e d i n (2 ) w ere d is s o lv e d i n a l k a l i n e 70^ e t h y l

a lc o h o l s o l u t i o n and p r e c i p i t a t e d b y th e a d d i t i o n o f g l a c i a l a c e t i c a c id * 4.

The f o u r t h r e c r y s t a l l i z a t i o n was made from a d i l u t e sodium h y d ro x id e

s o l u t i o n b y th e a d d i t i o n o f g l a c i a l a c e t i c a c i d .

A ll o f t h e s e r e c r y s t a l l ­

i z a t i o n s were c a r r i e d o u t i n a 15 m l. c e n t r if u g e t u b e , and th e p r e c i p i t a t e s washed by sh a k in g w ith d i s t i l l e d w a te r s e v e r a l tim e s fo llo w e d b y c e n t­ r i f u g a t i o n t o o b t a i n th e washed p r e c i p i t a t e .

T h is was d o n e , a s su c h a

sm a ll q u a n t i t y o f p r e c i p i t a t e i s d i f f i c u l t t o h a n d le s in te re d g la s s f i l t e r .

even u s in g a

F o llo w in g each r e c r y s t a l l i z a t i o n , sam ples o f t h e

43

th y r o x in e w ere m ounted on alum inum f i l t e r c u p s , d r i e d u n d e r i n f r a - r e d and w eig h ed .

The r e s u l t s f o llo w in g c o u n tin g a r e t a b u l a t e d b e lo w .

c o u n ts hav e b een c o r r e c t e d on th e b a s i s o f Ô.6 mg. o f t h y r o x in e .

AH On

c h e c k in g th e m ass a b s o r p tio n p r o p e r t i e s o f th y r o x in e sa m p le s, i t was fo u n d t h a t r e p r o d u c ib le r e s u l t s w ere o b ta in e d w ith sam ples w e ig h in g up t o 20 m g ., a s lo n g a s t h e sam ple was s p re a d e v e n ly o v e r th e s u r f a c e o f th e cup. 1 st re c ry s ta lliz a tio n . .

....

. . . . . . 3 3 c .p .m ./m g . o f th y r o x in e

2nd r e c r y s t a l l i z a t i o n ............................. . 3 0 c .p .m ./m g . o f th y r o x in e 3 rd r e c r y s t a l l i z a t i o n 4 th r e c r y s t a l l i z a t i o n ... . . . . . . . 3 2

. . 3 3 c .p .m ./m g . o f th y r o x in e c .p .m ./m g . o f th y ro x in e

T h is c o n s ta n c y o f s p e c i f i c a c t i v i t y i n d i c a t e s t h a t t h e r a d io ­ a c t i v i t y p r e s e n t i n t h e b u t y l a lc o h o l th y r o x in e f r a c t i o n i s th e same s u b s ta n c e vAiich u n d erw en t r e c r y s t a l l i z a t i o n , nam ely th y r o x in e . THE INTRAVENOUS INJECTION OF LABKLSD lODOGASEIN INTO SHEEP P r i o r t o b e g in n in g t h i s e x p e rim e n t, i t was n e c e s s a r y t o s e e i f id io le b lo o d e f f e c t s th e r a d i a t i o n from l a b e l e d io d o c a s e in by m ass a b s o r p tio n . One m i l l i l e t e r o f W iole b lo o d was adm ixed w ith 1 m l. o f r a d i o a c t i v e io d o c a s e in whose c .p .m ./ m l . was known.

A nother s o l u t i o n c o n ta in in g 1 m l.

o f t h e r a d i o a c t i v e io d o c a s e in b ro u g h t t o 2 m l. w ith d i s t i l l e d w a te r was made u p .

A 0 .1 m l. sam ple o f e ac h s o l u t i o n was m ounted on f i l t e r s t r i p s

i n th e u s u a l f a s h i o n . from b o th sa m p le s.

I t was fo u n d t h a t a n e q u a l co u n t was o b ta in e d

% u s w hole b lo o d d o e s n o t a f f e c t t h e r a d i a t i o n s b e in g

g iv e n o f f b y th e r a d i o a c t i v e io d o c a s e in even when a 0 .5 m l. sam ple o f

44 b lo o d was u s e d .

No e f f e c t was n o te d when a s o l u t i o n o f one p a r t o f

r a d i o a c t i v e io d o c a s e in t o 100 p a r t s o f b lo o d was s t u d i e d i n t h e same m a n n e r.

T h is i s t h e t y p i c a l c o n c e n tr a tio n t h a t w i l l b e fo u n d i n t h e

b lo o d o f t h e sh e ep fo llo w in g i n j e c t i o n . The sh e ep u se d i n t h i s e x p e rim e n t w ere b o rn i n t h e s p r in g o f 1947* Thus a t t h e tim e o f t h e e x p e rim e n t th e y w ere a b o u t i j y e a r s o l d .

They

w ere c a s t r a t e d m a le s , p re d o m in a n tly o f R am b o u iH et b r e e d in g and w eighed a p p ro x im a te ly 130 l b s .

T h e ir b lo o d volume was b etw een 2500 and 3000 ml*

As 30 m l. o f r a d i o a c t i v e io d o c a s e in i s t h e dosage o f c h o ic e f o r i n j e c t i o n , a 100 f o l d d i l u t i o n w iH ta k e p l a c e .

Thus t o g iv e a d e s i r a b l e c o u n tin g

v a lu e f o llo w in g i n j e c t i o n , t h e io d o c a s e in sh o u ld c o u n t a b o u t 1 0 0 ,0 0 0 c .p # m ./0 .1 m l. Ten m i n i c u r i e s o f io d in e -1 3 1 (3 m l.) c o u n tin g 440 m i l l i o n c .p .m ./m l. was added t o 20 g . o f c a s e i n .

T h is was t r e a t e d i n t h e u s u a l f a s h i o n t o

g iv e a s o l u t i o n o f r a d i o a c t i v e io d o c a s e in c o u n tin g 9 2 ,2 0 0 c . p . m . / O . l m l. Two sam p les o f 30 m l. each and two sam p les o f 50 m l. e ac h w ere g iv e n i n tr a v e n o u s ly v i a th e j u g u l a r v e i n , t o f o u r s h e e p .

B lood sam ples w ere

ta k e n from t h e same v e in a t a p p r o p r ia te tim e i n t e r v a l s , b e g in n in g im m e d ia te ly a f t e r a d m i n i s t r a t i o n .

F iv e one h u n d red s o f a m i l l i l e t e r o f

s a t u r a t e d o x a la te s o l u t i o n was added t o e ac h sam ple t o p r e v e n t c l o t t i n g . As shown i n Graph 3 . t h e d e c re a s e i n a c t i v i t y i s e x p re s s e d a s a p e r c e n t o f t h e a c t i v i t y o f t h e f i r s t b lo o d sam ple drawn im m e d ia te ly a f t e r admin­ is tra tio n .

From t h i s , i t i s se e n t h a t 99^ o f t h e io d o c a s e in i s rem oved

fro m th e b lo o d s tre a m i n 7 h o u rs and t h a t t h e l a r g e r dose o f io d o c a s e in shows a p e r c e n t d e c re a s e i n a c t i v i t y p e r h o u r t h a t i s p r o p o r t i o n a l l y

GPJiPH 3, Otirve 1 - 30 ml. o f 2 30 ml » o f V 3 - 50 ml^ o f H 4 - 50 ml. o f

r a d io a c tiv e ra d io a c tiv e r a illo :c tiv e r a d io a c tIv e

lodocaneln io d o ca sein io d o ca sein io lo c A se in

The intravenous in j e c tio n o f la b e le d l.o d o e a s e ir . I n t ) s h e e p r f c n r a n im a ls

8tudled«

3 Hour

4-

5

faXlo^'lng ad m in istrât Ion

45 h ig h e r th a n t h a t shown f o r th e s m a lle r dose# THE ABSQRPTiatJ OF LABELED IODOCASEIN FRCM THE ALIMENTARY TRACT OF THE SHEEP I n t h e s e s e r i e s o f e x p e r im e n ts , la b e le d io d o c a s e in was p r e p a r e d i n t h e u s u a l fa s h io n *

Where n e c e s s a r y , an io d o c a s e in o f h ig h e r s p e c i f i c

a c t i v i t y was made b y d e c re a s in g th e amount o f c a s e i n u se d f o r i o d i n a t i o n , a lo n g w ith d e c r e a s in g th e amount o f o r d in a r y i o d in e u se d a s c a r r i e r . By k e e p in g t h e amount o f io d in e -1 3 1 a t t h e same l e v e l , a p ro d u c t o f h ig h e r s p e c i f i c a c t i v i t y was o b ta in e d . F o u r r o u t e s o f a d m i n i s t r a t io n w ere s t u d i e d .

O ra l a d m i n i s t r a t i o n ,

v e n t r a l rumen s a c a d m i n i s t r a t i o n , s m a ll i n t e s t i n e a d m i n i s t r a t io n and cecum a d m in is tra tio n . t h e e a r l i e r work*

The sh e e p u se d w ere o f th e same g ro u p a s th o s e u se d i n Perm anent f i s t u l a s le a d in g i n t o t h e v e n t r a l rumen s a c ,

s m a ll i n t e s t i n e and cecum w ere s u r g i c a l l y e s t a b l i s h e d i n t h e a n im a ls a b o u t 9 m onths a f t e r b i r t h .

O ra l a d m i n i s t r a t io n was acco m p lish e d b y

p a s s in g a ru b b e r c a t h e t e r th ro u g h th e n o s t r i l and i n t o t h e e so p h a g u s. The m a t e r i a l was a ls o in tr o d u c e d i n t o b o th th e s m a ll i n t e s t i n e and cecum b y means o f a r u b b e r c a t h e t e r p a s s e d th ro u g h t h e perm anent f i s t u l a s l e a d ­ in g i n t o each o f th e s e o r g a n s .

A 12** m e ta l c a t h e t e r i n s e r t e d th ro u g h a

f i s t u l a i n t h e rum en w aH was u se d t o d e p o s it t h e m a t e r i a l i n t h e v e n t r a l rumen sac# The a n im a ls r e c e iv e d t h e i r u s u a l d i e t o f a l f a l f a hay and s h e l l e d c o rn d u rin g t h e e x p e rim e n ts .

Feed was n o t w ith h e ld a t any tim e , s i n c e i t

i s i m p r a c t i c a l t o a tte m p t t o t r e a t ru m in a n ts i n t h e p o s t—a b s o r p tiv e s ta te #

Ten sh e e p w ere u se d i n th e fo llo w in g e x p e rim e n ts on a b s o rp tio n #

46

1.

O ra l and s m a ll i n t e s t i n e a d m i n i s t r a t io n ; Ten gram s o f v ita m in —f r e e c a s e i n was i o d in a t e d w ith a m ix tu r e o f

2 .5 g . o f pow dered io d in e and 35 mil, 11 c u r i e s o f io d in e - 1 3 1 . c a s e i n form ed c o u n te d 3>734^000 c .p .m « /m l. and c o n ta in e d io d o c a s e in p e r m l. o f s o l u t i o n .

The io d o —

0 .0 3 2 g . o f

One h u n d red m l. o f t h i s m a t e r i a l was

g iv e n t o e ac h o f 8 s h e e p , f i v e o r a l l y and t h r e e th ro u g h f i s t u l a s i n t o t h e s m a ll i n t e s t i n e .

T hree m i l l i l e t e r sam ples o f b lo o d w ere rem oved from th e

j u g u l a r v e in a t a p p r o p r i a te tim e s o v e r a 100 h o u r p e r i o d . B lood sam ples o f 0 .5 m l. w ere p i p e t t e d o n to f i l t e r s t r i p s , d r i e d u n d e r i n f r a - r e d i r r a d i a t i o n , and c o u n te d . b ack g ro u n d and d e c a y .

A H c o u n ts w ere c o r r e c t e d f o r

As w i l l b e n o te d from G raph 4 * , t h e r e i s a h ig h e r

a b s o r p tio n fo llo w in g o r a l a d m i n i s t r a t i o n , a lth o u g h t h e m a t e r i a l a p p e a rs i n t h e b lo o d s tre a m m ore r a p i d l y fo llo w in g a n a H i n t e s t i n e a d m i n i s t r a t i o n . I n b o th c a s e s , t h e a b s o r p tio n p eak i s re a c h e d a t a b o u t 10 h o u rs f o l lo w ­ in g a d m i n i s t r a t i o n .

M easu rab le a c t i v i t y was s t i l l p r e s e n t a s l a t e a s

f o u r d ay s fo llo w in g a d m i n i s t r a t i o n . 2.

O r a l, v e n t r a l rumen s a c , sm a ll i n t e s t i n e and cecum a d m i n i s t r a t io n : The f o llo w in g e x p e rim e n t was c a r r i e d o u t i n a s i m i l a r f a s h i o n .

As

b e f o r e , 10 g . o f c a s e in was u s e d , b u t o n ly 15 m i l l i c u r i e s o f io d in e -1 3 1 was added t o t h e r e a c t i o n m ix tu r e .

T h is gave an io d o c a s e in w ith a lo w e r

s p e c i f i c a c t i v i t y c o u n tin g 1 ,4 0 0 ,0 0 0 c .p .j a ./ m l . and c o n ta in in g 0 .0 3 2 g . / m l. o f io d o c a s e in .

Each o f f o u r sh e ep w ere g iv e n 75 m l. o f th e m a t e r i a l .

F o u r r o u t e s o f a d m i n i s t r a t io n w ere s t u d i e d , o r a l , v e n t r a l rumen s a c , em ail i n t e s t i n e and cecum .

A f i f t h sh e e p was g iv e n 200 m l. o f io d o c a s e in ,

t o s tu d y t h e e f f e c t s o f l a r g e r dosage on t h e l e v e l o f a b s o r p t io n .

In

p e r 0*3 ml* o f mheefj 8 w hole b lo o d

O

« 07

O ,o

(5^ O .O

CD O O

O o o

ro O O

4^ O O

o p; 'I < a>

O 'r-* 'd O O y

3

P j-j

H« d t+ O (R o ef *-k 5 M * (9 O 5C o p. o 8 H* p m d '.t Hcm d » »• o 5{■ O a

A

(9

rt;

J-* i

4^

o o W )d p k* M O i—P o o B p C Od n> H* 0» d tf+ n rf P O c* M « (A O rr d

•s lO

^ •S o T* # a 0>

OS

■«

v>

lO

o' o

£ k

ed ■p u f-4 cd (d o •H

II (D OdC (D < CO + 3

CD @

0) Q) Q C,

2

3

4

5

Hours fo llo w in g a d m in is tr a tio n

Curve 1 -3 0 m l. o f la b e le d io d o c a s e in Curve 2 ” "

Curve 3 -5 0 m l. o f la b e le d io d o c a s e in Curve "

A ll in j e c t e d in tr a v e n o u s ly G R A T ^H 1

8 T h i r t y m i l l i l e t e r s o f io d o c a s e in c o u n tin g 9 2 0 ,0 0 0 c,p*m «/inl* and c o n ta in in g 0 .0 3 2 g ./ m l . o f io d o c a s e in was i n j e c t e d i n t o t h e j u g u l a r v e in o f two sh eep *

A n o th er tw o sh e e p w ere g iv e n 50 m l. b y t h e same r o u t e .

B lood sam p les w ere ta k e n f o r r a d i o a c t i v e a s s a y from t h e v e in a t a p p r o p r i a te in te rv a ls .

As shown i n G raph 1 . , t h e d e c r e a s e i n a c t i v i t y i s e x p re s s e d

a s a p e r c e n t o f t h e a c t i v i t y o f t h e f i r s t b lo o d sam ple draw n im m e d ia te ly a f t e r a d m in is tra tio n .

From t h i s i t i s se e n t h a t 90^ o f t h e i o d o c a s e in

i s ro n o v ed fro m t h e b lo o d s tre a m i n 7 h o u rs and t h a t th e l a r g e r d o se o f io d o c a s e in shows a p e r c e n t d e c r e a s e i n a c t i v i t y p e r h o u r t h a t i s p ro ­ p o r t i o n a l l y h ig h e r t h a n t h a t shown f o r t h e s m a lle r d o s e . THE ABSORPTION OF lA B S im IODOCASEIN FROM THE ALIMENTARY TRACT OF THE SHEEP F o u r r o u t e s o f a d m i n i s t r a t i o n w ere s t u d i e d , o r a l , v e n t r a l sa c o f rum en, s m a ll i n t e s t i n e and cecum .

The sh e ep u s e d w ere t h e same a g e ,

w e ig h t and b r e e d a s th o s e m en tio n ed above.

P erm anent f i s t u l a s le a d in g

i n t o t h e v e n t r a l sa c o f rum en, s m a ll i n t e s t i n e and cecum w ere s u r g i c a l l y e s t a b l i s h e d i n t h e a n im a ls 9 m onths a f t e r b i r t h .

O ra l a d m i n i s t r a t io n

was a c h ie v e d b y p a s s in g a r u b b e r c a t h e t e r th ro u g h t h e n o s t r i l and i n t o t h e e so p h a g u s.

The m a t e r i a l was in tr o d u c e d i n t o b o th t h e s m a ll i n t e s t i n e

and t h e cecum a l s o by means o f a ru b b e r c a t h e t e r p a s s e d th ro u g h t h e f i s t u l a s le a d in g i n t o t h e s e o r g a n s .

A 12" m e ta l c a t h e t e r i n s e r t e d

th ro u g h a f i s t u l a i n t h e rum en w a ll was u se d t o d e p o s it t h e m a t e r i a l i n t h e v e n t r a l sa c o f t h e rum en.

The a n im a ls r e c e iv e d t h e i r u s u a l d i e t

o f a l f a l f a h a y and s h e l le d c o rn d u rin g t h e e x p e rim e n ts*

F e e d was

n o t w ith h e ld a t an y tim e , s i n c e i t i s i m p r a c t i c a l t o a tte m p t t o t r e a t

C.P.M. per Ool m l. o f sheep blood

Ilf 0