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Studies on the in vivo metabolism of thyroxine

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Studies on the in vivo metabolism of thyroxine

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Content STUDIES O N T H E IN VIVO M ETABOLISM O P THYROXINE A D i s s e r t a t i o n P r e s e n te d t o The F a c u lty o f t h e G rad u ate S ch o o l U n iv e r s ity of S o u th e rn C a l if o r n ia I n P a r t i a l F u lf illm e n t o f th e R eq u irem en ts f o r th e D egree D o cto r o f P h ilo so p h y (B io c h e m istry and N u tr it io n ) by B u rto n J* K allm an June 1958 UMI Number: DP21582 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI DP21582 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 Ph. D Su ^ Kiif This dissertation, written by B u rto n J-. K allm an under the direction of h is Guidance Committee, and approved by all its members, has been pre sented to and accepted by the Faculty of the Graduate School, in partial fulfillment of re quirements for the degree of D O C T O R OF P H I L O S O P H Y Dean Guidance Committee f] I / Chairman I w ish t o e x p re s s my deep a p p r e c ia tio n h i s e n co u rag em en t, h e lp and f i n a n c i a l su p p o rt t h i s w o rk . I a ls o w is h t o th a n k D rs • John W* and Hans !»• F a lk f o r t h e i r a id and g u id a n c e . t o Dr • P a u l S t a r r f o r d u r in g th e c o u rse o f M ehl, B l l e r y C . S to w e ll TABLE O F CONTENTS PAGE I • HISTORICAL S H R V E Y ...................................................................... 1 The p re s e n c e o f tr i i o d o t h y r o n i n e in v iv o • • • • 1 The in v i t r o fo rm a tio n o f tr i io d o th y r o n in e and o th e r th y r o x in e a n a lo g u e s • • • • • ............................. • 5 s The d e io d ln a tio n th e o r y o f th y r o x in e a c t i v i t y 6 I I . STATEMENT OF THE PROBLEM ........................... 11 I I I . EXPERIMENTAL . . ....................................... 13 C hrom atographic s e p a r a tio n o f th y ro x in e and t r i io d o th y r o n in e ....................... 13 D e te c tio n and i d e n t i f i c a t i o n o f m a te r ia ls on chrom atogram s • • • • ...............................................• • 14 The d e t e c t i o n o f r a d i o a c t i v i t y on chrom atogram s • 15 P r e p a r a tio n and c o n c e n tr a tio n o f b u ta n o l e x t r a c t s o f serum ................................................................................ 16 P r e p a r a tio n and c o n c e n tr a tio n o f b u ta n o l e x t r a c t s o f t i s s u e s ................................................................................ 16 R eco v ery e x p e r i m e n t s .............................................................. 17 L ab eled com ponents i n human serum fo llo w in g 131 in tra v e n o u s th y r o x i n e - I • 20 L ab eled com ponents in human serum f o llo w in g 131 in tra v e n o u s th y r o x i n e - I and s u r g i c a l s t r e s s o r e p i n e p h r i n e ....................... 22 PAGE L ab eled com ponents i n r a t t i s s u e s fo llo w in g th y r o x i n e - I 24 131 E x c r e tio n o f I a f t e r t h e a d m in is tr a tio n o f la b e le d t h y r o x i n e ....................... 26 1 7 . DISCUSSION ...................................................................................... 28 V . SU M M A R Y ........................................................................... 46 ACKNOW LEDGEM ENTS ....................... 47 BIBLIOGRAPHY ........................................................................... 48 APPENDIX . ............................................................................ 55 LIST O F TABLES TABLE I . I I . I I I . IV . V . V I . V I I . V I I I . IX . PA® C hrom atographic A n a ly sis o f Human Serum A f te r T h y r o x i n e - I * ........................................................ 56 C hrom atographic A n a ly sis o f Human Serum A fte r 131 T h y ro x in e -I A d m in is tra tio n t o S u r g ic a l Cases • • 57 C hrom atographic A n a ly s is of Human Serum A f te r T h y ro x in e -I* ^ * and E p in e p h rin e • • • • • • • • • • 58 The E f f e c t o f E p in e p h rin e on th e Serum P r o te i n Bound I o d i n e ...................................... .............................................. 61 { Serum G lucose o f Group IV R a t s ........................................... 62 . | C hrom atographic A n a ly sis o f R a t T is s u e s F o llo w in g \ * 131 A d m in is tra tio n o f L -T h y ro x in e -I 63 C hrom atographic A n a ly sis o f R at T is s u e s F o llo w in g 131 A d m in is tra tio n o f L -T h y ro x in e -I • • • • • • . . 64; i C hrom atographic A n a ly sis o f R at T is s u e s F o llo w in g | i 131 ' A d m in is tra tio n o f L -T h y ro x in e -I and E p in e p h rin e • 65 i C h ro m ato g rap h ic A n a ly sis o f R at T is s u e s F o llo w in g j 131 ■ A d m in is tra tio n o f L -T h y ro x in e -I and E p in e p h rin e • 66 LIST O F FIGURES FIGURE PAGE 1 , S t r u c t u r a l F orm ulas o f Some D e r iv a tiv e s of T h y ro n in e • • 68 2 , R eco v ery E x p e r i m e n t s ........................................ 69 3 , P a t i e n t 4 . A th y r e o tic , e u th y r o id . Amyl A lc o h o l C h ro m a to g ra p h y ........................................ •............................................... 70 4 , P a t i e n t 5 , Myxedema - Amyl A lco h o l C hrom atography • , , 71 5 , P a t i e n t 5 , B i l i a r y P a th o lo g y , Amyl A lco h o l C hrom atography • * • » , ..................................................................................72 6 , P a t i e n t 6 . B i l i a r y P a th o lo g y , B u tan o l-D io x an e Chr omat o gr a p h y ........................................................................................... 73 131 7 , P a t i e n t 3 , T h y ro x in e -I and E p in e p h r in e , Amyl A lco h o l C h ro m a to g ra p h y ......................................................................................................... 74 131 8 , P a t i e n t 4 , T h y ro x in e -I and E p in e p h r in e , Amyl A lco h o l C hrom atography .......................................... 75 9 , Group I , R at 1 , Amyl A lco h o l C h ro m a to g ra p h y .......................... 76 1 0 , Group I , R a t 2 , Amyl A lc o h o l C h ro m a to g ra p h y .......................... 77 1 1 , Group IV , R a t 1 , Amyl A lc o h o l C h r o m a to g r a p h y ........................ 78 1 2 , Group IV , R a t 1 , B u tan o l-D io x an e C hrom atography , , , , 79 1 3 , Group IV , R a t 2 . Amyl A lco h o l C hrom atography • • , • • 80 1 4 , Group IV , R at 2 . B u tan o l-D io x an e C hrom atography • • , • 81 1 5 , Group IV , R at 3 , Amyl A lc o h o l C hrom atography • « « , • 82 1 6 , Group IV , R at 3 . B u tan o l-D io x an e C hrom atography . . • • 83 1 7 , Group IV , R at 4 , Amyl A lco h o l C h r o m a to g r a p h y ........................ 84 viii FIGURE PAGE 1 8 . Group IV , R at 4 , B u tan o l-D io x an e C hrom atography • • . 85 131 131 1 9 . U rin a ry E x c r e tio n o f I 9 £ —£ , U rin a ry I A f te r 131 T h y ro x in e A lo n e .® — 0 , U rin a ry I A f te r T h y ro x in e and E p in e p h rin e • • • • • • . . . . . . . 86 2 0 . F e c a l E x c r e tio n o f I ^ \ ® _ F e c a l A fte r 131 T h y ro x in e ,® —® , F e c a l I A f te r T h y ro x in e and E p in e p h rin e • • • • • • • • ........................................... 87 I . HISTORICAL SURVEY A . The P re s e n c e o f T r iio d o th y r o n in e in v i v o . The p re se n c e o f tr i i o d o t h y r o n i n e (F ig # l ) in b i o l o g i c a l ■ sy stem s was f i r s t o b serv ed by G ross eib a l • (1 ) i n 1 9 5 0 . T hese 131 j w o rk ers in j e c t e d I as io d id e i n t o r a t s and k i l l e d th e a n im a ls a t I 48 h o u r s • B u ta n o l e x t r a c t s o f th e th y r o id s w ere fo u n d t o c o n ta in j s e v e r a l r a d io a c ti v e com ponents s e p a r a b le by ch ro m ato g rap h y # These 1 in c lu d e d mono- and d i i o d o t y r o s i n e , th y r o x in e , io d id e and s e v e r a l unknown com pounds• One o f th e s e unknown s u b s ta n c e s was a ls o fo und i n mouse and r a t t i s s u e s by t h i s group (2 ) • The f o llo w in g y e a r , in i a p r e lim in a r y p a p e r , G ross and P i t t - R i v e r s (3 ) r e p o r te d th e p re s e n c e 131 o f tw o unknown r a d i o a c t i v e s u b s ta n c e s i n human p lasm a s if te r I tr e a tm e n t o f v a rio u s th y r o id d is o r d e r s * They l a t e r showed t h a t one o f th e s e unknowns was o h ro m a to g ra p h ic a lly i d e n t i c a l w ith s y n th e t ic t r i i o d o th y r o n in e (4 ) • T h is compound was so o n shown t o have th y r o i d hormone a c t i v i t y (5 -8 ) • ( L ab eled tr i i o d o t h y r o n i n e h a s b een r e p o r te d in th e serum o f 131 i humans t r e a t e d w ith I # D in g le d in e e t a l# (9 ) fo u n d i t bound t o serum alb u m in t o th e e x te n t o f 4 t o 20^ o f th e alb u m in -b o u n d th y r o x i n e • Benua and Dobyns d e te c te d c i r c u l a t i n g t r i i o d o t h y r o n i n e in 19 o f 31 p a t i e n t s ( 1 0 ) . T hese w o rk e rs b e lie v e t h e amount o f t r i io d o th y r o n in e i s r e l a t e d t o th e d e g re e o f th y r o t o x ic o s is # I n P it t- R iv e r s * la b o r a to r y t r i i o d o th y r o n in e was fo und i n an 131 I - t r e a t e d g o itr o u s c r e t i n ( 1 1 ) • A lp e rs and R a i l have r e p o r te d 2 l i t s p re s e n c e i n th e c e r e b r o s p in a l f l u i d i n t h r e e o f e le v e n p a t i e n t s , 131 t r e a t e d w ith I . T h y ro x in e was d e te c te d i n a l l e le v e n (1 2 ) . Benua e t a l • (1 3 ) fo u n d t r i io d o th y r o n in e in t h e b lo o d o f 22 o f 39 p a t i e n t s w ith G raves * d i s e a s e , one o f tw o w ith t o x i c n o d u la r g o i t e r s , tw o o f f o u r w ith t h y r o i d c a n c e rs w ith s m a ll rem n an ts o f th y r o i d t i s s u e and tw o o f sev en e u th y ro id c a r d ia c p a t i e n t s • The g r e a t e s t serum c o n c e n tr a tio n s o f t r i i o d o t h y r o n i n e w ere fo u n d a t ab o u t 72 h o u rs 131 a f t e r tr e a tm e n t w ith I , a lth o u g h t r a c e s w ere som etim es p r e s e n t as e a r l y as one h o u r a f t e r t h e r a p y . At t h e h ig h e s t l e v e l s o f t r i i o d o th y r o n in e , th e r a t i o th y r o x i n e s tr iio d o t h y r o n in e ra n g e d from f i v e t o f o r t y • T au ro g e t Al •» who had b een u n a b le t o d e m o n stra te c i r c u l a t i n g tr i i o d o t h y r o n i n e f o r some tim e , r e c e n t l y r e p o r te d on th e s u b s ta n c e s a p p e a rin g i n th e t h y r o i d v e in s o f la r g e an im a ls f o llo w in g t h e a d m in is - 131 1 t r a t i o n o f I in v e ry la r g e d o ses ( 1 4 ) . I n th e th y r o i d v e in plasm a 1 ' i o f th e s e a n im a ls r a d i o a c t i v i t y as t r i i o d o t h y r o n i n e am ounted t o 8 .5^ | \ o f th e th y r o x in e a c t i v i t y . No tr i i o d o t h y r o n i n e w as fo u n d in a r t e r i a l ; p la sm a . P a n c r e a tin - d ig e s te d sh ee p th y r o i d s w ere fo und t o c o n ta in ! a b o u t 9% o f t h e i r t o t a l as th y r o x in e and 1% as t r i i o d o t h y r o n i n e . O th er w o rk ers have r e p o r te d th e ab se n c e o f tr i io d o th y r o n in e i n 131 th e c i r c u l a t i o n f o llo w in g I t r e a t m e n t . W h ite and R e i l l y (1 5 ) in a s e r i e s o f 19 p a t i e n t s w ith G rav es1.d is e a s e and f o u r w ith a n g in a , fo u n d o n ly th y r o x in e , io d id e and a band a t th e s o lv e n t f r o n t . B lood sam ples f o r t h i s s tu d y w ere draw n a t tim e i n t e r v a l s b etw een one h o u r and 23 d ay s a f t e r t h e r a p y . L arso n e t a l . s u b je c te d th e serum o f p a t i e n t s g iv en I t o e le o t r o p h o r e s is on a s t a r c h b lo c k * They e x - , t r a c t e d th e r a d io a c ti v e p o r tio n s o f th e b lo c k w ith b u ta n o l* T hese i w o rk e rs fo u n d o n ly la b e le d th y r o x in e i n t h e i n t e r - a - g l o b u l i n zone ! and r e p o r te d th e alb u m in r a d i o a c t i v i t y t o be n o n - e x tr a o ta b le w ith i b u ta n o l ( 1 6 ) . C o n tra ry r e s u l t s w ere o b ta in e d by P i t t - R i v e r s e t a l * , ; who fo und b o th r a d io a c ti v e zo nes t o be e x t r a c t a b l e w ith b u ta n o l and fo u n d tr i i o d o t h y r o n i n e p r e s e n t (9 )* ; i j M oGirr and H u tc h in so n (1 7 ) s tu d ie d t h e la b e le d com ponents in th e ' I 1 3 i ■ b lo o d o f 12 g o itr o u s c r e t i n s g iv e n I * They o b serv ed a r a d i o a c t i v e , , p ro te in -b o u n d s u b s ta n c e w h ich w as e x t r a c t a b l e w ith b u ta n o l and in s o lu b le in B la u ’s r e a g e n t, as a l k a l i n e s o lv e n t w h ich d is s o lv e s io d o ty r o s in e s b u t d oes n o t d is s o lv e th e io d o th y r o n in e s • One o f t h e i r sam ples was a n a ly z e d by G ross and P i t t - R i v e r s and fo u n d t o c o n ta in n e i t h e r th y r o x in e n o r t r i i o d o t h y r o n i n e . The p re s e n c e o f a s u b s ta n c e w h ich was l a t e r d e m o n stra te d t o be t r i i o d o th y r o n in e in p e r ip h e r a l t i s s u e s w as r e p o r te d b y G ross and L eb lo n d (2 ) in i n t a c t and th y ro id e c to m iz e d m ice and i n t a c t r a t s • A nim als w ere k i l l e d IS h o u rs a f t e r t h e i n j e c t i o n o f la b e le d th y r o x in e 131 o r 48 h o u r8 a f t e r th e i n j e c t i o n o f I i n t o i n t a c t a n im a ls • The r e s u l t s w ere q u a l i t a t i v e l y t h e ,same in a l l c a s e s * T h y ro x in e i t s e l f i ! was fo u n d i n p lasm a, f e c e s and t h e o rg an s s tu d ie d ( l i v e r , k id n e y and j s k e l e t a l muse l e ) , as was an unknown s u b s ta n c e c a l l e d "Unknown Number 1*" T h is s u b s ta n c e was l a t e r shown t o be c h ro m a to g ra p h ic a l l y i d e n t i c a l w ith t r i i o d o t h y r o n i n e (4 ) • I n 1954 and 1955 K a la n t et^ a l • r e p o r te d f i n d i n g a c o n v e rs io n 1 ^ o f th y r o x in e t o t r i i o d o t h y r o n i n e (1 8 , 1 9 ) . I n t h i s s tu d y n orm al 4 and p r o p y l t h i o u r a c i l - t r e a t e d r a t s w ere i n j e c t e d w ith la b e le d t h y r o x i n e • T w e n ty -fo u r h o u rs l a t e r la b e le d tr i io d o th y r o n in e was fo u n d i n th e l i v e r and s k e l e t a l m u scle o f b o th g ro u p s • I n th e l a t t e r t i s s u e tr i i o d o t h y r o n i n e w as th e p r i n c i p a l r a d io a c ti v e s u b s ta n c e f o u n d . F lo c k and B oilm an have r e p o r te d t h a t t r i i o d o t h y r o n i n e a p p e a re d i n 1 1 a v e ry few ” p lasm a and s k e l e t a l m u scle sam p les o f a s e r i e s o f' norm al and e v is c e r a te d r a t s g iv e n la b e le d th y r o x in e ( 2 0 ) • D e io d in a - t i o n o c c u rre d i n b o th g roups as shown by th e p re s e n c e o f r a d i o a c t i v e i io d id e , b u t i t was m ark ed ly re d u c ed i n t h e e v is c e r a te d g ro u p . Sam ples j w ere s tu d ie d up t o 24 h o u rs a f t e r th e a d m in is tr a tio n o f th e la b e le d | m a t e r i a l . , ! - - - i t P i t t - R i v e r s e t a l • r e p o r te d th e d e t e c t i o n o f sm a ll am ounts o f j tr i io d o th y r o n in e in t h e p lasm a o f a th y r e o tic humans g iv e n in tra v e n o u s la b e le d th y r o x in e (2 1 ).• The la b e le d th y r o x in e used in th e s tu d y was c o n ta m in a te d w ith t r i i o d o t h y r o n i n e t o th e e x te n t o f 2%• The m axim al v a lu e s o f plasm a tr i io d o th y r o n in e in any o f th e s i x in d iv id u a ls t e s t e d w ere 5 .5 t o 6.5% o f th e t o t a l r a d i o a c t i v i t y p r e s e n t as th y r o x in e and t r i i o d o t h y r o n i n e • The a u th o rs r e p o r t t h a t th e a c t i v i t y b etw een th e tr i io d o th y r o n in e s p o t (w hich moves f u r t h e r th a n th y r o x in e ) and th e s o lv e n t f r o n t was alw ays T fc o n s id e r a b ly g r e a t e r th a n th e d e te c to r b ack ground co u n t .w Roche and M ich el (2 2 ) w ere u n a b le t o d e t e c t tr i i o d o t h y r o n i n e a f t e r a d m in is te r in g la b e le d th y ro x in e t o th y ro id e c to m iz e d r a t s » They a n a ly z e d p la sm a , b i l e and u r in e and .c o n clu d ed t h a t th e t h y r o i d g lan d i s th e o n ly s o u rc e o f t r i i o d o t h y r o n i n e . H ogness e t a l . (2 3 ) ad m in is te r e d la b e le d th y r o x in e t o r a t s and a n a ly z e d t i s s u e and plasm a 5 e x t r a c t s on a k ie s e lg u h r colum n* They w ere u n a b le t o d e m o n stra te t h e p re s e n c e o f t r i i o d o t h y r o n i n e • B# The in v i t r o F o rm atio n o f T r iio d o th y r o n in e and O th er T h y ro x in e A n a lo g u e s • The in v i t r o c o n v e rs io n o f th y r o x in e t o t r i i o d o t h y r o n i n e by e x t r a - t h y r o i d a l t i s s u e s h a s b e e n d e m o n stra te d b y s e v e r a l g ro u p s • A l b r i ^ i t e t a l . (2 4 ) r e p o r te d on a c y a n id e - s e n s iti v e e n zy m a tic c o n v e rs io n in r a t k id n e y s l i c e s • F u r th e r w ork b y t h i s group showed t h a t t h i o u r a c i l i n h i b i t e d t h e r e a c t i o n a s d id t h e a d d itio n o f sm a ll am ounts o f t r i i o d o t h y r o n i n e • An a d a p tiv e n a tu r e was p o s tu la te d f o r t h e en zy m e(s) s in c e k id n e y s l i c e s o f h y p e rth y ro id r a t s w ere m ore a c ti v e th a n th o s e o f norm al a n im a ls • The k id n e y s o f th y ro id e c to m iz e d r a t s e x h ib ite d th e l e a s t a c t i v i t y ( 2 5 ) . T his w ork h as b e en co n firm e d by V a n n o tti e t a l . ( 2 6 ) . E v id en ce f o r t h i s i n v i t r o c o n v e rs io n was a ls o p r e s e n te d by S p r o tt and M aclagan (2 7 ) u s in g a r a t l i v e r hom ogenate • M aolagan*s group h a s a d d i t i o n a l l y g iv e n p re su m p tiv e e v id e n c e f o r th e fo rm a tio n o f tr i i o d o t h y r o n i n e from th y r o x in e • T hese w o rk e rs h av e shown t h a t b u ty l- 4 - h y d r o x y - 3 ,5 - d iio d o b e n z o a te ( F i g . 1) i n h i b i t s th e m e ta b o lic e f f e c t s o f th y r o x in e w h ile au g m en tin g th o s e o f t r i i o d o t h y r o n i n e . The in v iv o d e io d in a tio n o f b o th compounds i s i n h i b i t e d b y b u ty l- 4 -h y d ro x y - 3 ,5 - d iio d o b e n z o a te • T hese w o rk e rs e x p la in t h e i r r e s u l t s by assu m in g t h a t t h i s s u b s ta n c e a c ts by r e d u c in g th e c o n v e rs io n o f th y r o x in e t o t r i i o d o t h y r o n i n e and p r e v e n tin g th e d e s t r u c t i o n o f th e l a t t e r by d e i o d i n a t i o n . T r iio d o th y r o n in e i s th u s assum ed t o be th e b i o l o g i c a l l y 6 a c tiv e compound (2 8 -3 0 ) • The a c e t i c a c id a n alo g u e s ( F i g . 1) o f th y r o x in e and t r i i o d o th y r o n in e a re known t o he a c ti v e (3 1 , 3 2 )• T r iio d o th y r o a c e tic a c id h a s b een r e p o r te d i n th e k id n e y s o f r a t s g iv e n la b e le d t r i i o d o th y ro n in e ( 3 3 ) . A lb r ig h t and h i s co -w o rk e rs h av e r e c e n t l y r e p o r te d on a s o lu b le enzyme sy stem p re p a re d fro m r a t k id n e y m ito c h o n d ria ( 3 4 ) . T h is sy ste m c o n v e rte d th y r o x in e and t r i i o d o t h y r o n i n e t o t h e i r c o r r e s p o n d in g a c e t i c a c id a n a lo g u e s » O th er w o rk e rs have shown t h a t b r a in t i s s u e s l i c e s and s u s p e n s io n s a re c a p a b le o f d e a m in a tin g th y r o x in e and tr i i o d o t h y r o n i n e t o t h e i r r e s p e c tiv e p ro p io n ic a n d /o r a c e t i c a c id an alo g u e s ( 3 5 ) . The enzyme sy stem in v o lv e d i s n o t s e n s i t i v e t o m ercu ry w h ereas th e d e io d in a tin g sy stem o f b r a in i s s e n s i t i v e . The a u th o rs r e p o r t th e p o s s ib le fo rm a tio n o f sm a ll am ounts o f t r i i o d o th y r o n in e from th y r o x in e » C . The D e io d in a tio n T heory o f T h y ro x in e A c t i v i t y . I t h as b een d e m o n stra te d t h a t tr i io d o t h y r o n i n e p ro d u c e s an in c r e a s e i n oxygen co n su m p tio n m ore r a p i d l y th a n th y r o x i n e • Many .i n v e s t i g a t o r s have a ls o r e p o r te d t h a t , on a m o lar c o m p ariso n , t r i io d o th y ro n in e i s m ore c a lo r ig e n i c th a n th y r o x in e (6 , 36, 37) • One s tu d y , i n w hich th e b a s a l m e ta b o lic r a t e o f m yxedem atous humans was o b s e rv e d , showed t r i i o d o t h y r o n i n e t o be. f i v e t o t e n tim e s as a c tiv e i as th y ro x in e . (3 8 ) • A th e o r y o f th y r o i d hormone a c ti o n h a s grown o u t o f th e body o f w ork m en tio n ed a b o v e . T h is th e o r y , in b r i e f , s t a t e s t h a t th y ro x in e i s th e m ajo r fo rm o f c i r c u l a t i n g th y r o i d hormone b u t i s n o t th e 7 p e r i p h e r a l l y a c ti v e horm one* D e io d in a tio n t o tr i io d o th y r o n in e i s p o s tu la te d , w ith t h i s compound a c t u a l l y e n t e r i n g th e c e l l s o f p e r i p h e r a l t i s s u e s (3 9 , 4 0 )* E v id en ce f o r t h i s th e o r y i s fo u n d i n th e serum b in d in g c h a r a c t e r i s t i c s o f th e tw o s u b s ta n c e s • T h y ro x in e i s f ir m ly bound by a s p e c i f i c p r o t e i n (4 1 , 42) w h ereas tr i io d o th y r o n in e i s d i s t r i b u t e d th ro u g h o u t th e serum p r o te in s (3 9 ) . From th e r e l a t i v e a b i l i t i e s t o d is p la c e bound la b e le d th y r o x in e fro m th e . s p e o i f i c serum p r o t e i n , R o b b in s and R a i l (4 3 ) have d e te rm in e d t h a t t r i i o d o th y r o n in e h a s 30 t o ‘ 35ft- o f th e b in d in g a f f i n i t y o f L - th y r o x in e • T h ere i s a r e p o r te d d if f e r e n c e in t h e d i s t r i b u t i o n o f la b e le d exogenous th y r o x in e and e n d o g en o u sly la b e le d th y r o id horm one among t i s s u e s , th e l a t t e r a g r e e in g more c l o s e l y w ith th e d i s t r i b u t i o n o f la b e le d t r i i o d o t h y r o n i n e ( 4 0 ) • Jo h n so n and A lb e rt s tu d ie d t h i s problem in young m ale r a t s m a in ta in e d on a s ta n d a rd com m ercial d i e t ( 4 4 ) • They fo u n d co m p arab le u r in a r y and f e c a l e x c r e tio n o f r a d i o a c t i v i t y a f t e r th e i n j e c t i o n o f **131 3 .9 ngn* o f D L -th y ro x in e -I , A f te r 96 h o u rs some 30ft o f th e i n - j i je c t e d a c t i v i t y had gone i n t o th e u r in e and 36ft in t o th e f e c e s • At ; 15 h o u rs th e g r e a t e s t c o n c e n tr a tio n was fo u n d i n th e sto m ach , w ith J s i g n i f i c a n t c o n c e n tr a tio n s a ls o p r e s e n t in l i v e r and k id n e y s* T hese a u th o rs d id n o t f i n d a c t i v i t y i n s t r i a t e d m u scle* A n a ly sis o f th e | g a s t r o i n t e s t i n a l t r a c t showed t h a t 30ft o f th e a c t i v i t y o f t h e stom ach j c o n te n ts was i n th e form o f th y r o x in e * The s m a ll and la r g e i n t e s t i n e j c o n ta in e d 90ft and 96ft r e s p e c t i v e l y as th y r o x in e (th e c h em ica l f r a c - | t i o n a t i o n u sed i n t h i s s tu d y d id n o t d i s t i n g u i s h among th y r o x i n e , 8 tr i io d o th y r o n in e and th e c o n ju g a te s o f th e s e compounds known t o he e x c r e te d in th e b i l e •) A lb e r t, Ford, and L orenz have s tu d ie d th e d i s t r i b u t i o n o f e n d o g en o u sly la b e le d th y r o id horm one i n young r a t s b u t, d iv id e d t h e i r a n im a ls in t o o n ly th r e e a n a to m ic a l p a r t s , v i z *s t h y r o i d gland* l i v e r , stom ach and i n t e s t i n e s * and th e re m a in in g c a r c a s s ( 4 5 ) • I n a n im a ls m a in ta in e d on a low io d in e d i e t f o r f o u r 't o s i x days a f t e r a s in g le i n j e c t i o n o f I ' ^ ^ # a slow d is a p p e a ra n c e o f a c t i v i t y in th e th y r o i d g la n d w as o b serv ed o v er a su b se q u e n t 11 day p e rio d * A c t i v i t y i n th e g a s t r o i n t e s t i n a l t r a c t re a c h e d a peak on th e second d ay and th e n f e l l o f f * From tw o - th ir d s t o t h r e e - q u a r t e r s o f th e t o t a l body a c t i v i t y rem ained, in th e t h y r o i d ; 5% t o 12% was i n th e g a s t r o i n t e s t i n a l t r a c t * 13% t o 31/C was in t h e c a r c a s s • U rin a ry a c t i v i t y was g e n e r a lly ab o u t tw ic e t h a t o f th e f e c e s , M a lth o u g h o c c a s io n a lly th e am ounts o f j xo± w ere th e sam e, or th e amount i n th e f e c e s w as g r e a te r t h a n . t h a t i n th e u r in e .w The a u th o rs d id c o n clu d e t h a t th e e x c r e tio n o f i ^ l f rom e n d o g en o u sly la b e le d hormone d i f f e r s fro m t h a t f o llo w in g th e a d m in is tr a tio n o f la b e le d th y r o x in e i n t h a t th e endogenous l a b e l i s p r im a r i ly e x c r e te d v ia th e u r in e * K e a tin g and A lb e rt (4 6 ) o b serv ed h ig h e r r a t e s o f g a s t r i c and u r in a r y s e c r e t i o n o f no n -h o rm o n al im m ature r a t s a f t e r th e a d m in is tr a tio n o f la b e le d t r i i o d o t h y r o n i n e , s u g g e s tin g a more r a p id r a t e o f d e io d in a tio n * At 15 d ay s 55% o f th y r o x in e a c t i v i t y was fo u n d in t h e l i v e r and ab o u t 40^ i n th e c a r c a s s * At t h e same tim e alm o st 100% o f th e re m a in in g t r i i o d o t h y r o n i n e a c t i v i t y w as in th e th y r o id or 9 th e c a r c a s s • Van A rs d e l a t a l • (4 7 ) s tu d ie d t h e d i s t r i b u t i o n o f la b e le d th y r o x in e and t r i i o d o t h y r o n i n e i n r a t s a t s h o r t tim e i n t e r v a l s • T r iio d o th y r o n in e was l e s s c o n c e n tr a te d i n p lasm a and l i v e r • I t was more c o n c e n tr a te d i n th e k id n e y f o r th e f i r s t 12 h o u r s , a f t e r w h ich th e th y r o x in e c o n c e n tr a tio n w as g r e a t e r * T r iio d o th y r o n in e was some w hat more c o n c e n tr a te d in s k e l e t a l m u s c le • T au ro g (4 8 ) h a s r e p o r te d t h a t e n d o g en o u sly la b e le d th y r o i d hormone i s e x c r e te d i n t h e b i l e i n a f a s h io n s i m i l a r t o exogenous th y r o x i n e • Roche and h i s c o -w o rk e rs have r e p o r te d t h a t la b e le d tr i i o d o t h y r o n i n e i s e x c r e te d i n th e b i l e ( 4 9 ) • Q u a n tita tiv e d i f f e r e n c e s in b i l i a r y e x c r e tio n w ere o b se rv e d b y t h i s group (5 0 ) • I n p h y s io lo g ic a l d o se s (1 t o 9 u g n .) m ost o f th e t r i io d o th y r o n in e i n r a t b i l e i s f r e e . At t h i s d o sag e m ost b i l i a r y th y r o x in e i s p r e s e n t as th e c o n ju g a t e . T h is c o n ju g a te h a s b e en shown by T au ro g (4 8 ) t o be l e s s r e a d i l y re a b s o rb e d fro m th e i n t e s t i n e th a n f r e e t h y r o x i n e « A ssum ing t h a t f r e e t r i i o d o t h y r o n i n e i s r e a d i l y re a b s o rb e d fro m th e i n t e s t i n e , i t s low f e c a l e x c r e tio n m ig h t be e x p la in a b le on th e b a s i s o f i t 8 r e l a t i v e l y low c o n ju g a tio n . O&ross (4 0 ) b e lie v e s t h a t th y r o x in e i s d i s t r i b u t e d e x t r a c e l l u l a r l y e x c e p t f o r th e t i s s u e s in v o lv e d in i t s e n te r o h e p a tic c i r c u l a t i o n , th e k id n e y and p e rh a p s t h e n eu ro h y p o p h y sis • V an A rs d e l e t a l . r e p o r te d t h a t s k e l e t a l m u scle c o n c e n tr a te s th y r o x in e , a t one h o u r a f t e r a d m in is tr a tio n , t o a p p ro x im a te ly t h r e e - q u a r te r s th e e x te n t t o w h ich i t c o n c e n tr a te s t r i i o d o t h y r o n i n e . At 10 t h r e e h o u rs t h i s r a t i o was a b o u t f o u r - f i f t h s and i t ap p ro ach ed u n ity a t 12 h o u rs « C h ro m ato g rap h ic a n a ly s is was n o t done so i t was n o t known i f th e l a t e in c r e a s e in a c t i v i t y w ith th y r o x in e r e p r e s e n te d th y r o x in e i t s e l f o r a m e ta b o lite ( 4 7 ) • A lth o u g h t h e c o n c e n tr a tio n o f th y r o x in e i n th e p e r f u s in g b lo o d w as alw ays h ig h e r th a n t h a t o f t r i i o d o t h y r o n i n e , th e d a ta do n o t p ro v e t h a t th y r o x in e i t s e l f does n o t e n t e r th e m u scle c e l l • D i f f i c u l t i e s e x i s t in th e i n t e r p r e t a t i o n of e x p e rim e n ts com par in g th e f a t e o f a s u b s ta n c e s lo w ly r e le a s e d fro m th e th y r o i d g lan d w ith t h a t o f one a d m in is te r e d i n a s i n g l e d o s e , how ever p h y s io lo g ic a l t h a t d o se may be • The b a s ic p ro b lem o f w h e th e r o r n o t th y r o x in e m ust j f i r s t be c o n v e rte d t o t r i i o d o t h y r o n i n e b e fo re e n te r in g th e p e r i p h e r a l | j c e l l s and e x e r t i n g a horm onal e f f e c t h a s n o t b een s o lv e d by th e s e j i d i s t r i b u t i o n s tu d ie s * I I . STATEMENT OF THE PROBLEM The w ork d e s c r ib e d i n t h i s d i s s e r t a t i o n was d e s ig ie d t o t e s t t h e h y p o th e s is o f th e e x t r a t h y r o i d a l c o n v e rs io n o f th y r o x in e t o t r i i o d o th y r o n in e in v iv o . S t a r r h a s s u g g e s te d t h a t t r i i o d o t h y r o n i n e may be p ro d u ced p e r i p h e r a l l y fro m th y r o x in e t o m a in ta in h o m e o sta s is i n an u n fa v o ra b le > e n v iro n m en t ( 5 1 ) • Thus t h e r a p id in c r e a s e i n m e ta b o lic r a t e r e q u ir e d f o r m a in te n an c e o f body te m p e ra tu re u n d er c o n d itio n s o f e x trem e c o ld m ig h t in c r e a s e th e r a t e o f c o n v e r s io n . A n o th er fo rm o f s t r e s s , th e a d m in is tr a tio n o f e p in e p h r in e , may have a s p e c i f i c e f f e c t on th y r o id hormone m e ta b o lism . S o f f e r e t a l . (5 2 ) h av e shown t h a t e p in e p h r in e p ro d u c e s an in c r e a s e d s e c r e t i o n o f th y r o tr o p h ic horm one i n b o th n o rm al and th y ro id e c to m iz e d d o g s . W illia m s e t a l . (5 3 ) r e p o r te d t h a t , i n th e r a t , e p in e p h r in e p ro d u ces a lo w e rin g o f t h y r o i d a l and serum p r o t e i n - 131 bound I , B o tk in and Je n s e n (5 4 ) fo u n d t h a t b o th th y r o tr o p h ic 131 131 horm one and e p in e p h r in e lo w er t h y r o i d a l I i n th e r a t . Serum I was fo u n d t o be in c r e a s e d s h o r t l y a f t e r th y r o t r o p h in a d m in is tr a tio n and d e c re a s e d s h o r t l y a f t e r e p in e p h r in e • T hese a u th o r s p o s tu la te d an in c r e a s e d p e r ip h e r a l u t i l i z a t i o n o f th y r o x in e due t o e p in e p h r in e . I ;E sk e ls o n ab a l • co n firm e d t h e s e f in d i n g s and s u p p o rte d t h e p o s tu la te d |i n c r e a s e in p e r i p h e r a l u t i l i z a t i o n o f th y r o i d horm one (5 5 ) • ! Swanson h a s shown t h a t th e e p in e p h r in e - in d u c e d in c r e a s e in m e ta b o lic r a t e i s d e p en d e n t on th e p re s e n c e o f th y r o i d horm one (5 6 ) • 12 T h y ro id e c to m iz e d r a t s g iv e n no exogenous th y r o x in e d id n o t e x h i b i t an in c r e a s e d oxygen co n sum ption when e p in e p h r in e was a d m in is te re d * In c o n tr o l r a t s and in th y ro id e c to m iz e d r a t s g iv e n th y r o x i n e , th e e p in e p h r in e - in d u c e d m e ta b o lic r a t e in c r e a s e was o b se rv e d w ith in one h o u r* The e f f e c t s o f e p in e p h r in e on th y r o x in e m e ta b o lism have been u t i l i z e d i n t h i s s tu d y * I f i t i s c o r r e c t t h a t th y r o x in e i s a c ti v e o n ly th ro u g h c o n v e rs io n t o t r i i o d o t h y r o n i n e , in c r e a s e d u t i l i z a t i o n o f t h y r o i d horm one w ould be e x p e c te d t o p ro d u ce an in c r e a s e d c o n v e rs io n t o tr i io d o t h y r o n i n e * T h is h as b een in v e s t i g a t e d by c h ro m a to g ra p h ic a n a ly s e s o f human serum and r a t t i s s u e s f o llo w in g th e a d m in is tr a tio n o f la b e le d th y r o x in e and e p in e p h rin e * I l l . EXPERIMENTAL A* C hrom atograph.ic S e p a r a tio n o f T h y ro x in e and T r iio d o th y r o n in e # The s e p a r a tio n o f th y r o x in e and t r i i o d o t h y r o n i n e w as a c h ie v e d by one d im e n s io n a l a s c e n d in g p a p e r ch ro m ato g rap h y on e i t h e r Whatman Number 1 o r Whatman Number 3 M M f i l t e r p a p e r* A v a r i e t y o f d e v e lo p in g s o lv e n ts w as u s e d , a l l b e in g f r e s h l y d i s t i l l e d arid p re p a re d b e f o r e e ac h c h ro m a to g ra p h ic ru n * * T h e 'b e s t and m ost c o n s i s t e n t r e s u l t s w ere o b ta in e d w ith t e r t i a r y amyl a lc o h o l s a t u r a t e d w ith ammonia ( c o n c e n tr a te d ammonia d i l u t e d 1*5 w ith w a te r ) ( 5 7 ) • T h is sy ste m a f f o r d e d th e b e s t s e p a r a tio n o f th y r o x in e and tr i i o d o t h y r o n i n e o f any used i n t h i s w ork* I t d id n o t p ro v id e a good s e p a r a tio n o f th y r o x in e and i o d i d e . Due t o i t s slo w r a t e o f m ovem ent, 48 h o u rs w ere r e q u ir e d f o r c h ro m a to g ra p h ic d ev elo p m en t • A sy ste m d e s c r ib e d b y A lb r ig h t , e t a l * (5 8 ) was a ls o u s e d , a lth o u g h w ith v a r i a b l e r e s u l t s • The m o b ile p h ase o f t h i s s o lv e n t c o n s i s t s o f n - b u ta n o l, 1 ,4 -d io x a n e and 2 N NH^OH (8 0 :2 0 :1 0 0 )* I t g e n e r a lly a ffo rd e d good s e p a r a t i o n o f io d id e , th y r o x in e and t r i i o d o th y r o n in e • On s e v e r a l o c c a s io n s , ho w ev er, sam p les w ere ch ro m ato g rap h ed in d u p l i c a t e w ith b o th th e amyl a lc o h o l and b u ta n o l-d io x a n e sy stem s • I t w as n o tic e d i n some o f t h e s e s tu d ie s t h a t more r a d i o a c t i v i t y w as lo c a te d a t o r n e a r th e s o lv e n t f r o n t o f ■the l a t t e r sy ste m * S y n th e tic la b e le d th y r o x in e , c h ro m a to g ra p h ic a lly p u re when d e v elo p e d w ith th e amyl a lc o h o l sy ste m , o f te n a p p ea re d g r o s s ly c o n ta m in a te d when d e v e lo p e d ! j w ith b u ta n o l-d io x a n e . T h is i s probably due t o th e d e g r a d a tiv e e f f e c t ; I o f p e ro x id e s form ed i n ev en f r e s h l y d i s t i l l e d d io x a n e • The h ig h ! J a c t i v i t y fo u n d n e a r t h e s o lv e n t f r o n t on chrom atogram s i s u s u a ll y due | . | t o th e p re s e n c e o f io d in a te d s o lv e n t ( 5 7 ) . T h ere i s , h o w ev er, some | e x p e rim e n ta l b a s is f o r b e lie v in g t h a t i t c a n .b e due t o . t h e p re s e n c e i o f an unknown m e ta b o lite o f th y r o i d hormone » I n c o n tr o l e x p e rim e n ts t o be d e s c r ib e d b e lo w , l i t t l e o r no f a s t m oving a c t i v i t y w as fo u n d when la b e le d th y ro x in e and tr i i o d o t h y r o n i n e w ere added t o t i s s u e hom ogenates and e x t r a c t e d im m e d ia te ly . ^When t h e s e s u b s ta n c e s w ere i n j e c t e d in t o r a t s and l a t e r e x tr a c te d fro m v a r io u s t i s s u e s , a c t i v i t y n e a r th e - s o l v e n t f r o n t was g e n e r a lly ,f o u n d ♦ O th er w o rk e rs (1 5 ) have d e s c rib e d - a f a s t m oving la b e le d m a te r ia l in t h e serum o f humans t r e a t e d n I ! w ith I - - i o d i d e . T hey b e lie v e i t t o be an io d in a te d l i p i d . The 2 , 4 , 6 - c o l l i d i n e , w a te r and ammonia sy ste m d e s c rib e d by T au ro g and h i s c o -w o rk e rs (5 9 ) was a ls o u sed t o some e x t e n t . E x tre m e ly v a r ia b le r e s u l t s w ere o b ta in e d . L arge am ounts o f s o lv e n t f r o n t a c t i v i t y w ere fo u n d in m ost a n a ly s e s a tte m p te d w ith t h i s d e v e lo p in g s o l u t i o n . _ ! B . D e te c tio n and I d e n t i f i c a t i o n o f M a te r ia ls on C hrom atogram s. v p u re s y n th e t ic L -th y ro x in e and L - tr iio d o th y r o n in e w ere used t o d e te rm in e th e c h a r a c t e r i s t i c m o b i l i t i e s o f th e s e compounds w ith th e i v a r io u s d e v e lo p in g s y s te m s . T h e ir lo c a t io n s on t h e p a p e r s t r i p s w ere j fo u n d by c o lo r d e v elo p m en t, u s in g e i t h e r n in h y d rin (6 0 ) o r d ia z o t iz e d J s u l f a n i l i c a c id ( 5 8 ) • About 75 jag o f r e f e r e n c e m a te r ia l was r e q u ir e d f o r a d e q u a te c o lo r w ith e i t h e r r e a g e n t . • 15 The l o c a t i o n o f added io d id e was d e te rm in e d by th e b la c k c o lo r form ed on s p r a y in g w ith P d C ^ * The R f v a lu e s o f th y r o x in e and tr i io d o t h y r o n i n e w ere fo und t o be v a r i a b l e from one t r i a l t o a n o th e r , b u t w ere c o n s ta n t w ith in each in d iv id u a l c h ro m a to g ra p h ic r u n . W ith th e amyl a lc o h o l sy ste m , th e R f ; o f th y ro x in e v a r ie d from 0 .1 t o 0 . 4 . T h at o f tr i io d o th y r o n in e ra n g e d from 0 .4 t o 0 . 6 . 1 I n m ost a n a ly s e s o f serum o r t i s s u e e x t r a c t s , no s t a b l e i I ; r e f e r e n c e m a t e r i a l was a d d e d . T h is was due t o th e te n d en c y o f th e e x t r a c t s th e m s e lv e s t o o v e rlo a d th e p a p e r and s p re a d from th e l i n e o f o r i g i n . T h is te n d e n c y was e x a g g e ra te d when r e f e r e n c e m a te r ia ls w ere a d d e d • C • The D e te c tio n o f R a d i o a c t i v i t y on Chrom atogram s . S e v e ra l in s tru m e n ts w ere used f o r th e d e t e c t i o n of r a d i o a c t i v i t y on th e chrom atogram s • When la r g e am ounts o f a c t i v i t y w ere p re s e n t th e i n t a c t s t r i p s w ere c o u n ted w ith a G eiger tu b e i n an a u to m a tic s c a n n in g d e v ic e (A c tig ra p h , IT u clear-C h icag o ) co n n ec te d t o an E s te rlin e -A n g u s r e c o r d e r . I n m ost o f th e s tu d ie s p e rfo rm e d , in - 1 s u f f i c i e n t a c t i v i t y was p r e s e n t f o r d e t e c t i o n by t h i s means . The , m a jo r ity o f s t r i p s w ere th e r e f o r e c u t in to one c e n tim e te r segm ents |w h ic h w ere co u n ted i n d i v i d u a l l y in one o f t h r e e d e t e c t o r s . A s c i n t i l l a t i o n w e ll c o u n te r , an end-w indow G eig er tu b e in a le a d I I | c a s t l e and an a u to m a tic sam ple c h a n g in g m achine w ith a gas flo w tu b e i ' w ere a l l u sed in o rd e r t o r a p i d l y a n a ly z e th e la r g e number o f s t r i p s | ch ro m ato g rap h ed in e ac h e x p e r im e n t. Each segpient was c o u n te d f o r a t 16 l e a s t 1280 co u n ts in th e w e ll c o u n ter (av erag e background: 4 co u n ts p er second) or 4 m inutes w ith th e .end window tu b e (av erag e back grounds 25 co u n ts p er m in u te )# A minimum of 80 co u n ts was ta k e n w ith th e gas flow tu b e (av erag e background: 15 co u n ts p e r m in u te) • Due t o th e m u ltip le in s tr u m e n ta tio n u sed , th e a b s o lu te amounts o f r a d i o - > a c t i v i t y on th e chromatograms p re s e n te d below cannot be d i r e c t l y c ompared • D* P r e p a r a tio n and C o n c e n tra tio n o f B utanol E x tr a c ts o f Serum* Serum sam ples w ere a d ju s te d t o pH 3 w ith d i l u t e HC1 and were e x tr a c te d th r e e tim es w ith ap p ro x im ately double volumes o f n -b u ta n o l • T his procedure was c a r r i e d o u t in a sm all b e a k e r, u s in g a g la ss s t i r r in g ro d t o a g i t a t e th e m ix tu re • A fte r c e n t r i f u g a t i o n , th e b u ta n o l la y e r s were p ip e tte d o f f and pooled • I n e a r l y work th e b u ta n o l e x t r a c t s w ere c o n c e n tra te d under an a i r j e t i n an open b e ak e r a t room te m p e ra tu re * T his p ro ced u re was e x tre m ely tim e consuming and l a t e r a vacuum d i s t i l l a t i o n method was adopted t o f a c i l i t a t e t h i s s te p * Samples w ere ta k e n t o n e a r d ry n ess o a t 55-60 under n itr o g e n * B . P r e p a r a tio n and C o n c e n tra tio n o f B utanol E x t r a c ts o f T iss u e s * In a l l oases t h e t i s s u e s were removed im m ediately a f t e r th e anim als had been k i l l e d by e x s a n g u in a tio n under l i $ i t e th e r or ch lo ro fo rm a n a e s th e s ia * They w ere p la ce d in an ic e c o ld s a li n e s o l u t i o n d u rin g th e b r i e f tim e r e q u ire d f o r d i s s e c t i o n and w ere th e n e i t h e r trimmed of f a t and f a s c i a , b l o t t e d d ry and homogenized 17 im m ediately or were f r o z e n u n t i l u s e d • The t i s s u e sam ples c o n s is te d of b o th k id n e y s, b o th su b m a x illa ry g la n d s, th e e n t i r e h e a r t , most o f th e l i v e r and as much s k e l e t a l m uscle as could be removed from one h in d l e g . T issu e sam ples were homogenized in 15 ml • of d i s t i l l e d w a ter in a S e r v a ll w Omnimixw hom ogenizer immersed in an ic e b a t h . The homogenates w ere th e n a c i d i f i e d and e x tr a c te d w ith b u ta n o l as d e s c rib e d above • The c o n c e n tra te d t i s s u e e x t r a c t s , e s p e c i a l l y th o s e of k id n e y and l i v e r , w ere v is c o u s and h ig h ly p ig n e n te d . The amounts which co u ld be p la ce d on th e chrom atographic s t r i p s w ere lim ite d t o th e o rd e r o f 20 ^ tl. by th e p re v io u s ly m entioned te n d e n c y of th e m a te r ia l t o sp read from th e o r i g i n . F . R ecovery E x p e rim e n ts. R ecovery ex p erim en ts were s e t up t o d eterm in e i f m inim al amounts of tr i io d o th y r o n in e w ere d e te c ta b le in th e p resen ce of t h y r o x i n e • In in v i t r o s t u d i e s , 70 gm. of b e e f k id n e y was homogenized in 80 m l. of w a t e r . To 10 m l. a liq u o ts o f th e homogenate was added 0 .2 4 p o of la b e le d th y ro x in e and v a ry in g amounts of la b e le d t r i i o d o t h y r o n i n e . The r a t i o s of t h y r o x i n e :t r i io d o th y r o n in e ran g ed from 1 .1 :1 t o 7 2 :1 . A n aly sis of b u ta n o l e x t r a c t s o f th e s e a liq u o ts showed, i n g e n e r a l, a g r e a te r r e l a t i v e re c o v e ry o f t r i io d o th y r o n in e th a n of th y r o x i n e . This was d e s p ite a la r g e contam inant in th e la b e le d t r i io d o th y r o n in e w hich m ig rate d as th y ro x in e or i o d i d e . At th e 72;1 18 r a t i o th e tr i io d o th y r o n in e 'a re a of th e chromatogram was n o t s i g n i f i c a n tl y h ig h e r th a n th e s t r i p background* .A t t h e 'n e x t h ig h e s t r a t i o , 14* 1 :1 , th e re c o v ere d a c t i v i t i e s on th e chromatogram w ere i n th e r a t i o of 13*5:1* L i t t l e or no f a s t moving a c t i v i t y was found i n th e s e e x t r a c t s • Two i n v iv o re c o v e ry ex p erim en ts were perform ed* I n th e f i r s t , t h r e e norm al male r a t s w ere in je c te d i n t r a p e r i t o n e a l l y w ith 60 ^ic of r a d io a c ti v e th y ro x in e combined r e s p e c t i v e l y w ith 6, 3, and 1 ^ie of la b e le d t r i io d o th y r o n in e * The anim als w ere k i l l e d a t seven h o u rs; serum , l i v e r , k id n e y , s k e l e t a l m uscle and h e a r t were an aly zed c h ro m a to g ra p h ic a lly * C o n sid erab le f a s t moving a c t i v i t y was found in a l l th e s e t i s s u e s e x ce p t kidney* S k e l e t a l m u scle, h e a r t and kidney c o n ta in e d th e g r e a te s t amounts of t r i io d o th y r o n in e * Poor r e s o l u t i o n was o b ta in e d w ith th e l i v e r e x t r a c t s • By com parison of th e r a d i o a c t i v i t y in th e th y ro x in e and tr i io d o th y r o n in e a r e a s , a l l but one of th e sam ples were r i c h e r in t r i i o d o t h y r o n i n e th a n th e i n j e c t e d m ix tu res • T his was t r u e o f serum as w e ll as of t h e p e r ip h e r a l t i s s u e s • I t m ight r e f l e c t a more r a p id uptake of tr i io d o th y r o n in e from th e p e r ito n e a l c a v ity * I t m ight a ls o i n d i c a t e t h a t th e a c t i v i t y o f unknown n a tu re lo c a te d a t th e o r i g i n o f th e s t r i p and i n th e a re a between th e tr i io d o th y r o n in e sp o t and th e s o lv e n t f r o n t was r e l a t i v e l y r i c h e r i n th y ro x in e or i t s m e ta b o lic p r o d u c ts • A second i n v iv o re c o v e ry experim ent was perform ed w ith th e same amounts of la b e le d m a te r ia ls • Female r a t s were used and w ere k i l l e d a t two h o u r s • T r iio d o th y ro n in e was d e te c te d i n a l l t i s s u e s 19 s tu d ie d * In th e anim al given 60 ^tc of t h y r o x i n e and 1 ^ic o f’ tr i io d o t h y r o n i n e , th e re c o v ere d r a t i o s th y r o x i n e :tr i io d o th y r o n in e w e r e ,- s k e l e t a l m uscle - 12*5, h e a r t - 22*7, serum - 2 4 * 7 ,-kidney - 44*0 and l i v e r - 65*0* The s p e c i f i c a c t i v i t y of th e in j e c t e d m a te r ia ls h as been o m itted from t h e s e c o n s id e r a tio n s * In work w ith u n la b e le d th y ro x in e and t r i i o d o t h y r o n i n e , chrom atographic s e p a r a tio n was achieved w ith i I 100 p.gn.m q u a n t i t i e s o f each* S in ce th e s e a re muchvl a r g e r amounts th a n would be p re s e n t in any of th e t i s s u e e x t r a c t s used in th e s e e x p e r i m en ts, i t was not f e l t t h a t chrom atographic r e s o l u t i o n o f th e t i s s u e e x t r a c t s would be a f f e c t e d by th e t o t a l amounts o f th y r o i d m a te r ia l p re s e n t * In o th e r re c o v e ry s tu d ie s la b e le d th y ro x in e and t r i i o d o th y ro n in e in v a ry in g amounts w ere given in tr a v e n o u s ly -to a th y r e o tic humans* The i n j e c t e d a c t i v i t i e s w ere ( th y r o x in e : tr iio d o th y r o n in e ) 1*1, 2 ;1 , 12:1 and 24:1* - The m inim al amount o f tr iio d o th y r o n in e i n j e c t e d was 10 n c • T riio d o th y ro n in e was d e te c ta b le a t a l l th e s e i l e v e l s b u t n o t as a d i s c r e e t peak in th e 2 4 :1 se ru m * * At t h i s l e v e l o f in je c te d a c t i v i t y , t r i i o d o t h y r o n i n e was found as a d e f i n i t e i i sh o u ld e r on th e th y ro x in e p e a k . From t h e d a ta o b ta in e d , c o n v e rsio n j t o tr i io d o th y r o n in e would be c l e a r l y d e te c ta b le , a f t e r t h e i n j e c t i o n o f 200 ^io of la b e le d th y r o x in e , i f th e c o n c e n tr a tio n o f c i r c u l a t i n g tr i io d o t h y r o n i n e reaohed about 8 ? o t h a t o f th e i n j e c t e d m a te r ia l* C onversion o f t h e same amount o f th y ro x in e t o a l e v e l would o f f e r d i f f i c u l t y in i n t e r p r e t i n g th e chrom atographic r e s u l t s but 20 d e te c ta b le a c t i v i t y would be p re s e n t i n th e tr i io d o th y r o n in e a r e a of th e c h ro m ato g ram -(F ig • 2 ) . G* L abeled Components i n Human Serum F o llo w in g In tra v e n o u s T h y ro x in e-I * L abeled th y ro x in e was given in tr a v e n o u s ly t o n in e in d iv id u a ls » The in j e c t e d a c t i v i t y ranged betw een 96 and 340 jxc and was in c o r p o r a t ed in 6 t o 15 ^agm* o f th y ro x in e * E ig h t of th e s e in d iv id u a ls w ere p a t i e n t s i n th e T hyroid C lin ic o f th e Los A ngeles County G eneral H o s p ita l* The n in th was presum ably e u th y ro id and was h o s p i t a l i z e d f o r ja u n d ic e and s u sp e c te d g a s t r o i n t e s t i n a l m alignancy* The T hyroid C lin ic p a t i e n t s c o n s is te d o f two w ith myxedema, two w ith a th y r e o s is m a in ta in e d a t a e u th y ro id le v e l w ith pure th y ro x in e and two o th e r * « * r a th y r e o tic s who w ere h y p o th y ro id * The rem ain in g two p a t i e n t s were h o s p i t a l i z e d f o r h y p e rth y ro id ism * Blood sam ples were drawn a t tim e i n t e r v a l s r a n g in g from 20 m inutes t o 24 hours a f t e r th e th y ro x in e i n je c tio n * In some o f th e c ase s chrom atographic a n a ly s is o f th e b u ta n o l e x t r a c t s was perform ed j ' ! w ith b o th th e b u ta n o l-d io x a n e and t e r t i a r y amyl a lc o h o l system s • Some t y p i c a l chromatograms a re shown in F ig u re s 3-6* The r e s u l t s a re ta b u l a t e d i n T able I , e x p re ssed as th e per c e n t o f th e t o t a l r a d io a c t i v i t y found as th y r o x in e , t r i i o d o t h y r o n i n e o r s o lv e n t f r o n t a c t i v i t y * On some s t r i p s peaks o f r a d i o a c t i v i t y were d e te c te d a t o th e r lo c a tio n s * These have been in c lu d e d in th e t a b l e s i n a p o s itio n ! r e l a t i v e t o t h e i r p la ce on th e chromatogreens, v iz *, betw een th y ro x in e j and t r i i o d o t h y r o n i n e • --------- J 21 I n f i v e in d iv id u a ls e s s e n t i a l l y pure th y ro x in e was found in th e e x t r a c t s # ' T his group c o n s is te d o f one myxedema, b o th a th y r e o tic and one h y p e rth y ro id p a t i e n t • The second myxedema case e x h ib ite d a s im ila r p i c t u r e in a l l b u t one blood sam ple* At n in e hours a sm all amount of tr i io d o th y r o n in e and co n sid erab le, s o lv e n t f r o n t a c t i v i t y were p r e s e n t; a t 24 hours o n ly th y ro x in e was found* In t h e second h y p e rth y ro id case la r g e amounts o f a c t i v i t y w ere found a t th e s o lv e n t f r o n t of a l l sam ples in b o th s o lv e n t system s* T riio d o th y ro n in e a c t i v i t y was p re s e n t in sm all amounts in two sam ples (20 m inutes and 5 .25 h o u rs )* An a th y r e o tic h y p o th y ro id in d iv id u a l ( p a t i e n t 6) p re s e n te d a complex p ic tu r e * S ix d i s t i n c t peaks o f a c t i v i t y were found w ith th e t e r t i a r y amyl a lc o h o l system * About 4$ of th e t o t a l a c t i v i t y was p r e s e n t as tr i io d o th y r o n in e a t 30 m inutes and a t 18*5 h o u rs* Only th y r o x in e , t r i io d o th y r o n in e and s o lv e n t f r o n t a c t i v i t y w ere d e te c te d , w ith th e b u ta n o l-d io x a n e sy stem , tr i io d o th y r o n in e r e a c h in g 1 6$ > a t 18*5 h o u rs* T his p a t i e n t had a p re v io u s h i s t o r y o f l i v e r and b i l i a r y p ath o lo g y * I t was f e l t t h a t th e more complex n a tu re of h e r serum chrom atographic p a t t e r n migjit be due t o a reduced a b i l i t y f o r h e p a tic c le a ra n c e o f th y ro x in e m e ta b o lite s • I n a second t e s t of t h i s s u b je c t e s s e n t i a l l y pu re th y ro x in e was found w ith b u ta n o l-d io x a n e , o n ly 2% of th e t o t a l a c t i v i t y b e in g as t r i io d o th y r o n in e a t f i v e h ours * P o o r1 r e s u l t s were o b ta in e d w ith th e amyl a lc o h o l system * A second p a t i e n t w ith h e p a tic d y s fu n c tio n was s tu d ie d * Only 22 th y ro x in e was found i n fo u r e a r l y sam ples u s in g both d e v e lo p in g sy ste m s . At 24 h o u rs fo u r peaks w ere p re s e n t w ith b u ta n o l-d io x a n e developm ent b u t no a c t i v i t y was found i n th e tr i io d o th y r o n in e a r e a . F ive peaks w ere found i n t h i s sam ple u s in g th e amyl a lc o h o l system w ith 11% o f th e t o t a l a c t i v i t y p r e s e n t a s t r i i o d o t h y r o n i n e . A second A th y re o tic h y p o th y ro id p a t i e n t p re s e n te d a complex chrom atographic p a t t e r n w ith th e t e r t i a r y amyl a lc o h o l s y ste m . F iv e peaks were found i n th r e e o f th e fo u r sam ples t e s t e d . The maximum amount o f tr i io d o th y r o n in e was 5% a t s i x h o u r s . Only th y ro x in e was p re s e n t i n th e b u ta n o l-d io x a n e a n a ly s e s . H . L abeled Components in Human Serum F o llo w in g In tra v e n o u s 131 T h y ro x in e-I and S u r g ic a l S tr e s s or E p in ep h rin e « The e f f e c t o f s u r g i c a l s t r e s s on c i r c u l a t i n g th y ro x in e was s tu d ie d in two p a t i e n t s . L abeled th y ro x in e was a d m in is te re d im m ediately a f t e r s u r g e r y . In b oth c a se s th y ro x in e was th e p rim ary component f o u n d . No t r i io d o th y r o n in e was p re s e n t in th e serum of e i t h e r in d iv id u a l (T able I I ) • V a ry in g amounts o f e p in e p h rin e w ere a d m in is te re d t o s i x s u b je c ts 131 fo llo w in g in tra v e n o u s th y r o x i n e - I . Three of th e s e in d iv id u a ls w ere s tu d ie d on two o ccasio n s • E p in ep h rin e was in j e c t e d s u b c u ta n e o u sly o r in tr a m u s c u la r ly . I t s p h y s io lo g ic a l e f f e c t s were fo llo w e d by s e v e r a l i n d i c e s • F u ls e r a t e , blood p r e s s u r e and th e p a tie n ts * s u b je c tiv e o p in io n s were r e c o r d e d . A m easure o f th e m e ta b o lic r a t e was o b ta in e d by s h o r t p e rio d s of b r e a th in g th ro u g h a b a s a l m e ta b o lic r a t e m ach in e. An in c re a s e in m e ta b o lic r a t e was observed in f i v e of th e sev en t e s t s i n w hich t h i s method was used* I n a d d itio n , serum p r o t e i n bound io d in e and in o rg a n ic io d in e d e te rm in a tio n s w ere o b ta in e d on th e blood sam ples o f seven of th e s e s tu d ie s • These d e te rm in a tio n s were perform ed in th e la b o r a to r y of th e T hyroid C lin ic * The p a t i e n t s used in t h i s ex p erim en t in c lu d e d f i v e a th y r e o tic e u th y ro id women and one man th y ro id ec to m iz ed f o r can cer and h a v in g m e ta s ta s e s in th e space o f B u rn s• The r e s u l t s o f th e s e s tu d ie s are shown i n T able I I I • T y p ical chromatograms may be found in F ig u re s 7 and 8* In sev en o f th e s e t e s t s no a c t i v i t y was found in th e tr i io d o th y r o n in e are a* These seven a n a ly se s re v e a le d p r im a r i ly th y ro x in e and some s o lv e n t f r o n t a c t i v i t y * I n th e two re m a in in g s tu d ie s a c t i v i t y in th e t r i i o d o th y ro n in e a r e a am ounting t o a maximum o f 4 % was found* The p r o te in bound io d in e d e te rm in a tio n s re v e a le d th e exp ected d e c re a se in f i v e o f th e seven cases so s tu d ie d (T able 17) • A t r a n s i t o r y r i s e in th e p r o t e i n bound io d in e , see n in two o f th e se c a s e s , has a l s o been observed b y Dr • S • Tanaka (61) fo llo w in g th e a d m in is tr a t i o n of e p in e p h rin e • I n two cases th e p r o t e i n bound io d in e in c re a s e d and was a s s o c ia te d w ith a v e ry h ig h in o rg a n ic io d in e * These two in d iv id u a ls w ere f u r t h e r d is tin g u is h e d i n h a v in g th e h ig h e s t m e ta b o lic r a t e in c re a s e s and in e x h i b i t i n g some t r i io d o th y r o n in e i n th e chrom ato g rap h ic a n a l y s e s • 24 131 1 . L abeled Components in R at T issu e s F o llo w in g T h y ro x in e-I . A s e r i e s of ex perim ents was perform ed in which la b e le d th y ro x in e was a d m in iste re d to r a t s * The m a t e r i a l was i n j e c t e d i n t r a - p e r i t o n e a l l y or i n t r a c a r d i a l l y ( i f th e l a t t e r , l i g h t e t h e r a n a e s th e s ia was used) • A t o t a l o f e le v e n r a t s was s tu d ie d in fo u r groups* The f i r s t group c o n s is te d of two norm al m ales given 65 j i c of la b e le d t h y ro x in e (4 ;ugm*) i n t r a p e r i t o n e a l l y * One anim al was k i l l e d a t two h o u rs , th e second a t f i v e hours » The rem ainder of th e a n im als had b een th y ro id e e to m iz e d by two 131 tre a tm e n ts w ith 500 jic o f I . H i s t o l o g i c a l s e c tio n s showed no th y r o id t i s s u e in th e necks o r c h e s ts o f th e s e r a t s * A ll th y r o i d e c - tom ized anim als e x cep t two i n Group I¥ were g iv en 8 jugn. o f L -th y ro x in e d a i l y by i n t r a p e r i t o n e a l i n j e c t i o n f o r two weeks p r i o r t o t h e i r use* Group I I c o n ta in e d th r e e fe m a le s, two of w hich were m a in ta in e d , _ o a t -5 f o r f o u r and e le v e n hours r e s p e c t i v e l y a f t e r th e i n t r a p e r i t o n e a l a d m in is tr a tio n of la b e le d th y ro x in e (75 ^ac, 4 jAgm*) • The t h i r d anim al in t h i s group was k ep t a t room te m p e ratu re and k i l l e d a t. th r e e h o u rs • i Group I I I c o n s is te d of two male r a t s w hich were given 80 ^igm. o f e p in e p h rin e , in tra m u s c u la rly ,, 30 m inutes a f t e r th e i n t r a p e r i t o n e a l a d m i n i s t r a t i o n 'o f 62 jio of th y r o x i n e - I 151 (3 >ign.) . These anim als w ere k i l l e d a t two and th r e e h o u rs r e s p e c tiv e ly * i The f o u r th group c o n ta in e d f o u r m ales* Two o f th e s e anim als were m a in ta in e d on th y r o x in e , two were n o t* These anim als were g£.ven 25 in tra m u sc u la r, e p in e p h rin e (80 jagpi* p er 100 gm. body w e ig h t) im m ediate ly a f t e r th e i n t r a c a r d i a l a d m in is tr a tio n o f la b e le d th y ro x in e (60 ^io, 5 jxgpi*) * They were k i l l e d a t 45 m in u tes* As a means of d e m o n stra tin g th e p h y s io lo g ic a l a c t i v i t y o f th e e p in e p h r in e , glucose d e te rm in a tio n s w ere o b ta in e d on th e s e r a of th e Group IV r a t s • A re a g e n t c o n ta in in g glu co se o x id a s e , p e ro x id a se and o - d ia n is id in e was used f o r th e s e d e te rm in a tio n s » The i n i t i a l blood sample was obtained, by h e a r t p u n ctu re im m ed iately p r i o r t o th e i n j e c t i o n of th e la b e le d t h y r o x i n e • In th e fo u r e x p e rim e n ta l anim als a marked in c re a s e In blood glucose was - observed fo llo w in g e p in e p h rin e * A c o n tr o l anim al given th e same tre a tm e n t w ith no e p in e p h rin e e x h ib ite d no change in g lu co se le v e l (T able V),* In norm al r a t s sm all amounts of t r i io d o th y r o n in e were found in t i s s u e s a t two and f i v e hours a f t e r th e a d m in is tr a tio n o f la b e le d th y ro x in e * The l a r g e s t c o n c e n tr a tio n of t r i io d o th y r o n in e was in a * " } k id n ey sample in w hich 83^ of th e t o t a l a c t i v i t y was i n th e th y ro x in e a re a and 10^ in th e t r i i o d o t h y r o n i n e a r e a . In o th e r t i s s u e s (serum , s k e l e t a l m u scle, h e a r t , s a l i v a r y gland and l i v e r ) th e amount o f tr i io d o th y r o n in e v a r ie d fro m 1 t o 9 ^ . There was no in c r e a s e i n t r i io d o th y ro n in e i n th e th y ro id e c to m iz e d r a t s m a in ta in e d a t b e lo w -fre e z in g te m p e ra tu re s * Most o f th e t i s s u e s i n t h i s group o f anim als w ere found t o have no d e te c ta b le t r i i o d o t h y r o n i n e • Many, chromatograms o f th e s e I two groups o f r a t s r e v e a le d c o n s id e ra b le amounts o f r a d i o a c t i v i t y a t th e s o lv e n t f r o n t . No in c r e a s e in tr i io d o th y r o n in e was se e n i n t h e anim als given 26 e p in e p h r in e . T riio d o th y ro n in e c o n c e n tr a tio n s in th e s e t i s s u e s v a r ie d from 0 t o 1276 w ith f a i r l y good c o r r e l a t i o n betw een th e amyl a lc o h o l and b u ta n o l-d io x a n e system s • Many of th e s e t i s s u e s c o n ta in e d a number of chrom atographic components n o t found i n th e c o n tr o l a n im a ls . Two such components see n most o f te n w ere a s u b sta n c e found betw een th e th y ro x in e and t r i io d o th y r o n in e a re a s and one found betw een th e o r i g i n and t h y r o x i n e . T y p ic a l chromatograms of th e s e ex p erim en ts are in c lu d e d i n B ig u res 9 t o 18 . ' The d a ta a re ta b u la te d i n T ables VI t o IX . 131 J . E x c re tio n of I A fte r th e A d m in is tra tio n o f L abeled T h y ro x in e . E x c re to ry s tu d ie s w ere conducted t o d eterm in e i f e p in e p h rin e cau ses an in c re a s e d c a ta b o lis m o f th y r o x i n e . F ive myxedematous m ale r a t s w ere in j e c t e d w ith la b e le d th y ro x in e (75 p c 9 4 ^igyn.) i n t r a - p e r i t o n e a l l y and w ere p laced in m etabolism c a g e s . U rine and f e c e s w ere c o lle c te d over 24 or 48 hour p e rio d s f o r a t o t a l of 96 hours and p o o le d . S e v e ra l weeks l a t e r th e same r a t s were given a s im ila r dose o f th y ro x in e fo llo w ed by in tra m u s c u la r e p in e p h rin e (80 . p er 100 g n . body w e i g h t ) . U rine and fe c e s w ere c o lle c te d f o r a b o u t.s i x days • Feces w ere homogenized in w a te r and made up t o volume in a v o lu m e tric f l a s k ; u rin e was f i l t e r e d and a ls o made up t o volum e. A liq u o ts were ta k e n f o r r a d i o a c t i v i t y d e te r m in a tio n and were compared t o a liq u o ts of a d ilu te d s ta n d a rd w hich c o n ta in e d th e amount of th y ro x in e g iv en t o one r a t . O ther a liq u o ts of fe c e s and u rin e were a c i d i f i e d and e x tr a c te d w ith b u ta n o l • The b u ta n o l e x t r a c t s were 27 j th e n washed w ith B la u 's re a g e n t and th e rem ain in g r a d i o a c t i v i t y was counted « I n s e v e r a l cases b u ta n o l e x t r a c t s w ere c o n c e n tra te d and chrom atographed w ith th e amyl a lc o h o l sy ste m . . I n 96 h o u r s , .27$ o f th e i n j e c t e d r a d i o a c t i v i t y was e x c r e te d , I I i p r im a r i ly as io d id e , in th e u rin e o f th e c o n tr o l r a t s • I n th e same j i n t e r v a l th e e p in e p h rin e t r e a t e d r a t s e x c re te d about 44$ o f th e i n j e c t e d i s o t o p e . F e c al e x c r e tio n t o t a l e d 53$ in th e c o n tr o l group and about 47$ ( a t 96 h o u rs) i n th e e p in e p h rin e t r e a t e d g ro u p . F e c a l ■131 I was m ain ly in th e form of t h y r o x i n e . Graphs of th e e x c r e tio n d a ta may be found i n F ig u re s 19 and 2 0 . IV . DISCUSSION i n th e p re s e n t work no evid en ce was found f o r an in c re a s e d p ro d u c tio n of t r i i o d o t h y r o n i n e from th y ro x in e under c o n d itio n s le a d in g t o in c re a s e d th y r o id hormone a c ti o n in th e p e r ip h e r a l t i s s u e s • E p in ep h rin e i s known t o d e c re a se th e serum p r o t e i n bound io d in e (53-55) and e f f e c t an in c re a s e d oxygen consum ption in v i v o * The l a t t e r e f f e c t has been shown t o r e q u ir e th e p re sen c e o f th y r o id hormone (56) • I f t h e horm onal a c t i v i t y o f th y ro x in e were dependent on i t s c o n v e rsio n t o t r i i o d o t h y r o n i n e * i t would be e x p ec te d t h a t e p in e p h rin e a d m in is tr a tio n would le a d t o in c re a s e d amounts of t r i i o d o th y ro n in e in th e t i s s u e s • I n th e human ex p erim en ts d e s c rib e d above, no s i g n i f i c a n t amounts of c i r c u l a t i n g tr i io d o t h y r o n i n e were observed a f t e r an e p in e p h rin e i n j e c t i o n w hich low ered th e p r o t e i n bound io d in e * The work of P i t t - B i v e r s e t a l •, (21) had in d ic a te d t h a t c i r c u l a t i n g t r i io d o th y ro n in e co u ld be found in humans a f t e r th e in tra v e n o u s adm ini s t r a t i o n of la b e le d th y ro x in e * In th e p re s e n t work v a r i a b l e amounts of t r i io d o th y r o n in e w ere found in th e serum of th e n in e human c o n tr o ls In fo u r in d iv id u a ls no t r i i o d o t h y r o n i n e was found a t any tim e ; i n a f i f t h , a sm all amount was d e te c te d a t n in e hours but had d isa p p e a re d a t 24 h o u rs* The serum of a s i x t h p a t i e n t c o n ta in e d a sm all amount o f tr i io d o th y r o n in e a t f i v e h o u rs (and a ls o a t 20 m inutes w ith th e amyl a lc o h o l a n a ly s is o n ly ) • In two o th e r p a t i e n t s tr i io d o th y r o n in e was found o n ly in th e sam ples analyzed w ith th e amyl a lc o h o l system ; 29 11^ o f th e t o t a l a c t i v i t y was found as tr i io d o th y r o n in e i n th e 24 i hour sample o f one of th e s e i n d i v i d u a l s • The n in th p a tie n t was found t o have c i r c u l a t i n g t r i io d o th y r o n in e a t 30 m inutes and a t 18*5 h o u rs when analyzed w ith b o th s o lv e n t system s* I n a l a t e r experim ent a sm all amount of tr i io d o th y r o n in e was found in one" blood sample o f t h i s in d iv id u a l* Two e a r l i e r sam ples c o n ta in e d none# I n c o n t r a s t t o th e complex p a tte r n s observed in some of th e c o n t r o l s , th e e p i n e p h r in e - tr e a t e d p a t i e n t s were fo und, in seven of n in e e x p e rim e n ts, t o have no c i r c u l a t i n g tr i io d o th y r o n in e • In two o f th e n in e t r i a l s * o r in two of th e s i x in d iv id u a ls used in th e s e e x p e rim e n ts, sm all amounts of t r i io d o th y r o n in e were found* Most of th e chromatograms c o n tain ed e s s e n t i a l l y pure th y ro x in e * E p in ep h rin e ap p ears t o e f f e c t , a r a p id d is a p p e a ra n c e from, th e c i r c u l a t i o n o f su b sta n c e s d e riv e d from th y ro x in e * T his o b s e r v a tio n i s n o t in c o n f l i c t w ith th e serum b in d in g p r o p e r tie s o f th e io d in a te d th y ro n in e s • -Thyroxine i s th e most f ir m ly bound o f th e s e compounds • Robbins and R a i l have c a lc u la te d th e c o n c e n tr a tio n o f unbound th y ro x in e in norm al human serum as 6*10 M (62)* Assuming a p r o t e i n bound io d in e of 6 )agm* per c e n t, th e norm al t o t a l serum th y ro x in e i s „7 about 8 -10 M* T riio d o th y ro n in e i s bound t o th y ro x in e b in d in g p ro t e i n w ith o n e - th ir d th e i n t e n s i t y o f th y ro x in e (43 )* U npublished d a ta o f Dr • S • Tanaka have in d ic a te d t h a t te tr a io d o th y r o p r o p io n ic a c id and t r i i o d o t h y r o a c e t i c a c id - a r e n o t bound by serum p r o te in s t o any a p p re c ia b le e x te n t • E p in ep h rin e alm ost c e r t a i n l y produces a rem oval of th y ro x in e * from th e c i r c u l a t i o n . The m ajor c i r c u l a t i n g th y r o id hormone i s th y ro x in e arid a r a p id d ro p i n serum p r o t e i n bound io d in e was g e n e r a lly observed a f t e r e p in e p h rin e a d m i n i s t r a t i o n . From th e d a ta p re s e n te d above, i t i s u n lik e ly t h a t t h i s e f f e c t i s m ediated th ro u g h a l t e r a t i o n of th e th y ro x in e m olecules i n th e blood t o more f r e e l y d i f f u s i b l e compounds. I f such were th e c a s e , one would e x p ect t o f in d th e s e su b sta n c e s in th e c i r c u l a t i o n a f t e r e p in e p h rin e a d m in is tr a tio n sin c e th e y appeared t o accum ulate i n some o f th e c o n tr o ls • I f such a c o n v ersio n occurs a t a l l , p r i o r t o th e e n tr y of th y r o id hormone in to th e p e r ip h e r a l c e l l s , i t w ould th e r e f o r e be ex p e cte d t o ta k e p la c e a t a s i t e from which th e th y ro -a c tiv © d e r iv a tiv e could n o t r e e n t e r th e c i r c u l a t i o n . The c e l l s u r f a c e m ight be th e s i t e of such a r e a c t i o n . A l t e r n a t i v e l y , th e e p in e p h rin e e f f e c t could be a n s a l t e r a t i o n in c e l l u l a r p e r m e a b ility a llo w in g a r a p id e n t r y of unbound th y ro x in e i t s e l f « A nother p o s s ib le e x p la n a tio n f o r th e e f f e c t of e p in e p h rin e on th y ro x in e le v e ls i s t h a t e p in e p h rin e a c ts in some e n t i r e l y d i f f e r e n t manner t o in c re a s e th e r a t e of c e l l u l a r o x i d a t i o n . T his could p ro duce e i t h e r a r a p id tu rn o v e r or a r e d i s t r i b u t i o n o f i n t r a c e l l u l a r th y r o id horm one. C i r c u l a t i n g th y ro x in e or a d e r i v a t i v e m ight th e n e n te r th e c e l l t o r e s t o r e th e c o n c e n tr a tio n o f c e l l u l a r th y r o id hormone • A r a p id tu rn o v e r w ould im ply t h a t t h y r o i d hormone is d e stro y e d w h ile f u n c tio n in g as a r e g u la to r o f o x id a tiv e rat© . A lthough no mechanism has been f i r m l y e s ta b lis h e d f o r th y r o id hormone a c t i v i t y 31 (se e b e lo w )/ i t would seem, from th e e x tre m e ly sm all amounts p r e s e n t i n t i s s u e , t o be c a t a l y t i c in n a t u r e . There i s some e v id e n c e , n e v e r t h e l e s s , t h a t th y r o id hormone i s d e stro y e d i n l a r g e r amounts d u rin g p e rio d s o f in c re a s e d o x id a tiv e m e ta b o lism . T hus, d e l Rey and de E scobar (63) re p o r te d a h ig h e r e x c r e tio n of I by t r a i n e d r a t s d o in g p ro lo n g e d 'm u sc u la r w ork th a n by t r a i n e d c o n tr o ls a t r e s t . Labeled th y ro x in e was given t o b o th groups and a c t i v i t y re m a in in g in th e c a rc a ss and organs was m easured a f t e r th e e x e r c is e p e r i o d . The e x c r e to r y s tu d ie s " d e s c r ib e d i n S e c tio n I I I of t h i s d i s s e r t a t i o n showed t h a t e p in e p h rin e (w hich, l i k e e x e r c i s e , in c re a s e s h e a t p ro d u c tio n and oxygen consum ption) produces an in c re a s e d u r in a r y io d in e e x c r e tio n and somewhat d e crea se d f e c a l i o d i n e . U rin a ry io d in e was found t o be p r im a r ily io d id e ; f e c a l io d in e was p r im a r i ly e x c re te d as th y r o x i n e . . . . 131 F le tc h e r has shown t h a t in humans g iv e n th e r a p e u ti c doses of I th e a c t i v i t y found in u rin e was m ain ly io d id e w ith some d iio d o ty r o s in e p re s e n t (64) • ’ T aurog has r e p o r te d t h a t th e f e c a l io d in e in r a t s i s m ain ly in th y ro x in e (48) • The above f i n d i n g s , a lth o u g h i n d i c a t i n g in c re a s e d c a ta b o lis m o f th y r o x in e , do n o t n e c e s s a r i l y in d ic a te t h a t th y ro x in e fu n c tio n s in a s to ic h io m e tr ic m anner. I n th e p r e s e n t work i t was found t h a t th e in c re a s e in th e r a t e o f u r i n a r y io d in e e x c r e tio n a f t e r e p in e p h rin e p e r s i s t e d f o r a t l e a s t 96 h o u r s . S in ce e p in e p h rin e is r a p i d l y i n a c ti v a te d in v iv o , i t i s im probable t h a t t h i s e f f e c t is due t o any prolonged a c tio n * I t ap p ears more l i k e l y t h a t e p in e p h rin e e f f e c t s a r a p id e n tr y of th y r o i d hormone in to th e p e r ip h e r a l c e l l s . The e x c re 32 t i o n o f io d id e may r e s u l t from a p h y s io lo g ic a l mechanism d e sig n e d t o r e s t o r e th e i n i t i a l c o n c e n tr a tio n o f th y r o id hormone in th e c e l l s * There i s e x p e rim e n ta l e v id en c e t h a t th e m ito c h o n d rio n i s th e s i t e of th y r o id hormone a c ti o n (see b e lo w ). A ll of th e th y r o i d hormone w hich e n te r s th e c e l l may n o t re a c h th e m ito c h o n d ria . DuToit (65) has I \ c i t e d u n p u b lish ed work by Crane w hich showed t h a t in j e c t e d la b e le d th y ro x in e i s e v e n ly d i s t r i b u t e d among th e n u c l e i , m ito c h o n d ria and s u p e rn a ta n t f r a c t i o n o f r a t l i v e r . D e io d in a tio n may r e p r e s e n t a r e g u la to r y mechanism f o r i n t r a c e l l u l a r th y r o id hormone c o n c e n tr a tio n j u s t as c o n ju g a tio n and b i l i a r y e x c r e tio n appear t o be one means of r e g u l a t i n g th e c i r c u l a t i n g th y r o i d hormone c o n c e n tra tio n * The in o re a se d io d id e e x c r e tio n a f t e r e p in e p h rin e p ro b a b ly r e s u l t s from a mechanism o f t h i s n a tu r e , w hich i s not d i r e c t l y r e l a t e d t o th e horm onal e f f e c t s o f th y ro x in e • No in c r e a s e i n th e d e io d in a tio n of th y ro x in e t o tr i i o d o t h y r o n i n e , d u rin g th e p e rio d o f e p in e p h r in e - induced in c re a s e d oxygen consum ption, was d em o n strated in th e p r e s e n t work (see b e lo w ). From t h i s f in d in g , and from th e pro lo n g ed in c re a s e in u r in a r y io d id e fo llo w in g e p in e p h r in e , i t i s p o s tu la te d t h a t th e in c re a s e d th y ro x in e c a ta b o lis m observed a f t e r e p in e p h rin e a d m in is tr a t i o n r e s u l t s from an a l t e r a t i o n in c e l l u l a r p e r m e a b ility t o th y r o x i n e . Numerous o th e r h y p o th e se s have been proposed t o e x p la i n th e c a l o r i g e n i c i t y of e p in e p h r in e . G r i f f i t h , i n a r e c e n t re v ie w , c i t e d n e a r ly 500 papers, p e r t a i n i n g t o t h i s to p i c (66) • There ap p ears t o be b u t one r e f e r e n c e t o th y r o i d hormone in t h i s v a s t l i t e r a t u r e - a 1955 r e p o r t t h a t t h y r o i d - s e n s i t i z e s th e e p in e p h rin e e f f e c t ( 6 7 ) . 33 ?*fork d esig n ed t o show t h a t ep in ep h rin e' i s a s p e c i f i c r e s p i r a t o r y c a t a l y s t a t th e c e l l u l a r le v e l has y ie ld e d r e s u l t s alm ost e v en ly d iv id e d in fa v o r of and a g a in s t t h i s h y p o th e s is • O th e r- proposed mechanisms have been based on th e s p e c i f i c dynamic a c ti o n o f c arb o h y d r a te , th e removal o f l a c t a t e and i t s r e c o n v e rs io n t o glycogen, th e c a r d io v a s c u la r e f f e c t s (in c re a s e d c a r d ia c a c t i v i t y , cutaneous v aso c o n s t r i c t i o n and r e s u l t a n t red u ced h e a t l o s s , v a s o c o n s tr ic tio n o f s k in and m uscle) and on e p in e p h rin e -in d u c e d in c re a s e d s k e l e t a l muscle tension® The ch rom atographic s tu d ie s on human serum l e f t unanswered th e q u e s tio n of w h eth er th y ro x in e is c o n v erted t o tr i io d o th y r o n in e i n t r a c e l l u l a r l y or a t some p o s itio n from w hich th e l a t t e r su b sta n c e i s unable t o r e e n t e r th e c irc u la tio n ® The anim al t i s s u e ex p erim en ts w ere u n d e rta k en in an a tte m p t t o answer t h i s question® The r e s u l t s d id n o t i n d ic a te any in c r e a s e i n th e t i s s u e c o n c e n tr a tio n of t r i i o d o th y ro n in e a f t e r th e a d m in is tr a tio n of. la b e le d th y ro x in e and e p in e p h rin e • S ix r a t s w ere s tu d ie d a f t e r epinephrine® Four of th e s e anim als w ere k i l l e d a t 45 m inutes • The re m a in in g r a t s , w hich were given a s m a lle r dosage o f e p in e p h r in e , were k i l l e d a t two and th r e e hours re s p e c tiv e ly ® Two o th e r r a t s w ere s u b je c te d to co ld s t r e s s a f t e r th e i n j e c t i o n o f la b e le d thyroxine® Low te m p e ra tu re s t r e s s was shown by Cannon e t a l ♦, t o produce a prompt in c r e a s e in a d re n a l e p in e p h rin e s e c r e t i o n (68)® Three c o n tr o l anim als were s tu d ie d and w ere k i l l e d a t tw o, 34 th r e e and f i v e h o u rs , r e s p e c t i v e l y . Sm all amounts of t r i io d o th y r o n in e were found in th e t i s s u e s o f th e s e a n im als, v a ry in g from 1 t o IC D ^ of t h e ' t o t a l a c t i v i t y re c o v e re d on t h e chrom atogram s. In -tw o o f th e c o n tr o ls some t r i io d o th y r o n in e was found in each t i s s u e a n a ly z e d . - The t h i r d c o n tr o l c o n ta in e d v e ry sm all q u a n t i t i e s o f t r i i o d o t h y r o n i n e (1 t o 2%) in serum , s a l i v a r y gland a n d * liv e r ; th e k id n ey and h e a r t •of I t h i s anim al c o n ta in e d n o n e . High l e v e l s o f s o lv e n t f r o n t a c t i v i t y w ere n o ted on th e chromatograms of th e t i s s u e s o f t h i s r a t • The t i s s u e s o f th e c o l d - s t r e s s e d anim als w ere found t o have l i t t l e or no t r i io d o th y r o n in e . These chromatograms w ere a l l r e l a t i v e l y sim p le , w ith a c t i v i t y c o n c e n tra te d o n ly in th e th y r o x in e , t r i i o d o th y ro n in e o r •s o lv e n t f r o n t a r e a s • The Group I I I r a t s , w hich w ere given 80 of e p in e p h r in e , p re s e n te d a somewhat s i m i l a r p i c t u r e . T r iio d o th y ro n in e was found i n o n ly one of th e c f i v e t i s s u e sam ples ( o th e r th a n 's e ru m ) a n a ly z e d . Ho o th e r peaks of r a d i o a c t i v i t y w ere - found e x ce p t a t th e s o lv e n t f r o n t , th e th y ro x in e a r e a and th e o r i g i n . A g r e a te r d egree o f co m p lex ity was o bserved on th e chrom ato grams o f th e Group IV r a t t i s s u e s . These anim als were given 80 ^iga. of e p in e p h rin e per 100 gms• o f body w e ig h t* F i f t e e n t i s s u e sam ples w ere a n aly zed w ith b o th ch ro m ato g rap h ic s y ste m s . In tw elv e of th e s e t r i i o d o t h y r o n i n e was found in b o th a n a ly se s w ith f a i r l y good c o r r e l a t i o n of t h e c o n c e n tr a tio n s , w hich ra n g e d -fro m 1 t o 12%* Of th e r e m ain in g t h r e e sa m p le s•an aly zed i n d u p l i c a t e , t r i io d o th y r o n in e was a b se n t i n one l i v e r sam ple*and was p r e s e n t o n ly w ith b u ta n o l-d io x a n e chrom atography i n a n o th er l i v e r and i n one h e a r t sa m p le . F iv e o th e r 35 t i s s u e sam ples w ere s u c c e s s f u lly chrom atographed w ith o n ly one s o lv e n t system * Some tr i io d o t h y r o n i n e was found t o be p re s e n t i n fo u r of th e s e * I t was ab sen t in one s k e l e t a l m uscle sam ple an aly zed w ith th e amyl 'a lc o h o l system * Peaks of r a d i o a c t i v i t y a t p o s itio n s o th e r th a n th y r o x in e , I tr i io d o th y r o n in e and th e s o lv e n t f r o n t w ere found on many of th e chromatograms o f th e Group-IV r a t s * Most o f te n found was a peak moving betw een th y ro x in e and tr iio d o th y r o n in e * T h is peak was found in tw elv e t i s s u e sa m p le s • C o r r e la tio n betw een th e two s o lv e n t system s was p o o r; t h i s peak was n o t found i n b oth a n a ly s e s o f any o f th e sam ples ru n in d u p lic a te - . -It was found in fo u r sam ples an aly zed w ith amyl a lc o h o l (two s k e l e t a l m uscle and two s a l i v a r y gland) and in i e ig jit a n aly z e d w ith th e b u ta n o l-d io x a n e system ( th r e e h e a rt* th r e e „ l i v e r and two k id n e y s a m p le s ) * * T his la c k of d u p lic a tio n r a i s e s th e p o s s i b i l i t y t h a t th e a d d i t i o n a l peaks found i n th e s e r a t s were a r t i f a c t s formed d u rin g e x t r a c t i o n or chrom atography* So f a r &6 i s known, how ever, c o n d itio n s w ere uniform th ro u g h o u t th e s e anim al ex p erim en ts • The ch rom atographic a n a ly se s were perform ed in -g ro u p s o f about t e n s t r i p s a t a t i m e . The t i s s u e e x t r a c t s o f r a t s 3 and 4 were chrom atographed sim u lta n eo u sly . I f ‘th e unknown peak b etw een * th y ro x in e and t r i i o d o t h y r o n i n e w ere an a r t i f a c t r e s u l t i n g from th e c o n d itio n s o f chrom atography, one would e x p ec t i t to appear in th e t i s s u e s o f b o th a n im a ls . I t was n o t fo und, how ever, in any* o f th e t i s s u e s o f r a t 4* - I t i s p o s s ib le t h a t t h i s peak r e p r e s e n ts two s u b s ta n c e s , each o f w hich is s e p a ra b le from th y ro x in e in one, b u t n o t th e o th e r . 36 s o lv e n t s y ste m . I f t h i s is th e t r u e i n t e r p r e t a t i o n , h a l f o f th e s k e l e t a l m uscle and s a l i v a r y gland sam ples analy zed c o n ta in e d one of th e s e unknown su b sta n c es • Three h e a r t , th r e e l i v e r and two k id n ey sam ples c o n ta in e d th e o t h e r . W hile one e f f e c t of e p in e p h r in e , th e n , appears to be an I in c re a s e d r a t e of th y ro x in e d e g ra d a tio n i n v iv o , i t ap p ears from t h e I ex p erim en ts j u s t d is c u s s e d t h a t t h i s i s n o t accompanied by an in c re a s e d , j I : t i s s u e c o n c e n tr a tio n of th y r o x in e - d e r iv e d t r i i o d o t h y r o n i n e • I t i s 1 ! assumed in i n t e r p r e t i n g th e s e r e s u l t s t h a t th e unknown su b sta n c e s formed t o an in c re a s e d d eg ree a f t e r e p in e p h rin e w ere not s o l e l y d e r iv a tiv e s of t r i i o d o t h y r o n i n e . T his assum ption i s based on th e chrom atographic m o b ility cf th e unknown most commonly found in t h i s w o rk . T his m o b ility in d ic a te s th a t th e s u b sta n c e may be e i t h e r t e t r a i o d o t h y r o a c e t i c or - p ro p io n ic a c i d . These t e t r a i o d i n a t e d com pounds a re known t o be form ed from th y ro x in e b o th _in v iv o and _in v i t r o . B arker has found t h a t t e t r a i o d o t h y r o a c e t i c a c id i s form ed more con s i s t e n t l y th a n t r i io d o th y r o n in e when th y ro x in e i s in c u b ated w ith r a t k id n ey s l i c e s (69) • F u rth e rm o re , a c a t a l y t i c mechanism i s m ost p ro - i b a b le f o r th y r o i d hormone a c t i o n . I t would n o t be ex p ected t h a t , i f j tr i i o d o t h y r o n i n e were th e a c tiv e a g e n t, i t w ould be d eg rad ed a t th e same r a t e a t w hich i t i s fo rm ed . The r e s u l t s of th e p re s e n t w ork do ! not su p p o rt th e th e o ry t h a t th y ro x in e must be c o n v erted t o t r i i o d o th y ro n in e f o r p e r ip h e r a l a c t i v i t y . j ■ I t has been m entioned above t h a t , d e io d in a tio n may r e p r e s e n t la mechanism t o r e g u la te th e i n t r a c e l l u l a r c o n c e n tr a tio n of th y r o id 37 horm one. Roche e t a l • have d e m o n strated th e r a p id fo rm a tio n of 3 ,3 * ,- d iio d o th y r o n in e from tr i io d o th y r o n in e i n v iv o and concluded t h a t th e t i s s u e d e io d in a tio n of th y ro x in e t o t r i i o d o t h y r o n i n e , " i f i t a c t u a l l y hap p en s” , r e p r e s e n ts o n ly an in te rm e d ia te s te p i n th e p ro d u c tio n o f d iio d o th y r o n in e , w hich has o n e - s ix th t o o n e -se v e n th t h e b i o l o g i c a l a c t i v i t y of t r i i o d o t h y r o n i n e (7 0 )• A lthough t r i i o d o th y ro n in e i s more a c ti v e th a n th y ro x in e as a rep lacem en t form o f th y r o i d hormone, in v i t r o s tu d ie s (se e below) have in d ic a te d t h a t th e s e two compounds a re e q u a lly a c tiv e i n t r a c e l l u l a r l y . I t i s th u s n o t i l l o g i c a l t h a t t r i i o d o t h y r o n i n e m igjit be th e f i r s t p ro d u c t of a s e r i e s of r e a c tio n s w hich degrade th y ro x in e t o an i n a c t iv e su b sta n c e* D e io d in a tio n i s n o t th e o n ly pathway a v a il a b le t o th y ro x in e * Two deam inated p ro d u c ts have been d em o n strated - th e p ro p io n ic a c id d e r i v a t i v e in v i t r o (35) and th e p y ru v ic a c id d e r i v a t i v e in v iv o (7 1 )* The fo rm a tio n o f th e a c e t i c a c id d e r i v a t i v e s has a ls o been d e m o n stra t ed w ith b o th th y ro x in e and tr i io d o th y r o n in e (3 4 , 7 0 )• I t is o f i n t e r e s t t h a t th e s e d e r i v a t i v e s have v a r y in g b i o l o g i c a l a c t i v i t i e s depending on th e a ssa y system used* In mammals, tr i io d o th y r o n in e ap p ears t o be th e most a c tiv e in v iv o i n a l l r e s p e c ts • The p ro p io n ic d e r i v a t i v e has 120 tim e s t h y r o x i n e Ts a c t i v i t y i n t a d p o le m etam orphosis (72) b u t i s le s s a c ti v e th a n th y ro x in e i n v iv o i n mammals so f a r as oxygen consum ption is concerned (7 3 , 74) • I t i s n o t known i f th e e f f e c t o f th y r o id hormone on ta d p o le m etam orphosis in v o lv e s in c re a s e d oxygen consum ption* T r iio d o th y r o a c e tic a c id ap p ears t o have d i s s o c i a t e d hormonal 38 e f f e c t s • In a sm all s e r i e s of p a t i e n t s i t h as been observed to lower serum c h o l e s t e r o l w ith o u t a f f e c t i n g t h e m e ta b o lic r a t e (3 2 )• Q uerido and co-w orkers have re p o r te d s im ila r r e s u l t s (75) • They a ls o observed in c re a s e d phosphate and c r e a t i n e e x c r e tio n and a change t o a norm al e le c tro c a rd io g ra m w ith t r i i o d o t h y r o a c e t i c a cid # T r o t t e r (76) has S found t h i s compound t o have o n e - f i f t h th e p o ten cy o f th y ro x in e .in lo w e rin g th e serum c h o l e s t e r o l of myxedematous p a t i e n t s # I n e u th y ro id p a t i e n t s w ith c o ro n a ry a r t e r y d is e a s e th e a c e tic a c id analogue low ered c h o l e s t e r o l s i g n i f i c a n t l y a t dose l e v e l s w hich d id n o t a f f e c t th e b a s a l m e ta b o lic r a te # B arker has " o b ta in e d e x c e l l e n t m e ta b o lic r e s ponses t o th e t e t r a i o d o - and tr i io d o th y r o p r o p io n i c and a c e tic a c id d e r i v a t i v e s ” , in mammals (69)* In view o f a r e p o r te d r a p id resp o n se t o th e a c e t i c a c id d e r i v a t i v e s , w ith o u t la g p e r io d s , i t has been th e o r iz e d t h a t th e s e su b sta n c e s are th e p e r ip h e r a lly a c tiv e th y ro id hormones (7 3 , 74) • B arker, how ever, has found a p p ro x im a te ly th e same a c t i v i t i e s f o r th y r o x in e , t r i i o d o t h y r o n i n e and t h e i r a c e t i c a c id d e r iv a tiv e s in re d u c in g th e r a t e of d e c re a se of oxygen consum ption by r a t k id n e y s l i c e s o v er a th r e e day p e rio d (78) • ¥fisw ell (79) has a ls o found th e in. v i t r o a c t i v i t y of v a rio u s th y r o id su b sta n c e s t o d i f f e r from in v iv o r e s u l t s • U sing a r a t h e a r t homogenate w ith s u c c in a te as s u b s t r a t e , he found t h a t a l t e r a t i o n s i n th e s id e c h a in o f th e th y ro x in e im o lecu le d id n o t im p air th e stim ulatory! e f f e c t on oxygen consum ption# Both L- and D -th y ro x in e w ere somewhat more a c tiv e th a n L - tr iio d o th y r o n in e i n t h i s system ; D -tr iio d o th y r o n in e was alm ost as a c tiv e as th e L -iso m e r• The a c r y l i c a c id analogue of 39 th y ro x in e was as a c tiv e as th y ro x in e • T his s u b sta n c e has about th r e e tim e s th e a c t i v i t y o f th y ro x in e i n s t i m u l a t i n g ta d p o le m etam orphosis b u t o n ly 1% i t s a c t i v i t y in s t i m u l a t i n g th e b a s a l m e ta b o lic r a t e of r a t s (80) • E x te n siv e work has been perform ed by s e v e r a l groups o f i n v e s t i g a to rs on th e r o l e of t h y r o i d hormone as an u n co u p ler of o x id a tiv e p h o s p h o ry la tio n . Much o f t h i s work has been done by Lardy and h i s c o -w o rk e rs . A b a s ic prem ise o f t h i s th e o r y o f t h y r o i d a c ti o n i s t h a t th e r a t e o f c e l l u l a r o x id a tio n i s n o rm a lly lim it e d by th e c o n c e n tra t i o n o f a v a il a b le phosphate and phosphate a c c e p to rs (8 1 ) • A lthough i t h as not been p o s s ib le t o in c re a s e t i s s u e r e s p i r a t i o n in v i t r o t o th e e x te n t observed i n i n t a c t anim als ( i t has been e s tim a te d t h a t th e oxygen consum ption o f s k e l e t a l m usole a t maximum e x e r tio n is in c re a s e d 100 f o ld over th e r e s t i n g v a l u e ) , m ito c h o n d ria l p r e p a r a tio n s o x id iz in g a v a r i e t y of s u b s t r a t e s have been s tim u la te d up t o 15 f o ld by th e a d d itio n o f e i t h e r in o rg a n ic phosphate o r a phosphate a c c e p to r system such as glucose and h ex o k in ase • The o x id a tio n o f s u b s t r a t e s in v o lv in g th e p y r id in e n u c le o tid e s produces about th r e e m olecules o f h ig h energy phosphate p e r atom of oxygen consum ed. These p h o s p h o ry la tio n s appear t o ta k e p la c e d u rin g th e o x id a tio n o f cytochrom e c by cytochrom e a and a t th e s te p s w here cytochrom e b and p y rid in e n u c l e o t i d e s , r e s p e c t i v e l y , a re o x id iz e d . T his l a s t a p p ea rs t o be th e r e a c t i o n most dependent on th e c o n c e n tra t i o n o f phosphate a c c e p to r ( 8 1 ) . A v a r i e t y of a g en ts have been shown t o uncouple p h o s p h o ry la tio n 40 from o x id a tio n * These su b sta n c e s in c lu d e v a rio u s n i t r o - and h a lo - p h e n o ls, a z id e and g ra m ic id in * They e f f e c t a d e c re a se i n th e r a t i o o f phosphate bound o r g a n ic a ll y t o oxygen consumed and g e n e r a lly , in v iv o , produce an o v e r - a l l in c re a s e i n oxygen consum ption* Some o f th e s e a g en ts cause m ito c h o n d ria t o h y d ro ly ze ad en o sin e tr i p h o s p h a te (81) . Lardy and h i s co -w orkers have s tu d ie d th e e f f e c t of v a rio u s th y r o - a c t i v e su b sta n c e s i n u n co u p lin g o x id a tiv e p h o s p h o ry la tio n in v i t r o (82) • At a c o n c e n tr a tio n of ICf® M th y ro x in e was found t o d e p re ss phosphate up tak e in a r a t k id n e y m ito c h o n d ria l system o x id iz in g glutam ate t o s-uccinate ( in th e p re sen c e of m alo n ate) • ,Some de p r e s s io n of r e s p i r a t i o n was s e e n i n t h i s system * T r iio d o th y r o n in e , i n s i m i l a r c o n c e n tr a tio n s , was fo u n d t o d e p re ss p h o s p h o ry la tio n s p e c i f i c a l l y . W ith th e m ito c h o n d ria of guinea p ig k id n e y , b u t n o t of r a t kidney o r l i v e r , tr i io d o th y r o n in e was o b serv ed t o in c r e a s e th e r a t e o f oxygen consum ption i n th e absence of ph o sp h ate a c c e p to rs * T h y ro x in e, tr i io d o th y r o n in e and t h e i r horm onal analogues w ere found t o a c c e le r a te th e breakdown o f adenosine tr ip h o s p h a te by m ito c h o n d ria . L i t t l e c o r r e l a t i o n was found betw een t h i s e f f e c t and in v iv o a c t i v i t y . D -th y ro x in e , L -th y ro x in e and L - tr iio d o th y r o n in e were found t o have eq u al a c t i v i t y . The a c r y l i c a c id analogue o f th y ro x in e was as a c ti v e as th e s e compounds • Lardy has th e o r iz e d t h a t -thyroid hormone r e g u la te s m e ta b o lic r a t e by v a ry in g th e e f f i c i e n c y o f o x id a tiv e p h o s p h o ry la tio n * I f th e u n co u p lin g of p h o s p h o ry la tio n occurs j u s t p r i o r t o a r a t e - l i m i t i n g r e a c t i o n i n a m e ta b o lic seq u en ce, i t is p o s tu la te d t h a t th e o v e r - a l l p ro cess w i l l be a c c e le r a te d and th e t o t a l number of h ig h en erg y p h o sp h ates form ed w i l l be in c re a s e d * T his o ccu rs a t th e expense o f I | e f f i c i e n c y , a~ p o in t which i s c o n s is te n t w ith th e known h ig h energy i I | c o st o f work perform ance by h y p e rth y ro id s u b je c ts * E x c e ssiv e doses i o f hormone e x e r t t h e i r to x ic e f f e c t s by u n c o u p lin g a t o th e r , le s s s p e c i f i c , r e a c t i o n s , a c c o rd in g t o t h i s t h e o r y . O ther w orkers have been unable t o f i n d an u n c o u p lin g e f f e c t w ith th y ro x in e o r t r i io d o th y r o n in e (83) • C o n firm ato ry r e s u l t s have been re p o r te d by Hoch and Lipmann (84) and by M artiu s and Hess ( 8 5 ) • Bain (86) has r e p o r te d t h a t p r e in c u b a tio n in c r e a s e s th e u n c o u p lin g e f f e c t of th y ro x in e and tr i io d o t h y r o n i n e when s u b s t r a t e i s su b se q u e n tly added* U ncoupling was re v e rs e d by in c r e a s in g th e magnesium c o n c e n tra tio n * Brody (87) concluded t h a t u n c o u p lin g may n o t be r e l a t e d t o t h e normal a c t i o n of th y r o id hormone b u t may be im p lic a te d T i n th e to x ic e f f e c t s o f l a r g e r d o s e s . L ehninger h as c o n tr ib u te d much work t o t h i s f i e l d (88, 89)* He has d e s c rib e d th r e e l e v e l s of o r g a n iz a tio n a t w hich p h y s io lo g ic a l c o n tr o l can be e x e r te d on m ito c h o n d ria l f u n c tio n s • These c o n tr o l l e v e l s a r e , f i r s t , a t a p a r t i c u l a r enzyme, by v a ry in g such f a c t o r s as c o n c e n tr a tio n s o f s u b s t r a t e s , p ro d u c ts or c o fa c to r s * The second l e v e l in v o lv e s th e i n t e r n a l geom etry o f th e m ito c h o n d ria . The m ultienzym e system s a re a p p a r e n tly arran g ed in a s p e c i f i c p a t t e r n ; s l i g h t changes in t h i s p a t t e r n m i g h t 'a f f e c t m e ta b o lic r a t e . A lte r a tio n s i n th e sem iperm eable membrane s u rro u n d in g th e m ito - 42 ch o n d rio n r e p r e s e n t th e t h i r d l e v e l of c o n tr o l (89) • j L ehninger and T ap ley have s tu d ie d th e e f f e c t s of th y r o i d hormone on th e membrane p e r m e a b ility and i n t e r n a l s t r u c t u r e o f th e | | m ito c h o n d ria . They confirm ed e a r l i e r fin d in g s t h a t th y ro x in e does i I no t produce an im m ediate u n c o u p lin g o f o x id a tiv e p h o s p h o ry la tio n in a r a t l i v e r m ito c h o n d ria p r e p a r a t i o n . They w ere a b le t o d em o n strate an immediate e f f e c t , how ever, i f th e m ito c h o n d ria w ere f i r s t in c u b a te d in h y p o to n ic s u c ro se and th e medium was low i n magnesium io n c o n ce n tra - ; t i o n . F u r th e r work showed t h a t spontaneous s w e llin g o ccu rred when m ito c h o n d ria were exposed t o s l i g h t l y h y p o to n ic s u c r o s e . T his s w e ll in g was p re v e n te d by a v a r i e t y of a g e n ts , i n c lu d in g ad en o sin e t r i p h o sp h ate , magnesium and manganous io n s and c i t r a t e . Calcium io n was | found t o promote s w e lli n g . D in itro p h e n o l and d ic u m a ro l, a t c o n c e n tra ti o n s w hich c o m p le te ly uncouple o x id a tiv e p h o s p h o ry la tio n , i n h i b i t e d m ito c h o n d ria l s w e llin g , w h ile g ra m ic id in , a t u n c o u p lin g c o n c e n tr a tio n s , d id n o t a f f e c t s w e llin g . Thyroxine and tr i io d o th y r o n in e promoted s w e llin g and d id so even a t a c o n c e n tr a tio n (10“ m) a t w hich th e y d id n o t u n c o u p le . The s w e llin g produced by th y ro id hormone was p re v e n ta b le w ith magnesium, a d e n o sin e tr ip h o s p h a te or d in itr o p h e n o l (89) . I t w a s ’d i s c o v e r e d ,1 in L e h n in g e rfs la b o r a to r y , t h a t d i g i t o n i n e x t r a c t s . o f r a t l i v e r m ito c h o n d ria c o n ta in e d an enzyme system cap ab le o f o x id iz in g J3-hydroxybutyrate t o a c e to a c e ta te w ith an o bserved PsO r a t i o o f 2 .8 when phosphate and ad en o sin e d ip h o sp h ate w ere ad d ed . T his enzyme complex was found t o be i n s e n s i t i v e t o p ro lo n g ed exposure 43 t o d i s t i l l e d w a te r , s u g g e s tin g i t is n o t surro u n d ed by a sem i- perm eable membrane. D in itro p h e n o l and o th e r a g e n ts which i n h i b i t m ito c h o n d ria l s w e llin g , and g ra m ic id in , were found t o uncouple th e o x id a tiv e p h o s p h o ry la tio n o f t h i s com plex. T h y ro -a c tiv e su b sta n c es ( th y r o x in e , tr i io d o th y r o n in e and t h e i r a c e t i c a c id a n a lo g u e s) had no u n co u p lin g e f f e c t ( 8 9 ) . These f in d in g s i n d ic a te th e p o s s i b i l i t y t h a t th e u n co u p lin g I j e f f e c t of th y r o id hormone i s secondary t o an e f f e c t on m ito c h o n d ria l i 1 ■ s t r u c t u r e or membrane p e r m e a b il ity . L ehninger co n clu d es t h a t i t is i I prem ature t o a c c e p t th e u n co u p lin g a c tio n o f th y r o i d hormone as i t s p h y s io lo g ic a l r o l e . However, i t is p o s s ib le t h a t , a lth o u g h i t i s a k seco n d ary e f f e c t dependent on a l t e r a t i o n s i n m ito c h o n d ria l s t r u c t u r e , u n co u p lin g o f o x id a tiv e p h o s p h o ry la tio n i s th e mechanism b y w hich th y r o id hormone e x e r ts i t s r e g u la to r y e f f e c t on c e l l u l a r m e ta b o lism . There i s as y e t i n s u f f i c i e n t evid en ce t o d e c id e on th e im por ta n c e of th e d e r i v a t i v e s of th y r o x i n e . I t c an n o t be s a i d d e f i n i t e l y t h a t th e known m e ta b o lite s of th y ro x in e and t r i i o d o t h y r o n i n e f u n c tio n p h y s io lo g ic a ll y t o a s i g n i f i c a n t e x t e n t . These s u b sta n c e s a re I g e n e r a lly much le s s a c ti v e Ln v iv o th a n t h e i r p a re n t horm ones. T riio d o th y ro n in e i t s e l f cannot be given a d e f i n i t e p h y s io lo g ic a l r o l e . A p p a ren tly more a c ti v e th a n th y ro x in e on a m olar b a s i s , i t s p re sen c e as a s i g n i f i c a n t s e c r e t o r y p ro d u ct of th e th y r o i d gland i s as y e t un c e r t a i n , a lth o u g h r e c e n t work te n d s t o e s t a b l i s h t h i s p resen ce ( 9 0 ) . T r iio d o th y ro n in e has been v a r i a b l y found as a m e ta b o lite of th y ro x in e b o th in v iv o and i n v i t r o * B arker has found i t p re s e n t in t h e I 4 4 A lb rig h t r a t k id n ey s l i c e system i n c o n s i s t e n t l y , w ith a more u su a l appearance o f t e t r a i o d o t h y r o a c e t i c a c id (69) • | The d i f f e r e n t in v iv o a c t i v i t i e s o f th e s e compounds m ight be due t o d if f e r e n c e s i n such f a c t o r s as a b s o r p tio n , c e l l u l a r p e rm e a b ility * and r a t e s o f d e s t r u c t i o n . T h e ir i n v i t r o a c t i v i t i e s appear t o be more s im ila r t o each o t h e r . A ttem pts t o c o r r e l a t e t h y r o - a c t i v i t y w ith i m o lecu lar s t r u c t u r e have been based on in v iv o a c t i v i t y . I t would seem t h a t th e more v a l i d system f o r t h e d e te r m in a tio n o f th y r o - a c t i v i t y is th e in v i t r o , u t i l i z i n g th e as y e t unknown (o r unproven) ! I s im p le s t sy stem w hich responds t o th y r o id horm one. I n th e a ssa y system w hich most c lo s e ly approxim ates t h i s to d a y (m ito c h o n d ria l un c o u p lin g and th e more r e f in e d m ito c h o n d ria l s w e llin g s y ste m ), th y r o x in e , tr i io d o th y r o n in e and t h e i r d e r i v a t i v e s s tu d ie d t o d a te a re e q u a lly a c tiv e • Q u a n tita tiv e d e te rm in a tio n s of d if f e r e n c e s in in v i t r o a c t i v i t y , i f such d if f e r e n c e s e x i s t among th e t h y r o - a c t i v e su b sta n c e s found in t i s s u e , may depend on th e f u t u r e i s o l a t i o n o f more s e n s i t i v e a ssa y s y s te m s , These system s w ould presum ably be more l i k e l y t o in v o lv e th e p h y s io lo g ic a l mechanism th ro u g h w hich th y r o id hormone f u n c t i o n s . I t i s p ro b a b ly s i g n i f i c a n t t h a t th e u n c o u p lin g o f o x id a tiv e p h o s p h o ry la tio n in v i t r o occurs a t th y ro x in e c o n c e n tra t i o n s about 104 tim e s g r e a te r th a n found i n t i s s u e s (9.1) • There i s o b v io u sly much work t o be done b e fo re th e p e r i p h e r a l l y a c ti v e form o f th y r o id hormone and i t s s i t e and mechanism of a c tio n a re known. The d a ta c o lle c te d in th e p re s e n t work do n o t su p p o rt th e c o n te n tio n t h a t th y ro x in e m ust be d e io d in a te d t o t r i io d o th y r o n in e t o e n t e r th e c e l l s and e x e r t a horm onal e f f e c t * T hyroxine i s g e n e r a lly p re s e n t i n t i s s u e s i n l a r g e r amounts th a n any o f i t s d e r i v a t i v e s and may w e ll be th e most S i g n i f i c a n t th y r o id hormone t o th e body economy* I t s d e r i v a t i v e s do, how ever, ap p ear t o be e q u a lly a c tiv e on a m olar b a s is in v i t r o , and i t i s p ro b ab le t h a t , in B a r k e r’s w ords, w we should c o n sid e r a g alax y o f th y r o - a c tiv e m a te r ia l r a t h e r th a n a s in g le m a t e r i a l 1 * ( 9 2 ) . V . .SUMMARY i S tu d ie s w ere made on th e ap pearance of tr i io d o th y r o n in e d e riv e d from th y ro x in e i n human serum and v a rio u s r a t t i s s u e s under d i f f e r e n t c o n d i t i o n s • The e f f e c t s of e p in e p h rin e on t h i s c o n v e rsio n w ere observed s in c e i t has been r e p o r te d t h a t t h e c a l o r i g e n i c i t y of e p in e p h rin e i s th y r o id - d e p e n d e n t« The e f f e c t s o f exposure t o co ld and o f m ajor s u rg e ry were a ls o determ in ed * T his work was d e sig n ed t o in d ic a te i f s t r e s s and in c re a s e d c a l o r i c need a re accompanied by an in c re a s e d fo rm a tio n of t r i io d o th y r o n in e from th y ro x in e * T r iio d o th y ro n in e and o th e r , u n i d e n t i f i e d , d e r i v a t i v e s of th y ro x in e w ere found in some human serum and r a t t i s s u e samples « A fte r e p in e p h r in e , th y ro x in e alo n e was g e n e r a lly found i n th e serum* T here was no in c r e a s e i n th e t i s s u e c o n c e n tr a tio n o f t r i io d o th y r o n in e a f t e r e p in e p h r in e , s u rg e ry or exposure t o co ld* The c a ta b o lis m of th y ro x in e was in c re a s e d by e p in e p h rin e a d m in is tr a tio n * I t is p o s tu la te d t h a t e p in e p h rin e e f f e c t s an in c re a s e d c e l l u l a r p e r m e a b ility to th y ro x in e and i t s d e r i v a t i v e s * The s i g n i f i cance o f th e v a r io u s t h y r o - a e t i v e su b sta n c e s i s d is c u s s e d i n th e l i g h t o f r e c e n t in v iv o and in v i t r o work* ACKN CW LED GEM ENT S 1 . 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Biochem . B io p h y s., 33, 486 (1951) |86. B ain, J . A ., J . P h a rm a c o l. E xp. T h e ra p ., 110, 2 (1954) i j87 • Brody, T . M ., P h a rm a c o l. R ev ., 7, 335 (1955) 8 8 . L e h n in g er, A . L ., H arvey L e c tu r e s , 49, 176 (1953-54) 8 9 . L eh n in g er, A . L ., in H enry Ford H o s p ita l I n t e r n a t i o n a l Symposium on Enzymes ( 0 . H . G aeb ler, e d . ) , page 217, Academic P r e s s , i ! _1956 ________________________ __________________________ ____ 54. 9 0 o M aclagan, N. P*, Biochem. J . , 67, 5 (1957) 9 1 . K lem perer, H . G ., Biochem. J . , 60, 122 (1955) 9 2 . B ark er, S • B ., in Ciba F o u n d atio n C o llo q u ia on E n d o crin o lo g y , 10, 1957, page 178 A P P E N D I X T A B IE I C H R O M A T O G R A P H I C A N A L X S 3 5 O F HUM S E R U M A F T E R T H Y R O X IH E -I 1 ^ 1 .Patisnt Condition , Thyroxine T im e C hrom atographic com ponents in percent of radioactivity recovered Butanol-dioxane - N H 3 Tert. Anyl Ale. - N H 3 T x TrlT S. F,1 • T x TrlT S. F. 1 Athyreotic, euthyroid ' 260 p s} 7 jigm . 3, 7, 2 1 1 Hrs. 100 2 M yx ed em a 3b 0 juej 15 jigm . 3.5> 2b Hrs. 100 3 Hyperthyroid 3b0 ps} 15 pm* 3.5, 2b Hrs. 100 b Athyreotic, euthyroid 2 1 * 0 pe*, U pm* 20 Mins. 9b.0 1.8 81 Mins. 100 2 Hrs. 100 5 M yxedem a, tuberculosis 200 jiej 11 ( jigm . 1 (2 M ins. 100 100 2.5 Hrs. 100 8b. 6 6.b 6.5 Ifrs* 100 100 9.0 Hrs. 5b.3' 2.9 19.7 b O .O 5.3 15.7 2b.5 Hrs. 100 100 6 Athyreotic, Hypothyroid, 95 p e > 6 ^gm . 30 M ins. 50.5 5.5 23.9 15.1 27.1 21.7 5.5 12.8 15*9 biliary pathology 18.5 Hrs. 32.6 16.3 28.1 9.8 29.b 18.8 b.5 9.2 28.2 150 ;acj 9 pm* 30 Mins. 87.7 2.9 2.5 Hrs. 92.0 2.0 b.75 Hrs. 72.1 2.0 b.9 7 Hyperthyroid 200 pe; 13 /ig m * 20 Mins. 67.7 15.6 56.6 3»b b.b 28.b 1.25 Hrs. 3.8 7b*0 6.b bb.5 16.3 3.25 Hrs. v 8.8 57.3 16.3 5.25 Hrs. 65-5 3.9 10.7 6.1 16.1 6.0 l.b 3 * .b b9.2 8 Athyreotic hypothyroid 200 jicj 13 pm* b0 Mins. 100 lb.7 38.8 2b.3 1.8 12.8 1.67 Hrs. 100 6.5 11.6 65.3 3.0 3.6 3.67 Hrs. 100 lb.7 22.1 50.0 2.0 b.8 5.67 Hrs. 100 b.6 27.2 65.9 5.1 12.1 9 Jaundice, i5Q pc; 9 pm* 30 Mins. 100 86.7 2.3 gastrointestinal cancer 2.5 Hrs. 100 1.8 86.5 1.5 b.75 Hrs. 100 86.7 2lt Hrs. 6.1 57.0 lb. 8 5.0 8.9 13.9 38.9 11.0 n .o 1 , / T x - thyroxine j TrH - triiodothyronine j S. F. - solvent front. Table TA BLE II 1^1 CH RO M A TO G RA PH IC ANALYSIS OF H U M A N SERUM AFTER THYROXINE-1 ADMINISTRATION TO SURGICAL CASES - P a tie n t Condition Thyroxine Time Chromatographic components in percent of r a d io a c ti v ity recovered Tx TrlT S .F . P r o s ta tic surgery 320 ^ c , 11 ;Agm, 5 Mins • 72 Gastro i n t e s t i n a l surgery 335 jict 13 jugm. 25 Mins • 8 89 2 H rs. 90 35 Mins • 10 86 1 H r. 10 81 5 Hrs • 94 24 H rs • 8 85 1 / Analyzed w ith butanol-dioxane system . T A B L E III C H R O M A T O G R A P H IC ANALYSIS O F H U M A N S E R U M A F T E R TH YRO XINE-1 ° A N D EPINEPHRINE P atien t thyroxine Epinephrine Epinephrine Time Chromatographio components in response % of r a d io a c tiv ity recovered Tx TrlT S «F« i h S / 500 p o , 29 0*3 M gjn. Pulse? 92+104 15 Mins* at 75 mins • Subjective 80 Mins • 5 *5 Hrs • 2 528 ^ 0 , 39 ;ugm* 0*6 M gm * Pulse: 68+100 24 Mins* at 25 mins* Blobd pressure: ISO 140 42 Mins * 80 66 Subjective 4*3 Hrs* 200 jio, 7 ;ugn. 0 .5 Mgn. P ulse: 64+80 9 Mins at 6 Mins* Oxygen uptake: No change 40 Mins Blood pressure: 120 . 138 84 Mins* Subjective 104 Mins• 200 7 /igm* 0.5 Mgn* Pulse: 80+100 6 Mins* at 5 m ins. Oxygen uptake: No change 38 Mins • Subjective 76 Mins* 103.Mins• 100 100 100 100 100 100 71.9 15.0 43.6 35 .6 85.4 4.1 70.4 8*2 73.4 10.9 80.7 4 .8 83.8 4 .9 84.3 3.0 Table XII (Continued) P atien t Thyroxine Epinephrine 240 p o 9 6 pga> 4 240 p o 9 6 p gpi. 0 #5 m gu.9 at 16 and 22 mins* 0.5 at 10 and 16 m ins« 236 p o , 11 ^igm. 1 .0 M gm * at 26 and 72 m ins. Epinephrine response Time Chromatographic components in % of r a d io a c tiv ity recovered Tx TrlT S .P. Pulse? 84-*120 Blood pressure? 110 160 IS'-"* “35 Oxygen uptake? Increased Pulse? 88*96 Blood pressure? 120 130 70'*' 60 Oxygen uptake? Increased Pulse? 70*98 Blood pressure? 112 132 Oxygen uptake? Increased 7 Mins • 79.2 13.9 43 M ins• 65.4 21.8 100 Mins• 81.4 1.8 149 Mins• 84.0 2.3 8 Mins * 92 .1 2 .4 29 Mins• 96.3 1.7 94 M ins. 92.2 2.1 165 Mins. 88.3 2 .7 22 M ins. 89.6 3.2 37 Mins. 90.4 2 .6 62 Mins• 89.3 2.5 83 Mins. 89.9 1.6 114 M ins• 89 .9 2.2 tn < o Table III (Continued) P atien t Thyroxine Epinephrine Epinephrine response Time Chromatographic components in % of r a d io a c tiv ity recovered Tx TrlT S.P# 5 250 jxo9 18 /xga# 1*0 Mga # Blood pressures 22 Mins# 76*8 2 .6 2.7 at 36 m ins• 156 163 T T o - * - ^ Oxygen uptakes 71 Mins# 74*4 3.5 7.6 Increased 141 Mins. 75 .1 3.9 2.2 6 250 18 jxgo.» 1*0 'M gpw . Pulses 78^104 24 Mins« Sample lo s t at 34 mins • Blood pressures 0*4 M gm * 111 168 49 Mins• 82 *6 2*5 at 66 m ins« 61 90 Oxygen uptakes 0*3 M gm # Increased at 93 mins • Subjective 141 Mins* 65.9 3 #8 Analyzed w ith butanol-dioxane system# A lb'other samples analyzed with te r t# amyl alcohol system# Thyroid cancer patient® 61 TABLE 17 TH E EFFECT O F EPINEPHRINE O N TH E SE R U M PROTEIN B O U N D IODINE Tim© a fte r P a tie n t Epinephrine epinephrine (mins •) P *B .1 . JJgm. % % change Serna p m 2 0*5 mgn. 3 7 .6 2 34 8 .0 + 5 4 78 7 .5 - 1 3 98 6 .9 - 9 3 3 Oo5 mgm* 1 7 .3 4 33 8 .5 +15 4 71 6.7 - 8 3 98 6 .1 -16 4 4 0*5 mgm* 0 7 .8 8 (tw ic e ) 19 , 13 6 .0 -2 3 10 84, 78 5 .8 -3 6 8 155, 149 - - 8 3 0 *5 "m gm * 0 6 .6 8 (tw ic e ) 2 7 , 21 - - - 84, 78 5 .8 -12 10 133, 127 5 .4 -1 8 8 5 1 *0 mgm • 0 7 .0 30 35 8 .0 +14 60 105 8 .2 +17 30 4 1 *0 mgm* 0 3 .4 4 (tw ic e ) 11 2 .4 -2 9 6 36 1 .8 -47 4 57 , 11 2 .0 -41 6 8 8 , 42 2 .0 -41 7 6 1*0 m gn. • 0 4 .2 30 0*4 mgm* 15 4 .6 +10 30 0*3 mgn. 105, 7 5 , 48 4 .6 +10 30 62 SERUM GLUCOSE OF GROUP IV RATS TABLE V Rat I n i t i a l glucose F in a l glu cose mg. % mg» % C ontrol 159 162 1 170 254 2 104 232 3 118 237 4 97 231 63 TABLE VI CHROM AT0 G R APHIC ANALYSIS OP RAT TISSUES FOLLCWINO 1 , 2/ ADMINISTRATION OF L-THYROXINE-I151 Group I - R a t 1 (2 h o u rs ) T is s u es Serum S k e l e t a l m u scle H e a rt S a liv a r y g la n d L iv e r K idney Tx 61 36 39 52 T rlT 2 5 6 9 S *F • 4 20 13 p o o r r e s o l u t i o n l o s t R a t 2 (5 h o u rs ) Tx 77 36 36 92 82 83 T rlT 1 9 9 3 4 10 S._F 2 13 17 l / A n aly zed w ith t e r t i a r y amyl a lc o h o l sy ste m 2/ F ig u r e s r e p r e s e n t p e rc e n ta g e o f r a d i o a c t i v i t y re c o v e re d TABLE VII C H R O M A T 0G R APHIC ANALYSIS O P RAT TISSUES FOLLOW ING 1 2 / ADMINISTRATION OF L-THYROXINE-I131 Group I I R at 1 (c o n tro l) R at 2 ( - 5 ° , 4 h r s .) R at 3 ( - 5 ° , 11 h r s .) T issu e Tx TrlT S JF'. Tx TrlT S JF. Tx T rlT S .F • Serum 44 1 31 62 1 20 47 24 H eart 40 21 33 4 15 54 S a liv a ry gland 42 37 70 4 15 62 6 17 L iv er 51 40 13 67 52 25 Kidney 58 29 50 26 73 7 l / A nalyzed w ith t e r t i a r y amyl a lc o h o l system 2/ F ig u re s r e p re s e n t p e rc en ta g e o f r a d i o a c t i v i t y reco v ered TABLE VIII CH RO M A TO G RA PH IC MALYSIS OF RAT TISSUES FOLLOW ING ADMINISTRATION O F L-THYROXINE-I151 A N D EPINEPHRINE Group I I I -■ R at 1 (2 h rs ») R at 2 (5 h rs> ) T issu e Tx TrlT S*F* Tx TrlT Serum 53 15 43 S k e le ta l L ost 46 7 m uscle H e art 62 68 L iv er L ost Poor r e s o lu tio n K idney 62 16 81 2 / A nalyzed w ith t e r t . amyl a lc o h o l system® 2/ F ig u re s re p re s e n t p erc en ta g e o f r a d i o a c t i v i t y reco v ered * T A B L E IX CHRO M A TO G RA PH IC ANALYSIS OF RAT TISSUES FOLLOW ING ADMINISTRATION O F L-THYROXINE-I131 A N D EPINEPHRINE Group IV R at 1 R at 2 T issu e Chromatog* system Tx tr I T S .F . Tx TrIT S J . Serum T e rt • amyl 89 82 2 BuOHjDiox. 81 82 S k e le ta l T e rt • amyl 71 2 33 6 7 7 m uscle BuOHjDiox. 95 3 77 2 H eart T e rt • amyl 50 10 54 1 14 BuOHjDiox. 44 36 7 8 40 17' 3 10 S a liv a ry T e rt • amyl 34 10 7 35 43 11 12 gland BuOHjDiox. 43 7 12 Lost L iv er T e rt • amyl 69 5 52 BuOHjDiox • 45 31 29 33 9 Kidney T e rt • am yl• 24 59 5 4 69 6 5 78 7 45 19 12 8 TA BLE IX (Continued) Group IV R at 3 . R at 4 T issu e Chrom atog. system Tx TrIT S .F . Tx TrIT S *F. Serum T e rt • amyl 86 90 2 BuOHjDiox • 80 73 4 S k e le ta l T e rt • amyl 23 28 5 4 62 6 5 m uscle BuOHjDiox • Poor r e s o lu tio n 21 49 4 H eart T e rt • amyl Low a c t i v i t y 66 1 24 BuOHjDiox. 13 16 3 21 24 22 3 21 S a liv a ry T e rt • amyl 60 7 9 3 31 8 28 gland BuOHjDiox• 62 5 11 Low a c t i v i t y L iv er T e rt • amyl 73 2 2 41 2 BuOHjDiox* 7 59 25 4 4 Poor r e s o lu tio n Kidney T e r t • amyl 54 2 4 7 64 2 8 BuOHjDiox* 20 17 2 38 32 26 37 4 14 y F ig u re s re p re s e n t p ercen tag e of r a d io a c ti v ity reco v ered * 2/ R ats 1 and 2 m ain tain ed on th y ro x in e * 68 3 2 3 2 - Q ° © CHg .CHNH2 «C02 H T h y r o n in e 5 6 5 6 HOC >OC > CH2 #CHNH2 .C 02H T h y r o x i n e ( T x ) HO o ° o CH2 .CHHHg .C 02H 3 , 5 , 3 * - T r i i o d o t h y r o n i n e ( T r I T ) I HO C H 2 tCEg .C O gH I I T e t r a i o d o t h y r o p r o p i o n i c a c i d ( d e a a m l n o t h y r o x i n e , t e t r a p r o p ) I I b d / N o / N ch2 ,ch2 . c o ^ T r i i o d o t h y r o p r o p i o n i c a o i d ( t r i p r o p ) I I h o <P°q ch2 *co2 h T e tra io d o th y ro a c e tic aoid ( te tr a o ) I I HO O O CHj vCOgH T r i i o d o t h y r o a c e t i c a c i d ( t r i & c ) “O C - 0 -(C H 2 ) j CH3 B u t y l - 3 , 5 - d i i o d o - 4 - h y d r o x y b e n z o a t e (BDHB) F I G . 1 . S t r u c t u r a l f o r m u l a s o f so m e d e r i v a t i v e s o f t h y r o n i n e C t s ./W n . above bkgrnd • Cts*/^nin* above bkgrnd* 600 v c In V iv o , H eart TxtTrIT - 6 0 i l 200 o £ 100 o r ig in 600 400 Human Plasm a TxtTrIT ° 24*1 200 2 t & < B c 100 20 o r ig in D ista n c e (c m s .) 300 200 TxtTrIT - 1 4 :1 1Q 0 S J , o r ig in D ista n o e (e m s•) 600 200 In V iv o , S k e le t a l m usole TxtTrIT ■ 6 0 t l 100 H o r ig in D ista n o e (c m s.) P ig * 2 » R eoovery e x p e r im e n ts . o > c o C ts.A iin. above bkgrnd 10,000 7 ,5 0 0 1,200 20 Mins 800 600 o r ig in % « 1Q 0 B 4 » U o r ig in S .F D ista n o e (oms b 100 122 M in s. o r ig i n 8 10 Distanoe (oms.) *5 &JP* F ig . 3 . P atien t 4 . A thyreotio, eu thyroid. Amyl aloohol ohromatography. O n •e c 4 0 0 & © t x > © 0 ,6 7 H ours 200 -p o o r i g i n 10 20 D i s t a n c e (om s *) S . F . • tj c h M ja © % * 200 6 ,5 H ours 100 • H C O -P o 20 o r i g i n D i s t a n c e ( o m s ,) F i g , 4 , P a tie n t 6 , Myxedema C t s ,/ m i n , above b k g r n d , C t s ./ m i n , above b k g rn d 100 2 ,5 R o u rs .5 0 o r i g i n D i s t a n c e ( c m s .) S «F o 2. 0 9 , 0 H o u rs U O 10 20 D is t a n c e ( o m s , ) o r i g i n S . F . -a h -1 - Amyl a lc o h o l chrom atography. Cts . / m i n • above b k g r n d 30 M ins 4 0 30' o r i g i n S .F D is t a n o e ( c m s .) 1 8 .5 H rs • fi 4 0 o r i g i n 10 20 D is t a n c e (era s# ) S J . F i g . 5 . P a t i e n t 6 . B i l i a r y p a t h o lo g y . Amyl a l o o h o l c h r o m a to g r a p h y # Cts ,/m in • above b k g rn d 100 30 M ins 4 0 o r i g i n S .F D is t a n o e (o m s* ) 18 *5 H rs • c k b x & £ o & C D • C C O -p o 10- D is t a n o e (c m s* ) 20 30; o r i g i n F i g . 6 . P a t i e n t 6 . B i l i a r y p a t h o lo g y . B u t a n o l-d io x a n e c h r o m a to g r a p h y . -a C A Cts ./s e o • above b k g r n d . Cts . / s e c . above b k g r n d 120 ao 6 I/*ins S . F . D is t a n c e ( c m s .) © > o X > c t J 38 W ins © ® CO ( 0 -P o o r i g i n D is t a n c e (cm s .) 7 0 76 w in s S .F D i s t a n c e (om s .) © Z 3 0 © . o © 03 . to -P o o r i g i n SoP 131 F ig . 7 . P a tie n t 3, th y r o x in e -I and e p in e p h r in e . Amyl a lc o h o l chrom atography. - ■ 3 C t s ./m in . above bkgrnd* Cts ./s e c • above bkgrnd 100 50 *o 40 29 Mina « W O 10 o r ig in Distanoe (om s*) 60 50 4 0 40 94 Mins 104 Mins 20 t> o r ig in o r ig in Distance (o m s.) 131 F ig . 8 . P a tien t 4, thyroxine-I end ep in ep hrin e. Amyl alcohol chromatography. -3 O l Cts »/mi n • 9bove b k g r n d . 46 30 15 S k e l e t a l m u scle M * o r ig i n 10 tO D is ta n c e (c m s .) * 20 tj a « n I £ > ® 10 e £ fy S a liv a r y gland J1 u t- O u u v L r T - ~ o r ig i n 10 $0 D istanoe (oms.) 8J . 8,000 6,000 I 2.°00 £ o fi « c Serum 1,000 £ o r ig in 7 £ I Jg 6 0 H eart i— o r ig i n 10 20 D ista n o e (oms •) r v T | S .F . F ig . 9 . Group I , Rat 1 , Amyl alcohol ohromatography. 05 C t s ./m in . above bkp*nd. C ta ./m in . above b k g r n d . 1,800 L iv e r 4 0 0 .200 o r ig i n D is ta n o e (o m s•) 160 100 60 S a liv a r y gland I o r ig i n 10 20 D istanoe (om s.) S .F , *46 fc b -30 15 o r ig i n K idney 10 20 D ista n o e (o m s•) 3 0 , S J . c H eart b > I & 0 . a SO M o r ig in S .F P ig . 1 0 , Group I , Rat 2 , Amyl alcohol ohromatography, — 5 C t s ./m in . above b k grn d* Serum 800 4 00 o r ig i n D is ta n c e (o m s•) 1-50 c S k e l e t a l m u scle 100 a > > o & < s c 50 2 t) D ista n o e (om s*) o r ig in 50. 50 S a liv a r y gland 10 o r i s in 10 20 D ista n o e (om s*) S *F • fo 3 ,2 0 0 1 ,6 0 0 o r ig in K idney 10 20 Distanoe (oms •) T 8*F. F ig . 1 1 . Ooup TV, Rat 1 . Amyl aloohol ohromatog*aphy. 0 0 Cts ./m in * above bkgrnd# Cts . / s e c . above b k g r n d 50. 4 0 Serum 20 ) 20 D i s t a n c e ( c m s . ) o r i g i n S .F 8 0 S a l i v a r y g la n d 4 0 20 J S i S .F o r i g i n D is t a n o e (c m s# ) 3 00 ® o <0 c S k e l e t a l m u s o le 200 to • P o 100 M o r i g i n D is t a n o e (c m s# ) • o c tb > S » 0 © ( ► o x > 2*000 1,000 K id n e y 1 K E h U h . . 10 20 o r iSin D istanoe (cm s.) S . F . ^ 3 F ig . 1 2 . Group IV, Rat 1# B u tanol-dioxane chrom atography. 50 C t s . / m i n . above b k g r n d . Cts . / m i n . above b k g r n d 40 S e r u m 3 0 10 - D i s t a n c e ( c m s . ) o r i g i n S J H e a r t 200 7 0 S J . o r i g i n D i s t a n c e ( c m s . ) P ig, IS, Group TV, Rat 2 . C t s . / m i n . above bkgjrnd . Cts ./m in • above b k g r n d . 40 S k e l e t a l m u s c l e 20 M S . F o r i g i n D i s t a n o e ( c m s . ) 8 0 0 K id n e y .100 10 20 D i s t a n c e ( o m s . ) S J . o r i g i n Amyl a lc o h o l ch rom atograp h y* o o O Cts ,/n in • above b k g r n d , Cts , / s e o » above b k g r n d 4 0 S e r u m 20 o r i g i n S J D i s t a n o e (o m s * ) 100 H e a r t .4 0 20 20 o r i g i n D i s t a n c e ( c m s . ) F ig , 14, Group IV, Rat 2 . C t s . / m i n , above b k g r n d , Cts ./m in * above b k g r n d . 8 0 S k e l e t a l m u s c l e 4 0 S J . o r i g i n D i s t a n c e (o m s • ) 4 0 K id n e y S J . o r i g i n D i s t a n o e (c m s Butanol-dioxane chromatography. o o C t s ./4 n in • above bkgrnd* Cts . / s e c . above b k grn d 150 100 L iv e r 50 _ S b y = jr r D * T~. 10 20 o r i g i n D is t a n c e (c m s .) S J . 50 0 Salivary gland 300 100 20 S J o r i g i n D is t a n c e (o m s.) 40 a b > Seruui ► o at 20 o a t b a 4 > O o r ig i n S J . D is t a n c e ( c m s .) • o a K id ney o (D CO o r ig i n S .F D is t a n c e ( c m s .) F ig . 1 5 . Group IV, Rat 3 . Amyl alcoh ol chromatography. 60 L iv e r T D c & 4 0 m £ > © > o £ > OS u © to 20 20 o r ig i n D is t a n o e (cms S a l i v a r y g la n d « 100 o o r ig i n F ig . 1 6 . Group IV, Eat 3 . C ts ./see . above bkgrnd. C ts ./s e c . ebove bkgrnd H ea r t 30 2Q 10 o r ig i n Distance (cms «) 20 K id ney 10 20 D istance (cms .) o r ig i n B utanol-dioxane chromatography. 0 0 0 3 C t s . / m i n . above b k g r n d . C t s . / m i n . above b k g r n d . H e a r t o r i g i n S «F • D i s t a n c e ( c m s .) 100 Salivary gland 6 0 20 o r i g i n S . F . D i s t a n c e ( c m s . ) . 200 c lb * I S 100 c S k e l e t a l m u s c le a r t e £ M origin S J D i s t a n c e (om s • ) 1 4 0 0 Kidney •° 6 0 0 200 u origin D i s t a n o e ( o m s .) F i g . 1 7 * G roup I V , R e t 4 . Am yl a l o o h o l o h r o m a to g y a p h y • o o C t s . / m i n . above b k g r n d . Cts . / s e c . above b k g r n d 10 H e a r t or i g in D i s t a n c e (cm s . ) 4 0 S k e l e t a l m u s c le 20 20 S J . o r i g i n D i s t a n c e ( o m s . ) •o c kb X > 20 S eru m c £ > « O © m x C 3 C - « 20 o r i g i n D i s t a n o e (om s .) K id n e y tb 4 0 0 o r i g i n 10 20 D i s t a n c e ( c m s .) S J F i g . 1 8 . Group IV , Rat 4 . B u ta n o l-d io x a n e ch rom atograp hy. CD • C J l 86 6 0 Q 8 4 0 w g > c w w 8 30 a 10 0 2 4 1 4 4 72 120 9 6 4 8 HOURS F i g * 1 9 . U r i n a r y e x c r e t i o n o f ^ ® , u r i n a r y a f t e r t h y r o x i n e a l o n e . > £| * u r i n a r y l ! 3 1 a f t e r t h y r o x i n e and e p i n e p h r i n e . 87 6 0 4 0 < £ 6 B « 8 a I B 10 120 1 4 4 72 9 6 4 8 2 4 0 H O U R S F i g . 2 0 # F e c a l e x c r e t i o n o f f e c a l 1 * ^ a f t e r t h y r o x i n e • f e c a l 1 ^ 3 1 a f t e r t h y r o x i n e and e p i n e p h r i n e . Gaiversity of Southern GaliSotsSl

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Asset Metadata

Creator Kallman, Burton J (author)

Core Title Studies on the in vivo metabolism of thyroxine

Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-1785

Unique identifier UC11348180

Identifier DP21582.pdf (filename),usctheses-c17-1785 (legacy record id)

Legacy Identifier DP21582.pdf

Dmrecord 1785

Document Type Dissertation

Rights Kallman, Burton J.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA