The effect of thyroxine on the rate of incorporation of P32 into the phosphorus-compounds of rat diaphragm in vitro

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Content th e e f f e c t o f th y ro x in e on th e r u e o f in c o rp o ra tio n o f p 3 2 INTO THE PHOSPHORUS "•C O M PO U N D S OF RAT DIAPHRAGM in v itr o A T h esis P re se n te d to th e F a c u lty o f th e D epartm ent o f B io ch em istry U n iv e rs ity o f S o u th ern C a lif o r n ia School o f M edicine In P a r t i a l F u lfillm e n t o f th e R equirem ents f o r th e Degree M aster o f S cien ce ■William C Ja n u a ry 1948 i/werkheiser UMI Number: EP41287 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. UMI EP41287 Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. Dissertation: ftb fe tiiig 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 7 tA j j thesis, written by , ................ WILLIAM CLABK WEBKHIISEB...................... under the guidance of h.is ... Faculty Committee, and a p p ro ved by all its m em bers, has been presented to and accepted by the Council on Graduate Study and Research in partial fulfill ment of the requirements for the degree of M ASTER OF SCIENCE Secretary D ate.... JAN.UARX...194.a. Faculty Committee iC K W C W LSIO JEirPS I w ish to e x p re ss my g r a titu d e to Dr* R ich ard J . W in zler f o r h is i n t e r e s t and su g g e stio n s about th e work. I w ish a ls o to th an k th e E li L i l l y Company f o r making t h i s work p o s s ib le . TABLE OF CONTENTS CHAPTER PACE I , INTRODUCTION ...................... * * 1 I I . HISTORICAL REYIEU........................... 3 I I I . PROCEDURE AM) M ETH O D S ........................ 7 A/ T reatm ent o f Animals . . . . . . . . . . . . . . 7 B. In c u b a tio n Media ..................... 7 C. Removal o f T issu e and In c u b a tio n . . . . . . . . 9 D» F r a c tio n a tio n o f T issu es . . . . . . . . . . . . 11 E. A n a ly sis f o r P 3 2 and P 3 1 ....................................... 16 F . H andling o f R e su lts • 18 G. C o n sid e ra tio n o f E rro rs . . . . . . . . . . . . 18 IF . EXPERIMENTAL DATA ........................... 20 ¥ . DISCUSSION ........................... . . . . . . . . . . . . . . 32 ¥ 1. SU M M A R Y ...................................................33 B ib lio g rap h y 34 LIST O F TABLES TABLE PAGE I . COMPOSITION O B ’ STOCK D IE T ........................................................................ 8 I I . COMPOSITION OF INCUBATION MEDIA....................................................... . 10 I I I . PHOSPHORUS METABOLISM IN DIAPHRAGM S’ FROM NO RM A L, HYPOTHY* ROID, A N D HYPERIHYROID RATS—P3 2 Uptake by P hosphorus- C o n tain in g Compounds 25 IV. PHOSPHORUS METABOLISM IN DIAPHRAGM S IROM NORM AL, HYPOTHY ROID AN D HYPERTHYROID RATS—Phosphorus D is tr ib u tio n in P h o sphorus-C ontaining Compounds . . . . . . . . . . 24 V. THE EFFECT OF THYROXINE in v i t r o O N PHOSPHORUS METABOLISM OF DIAPHRAGM S FRO M RATS TREATED WITH THIOURACIL—P3 2 U ptake by P hosphorus-C ontaining Compounds . . . . . . 26 V I. THE EFFECT OF THYROXINE in v i t r o ON PHOSPHORUS METABOLISM OF DIAPHRAGM S FRO M RATS TREATED WITH THIOURACIL— Phosphorus D is tr ib u tio n in P h o sp h o rus-C ontaining Com pounds . . . . . . . . . . . . . . . . . . . . . . . . . 27 V II. THE EFFECT OF THYROXINE in v itr o O N PHOSPHATE METABOLISM IN DIAPHRAGM S OF THIOURACIL-TREATED RATS PREVIOUSLY TREATED WITH P3 2 —P 3 2 T urnover in P h o sp h o ru s-fto n tain - in g Compounds O ccurring during In c u b a tio n . . . . . . 29 V III. THE EFFECT OF THYROXINE in v i t r o ON PHOSPHATE METABOLISM W DIAPHRAGM S OF THIOURACIL-TREATED RATS PREVIOUSLY TREATED WITH P32—Changes in Phosphorus D is tr ib u tio n in P h o sp h o rus-C ontaining Compounds O ccurring d u rin g In c u b a tio n 3 0 CHAPTER I INTROIXJCTIC® The e f f e c t s o f th y ro x in e , h y p e rth y ro id ism , and hyp o th y ro id ism on anim als and on tis s u e s h as been th e s u b je c t o f many in v e s tig a tio n s over th e p a s t f i f t y y e a r s . I t i s w ell-know n, f o r in s ta n c e , th a t th e le v e l o f th y ro id a c t i v i t y i s la r g e ly re s p o n sib le f o r th e le v e l o f r e s p ir a to r y m etabolism o f th e whole body and o f is o la te d t i s s u e s . More r e c e n tly , i t h as been shown by many in v e s tig a to r s t h a t a d m in is tra tio n o f th y ro x in e in c re a s e s th e c o n c e n tra tio n o f r e s p ir a to r y enzymes in th e t i s s u e s . I t h as lik e w is e been shown, s t i l l more r e c e n tly , t h a t a d m in is tra tio n of th y ro x in e e f f e c t s changes in o th e r m etab o lic fu n c tio n s such as p ro te in s y n th e s is , th e up tak e o f phosphorus by t i s s u e s , and th e a b so rp tio n o f g lu co se from th e i n t e s t i n a l t r a c t . I t i s v e ry p ro b ab le th a t th e s e - e f f e c ts a re r e f l e c t i o n s o f th e in c re a s e d m etabolism o f th e tis s u e s in v o lv e d . There i s a ls o a sm all amount o f work which perhaps in d ic a te s th a t e i t h e r th e th y ro id hormone o r th y ro x in e i s a b le to cause an in c re a s e in t i s s u e r e s p ir a tio n in v i t r o . . However, none o f th e evidence as y e t a v a ila b le o f f e r s more th a n a c lu e to th e im m ediate a c tio n o f th y ro x in e which i s re s p o n s ib le f o r th e in c re a s e d r e s p i r a t i o n observed under i t s in flu e n c e . I t has been th e purpose o f t h i s in v e s tig a tio n to stu d y th e e f f e c t , b o th in v iv o and in v i t r o , o f th y ro x in e on th e tu rn o v e r of ra d io a c tiv e phosphorus (P3 2 ) in th e phosphorus-com pounds o f r a t diaphragm . I t was hoped th a t from th e r e s u l t s some in d ic a tio n o f th e p rim ary a c tio n of 2 th y ro x in e m ight he observed. S ince th e r a t e a t w hich P3^ accum ulates in th e v a rio u s p h osphate f r a c tio n s o f tis s u e is- a m easure o f th e m etab o lic a c t i v i t y o f th o se f r a c tio n s (B ark er, F u rc h g o tt, and S c h o rr, 1941; B ru es, T racy , and Cohn, 1944), th e u se o f t h i s tr a c e r i s p a r t i c u l a r l y advantageous* R at diaphragm m uscle has been chosen as th e tis s u e fo r s tu d y be cause ( 1 ) i t p ro v id e s a m uscle t i s s u e which i s s u f f i c i e n t l y t h i n f o r t i s sue s l i c e stu d y , w ith o u t th e damage r e s u ltin g from s l i c i n g (U m breit, e t a l , 1945), (2) th e e x tr a c e llu la r f l u i d c o n tain ed in i t i s sm all and can be e a s ily removed by sim ple im m ersion in a b u ff e r (F u rc h g o tt and S c h o rr, 1942), (3) i t h a s, among m u scles, a v e ry h ig h m e ta b o lic r a t e (C ra ig , B a s s e t, and S a l t e r , 1941), and (4) i t i s v e ry s e n s itiv e to th y ro x in e (S m elser, 1944)* CHAPTER. I I HISTORICAL REVIEW S in ce Magnus-Levy (1895) f i r s t observed th e e f f e c t o f th y ro id on b a s a l m etab o lic r a t e s , many in v e s tig a to r s have extended and a m p lifie d t h i s ev id en ce. Gaddum (1930) showed th a t th y ro g lo b u lin and th y ro x in e , b u t n o t th y ro n in e and d iio d o ty r o s in e , were e f f e c tiv e in e le v a tin g th e B M R o f r a t s . That t h i o u r a e i l fe e d in g r e s u l t s in a m arkedly low ered B M R was. shown by many w orkers (Astwood e t a l , 1943; B ark er, 1945). B ark er, how ever, showed th a t th e th i o u r a e i l le v e l s u sed in m ost p re v io u s work w ere n o t s u f f i c i e n t l y h ig h to y ie ld c o n s is te n t r e s u l t s . I t was th e n f u r th e r shown by many w orkers t h a t th e Q q^ o f su rv iv in g ti s s u e was In c re a se d when th e anim al had b een tr e a t e d w ith th y ro id (G erard and M cIn ty re, 1933; Palm er and L elan d , 1935; Cohen and G erard, 1937) o r th y ro x in e (M cEachern, 1935; S m elser, 1944; Gordon, 1944), and was low ered when th e anim al was th y ro id ecto m ized (S m elser, 1944) o r tr e a te d w ith t h i o u r a e i l (Ja n d o rf and W illia m s, 1944). T ip to n and co-w orkers ( 1946a,b) showed th a t c o n c u rre n t w ith th e changes in Q q , th e re was a p a r a l l e l change i n th e a c t i v i t y o f su cc in ic ' o x id ase and cytochrom e o x id ase o f tis s u e s .f r o m r a t s tr e a t e d w ith th y ro x in e , d e s ic c a te d th y r o id , th y ro tro p ic hormone, and t h i o u r a e i l . T is s ie r e s (1946) found th a t th e cytochrom e C o f r a t m uscle was lik e w ise reduced a f t e r th i o u r a e i l tre a tm e n t and e le v a te d by th y ro x in e tre a tm e n t. T his in c re a s e d m etabolism caused by h y p e rth y ro id ism i s r e f le c te d in o th e r fu n c tio n s o f th e organism . A lth au sen and Stockholm (1938) showed t h a t in, t h i s c o n d itio n th e r e i s a m arked in c re a s e in th e r a t e o f a b so rp tio n o f su g ars from th e in t e s t i n e and concluded th a t th e e f f e c t was due to enhanced p h o sp h o ry la tio n . Tweedy, C h ilc o te , and P e te r s (1946) found th a t th y ro p a ra th y ro id e c to m y causes a re d u c ed e x c re tio n o f adm in- 52 i s t e r e d p , w hich may p o s s ib le be r e l a t e d to d ecreased phosphate tu r n over in th e t i s s u e s . G reenberg, P ra e n k e l-C o n ra t, and G lendening (1947) found th a t h y p e rth y ro id ism cau ses an in c re a s e d t r a n s f e r o f P 3 2 a c ro ss * c e l l membranes*. S tern h eim er (1939) showed th a t in je c tio n o f th y ro x in e causes an in c re a s e i n th e p r o te in c o n te n t o f liv e if and an a c tu a l grow th o f l i v e r as m a n ife ste d by an in c re a s e d p ro p o rtio n of m ito tic c e l l s . Rubino (1 9 4 1 ), in a c l i n i c a l stu d y , o b serv ed an in c re a s e d p u rin e e x c r e tio n a s s o c ia te d w ith th y ro id tre a tm e n t, and concluded th a t i t re p re s e n te d in c re a s e d n u c le o - p r o te in m etabolism in th e tis s u e s * The evidence c ite d th u s f a r seems q u ite c o n c lu s iv e ly to a s s o c ia te th e e f f e c t o f th y ro id tre a tm e n t w ish p r o te in and enzyme s y n th e s is . P r o te in s y n th e s is a ls o seems to be r e l a t e d to n u c le o p ro te in a c t i v i t y . Thus B rues, 32 T racy, and Cohn (1942, 1944) found th a t P up tak e by d eso x y rib o n u cle ic a c id , and, to a le s s e r e x te n t, r ib o n u c le ic a c id , i s g r e a tly in c re a s e d in re g e n e ra tin g r a t l i v e r and in hepatom a, b o th re p re s e n tin g c o n d itio n s i n which p r o te in s y n th e s is i s p ro ced in g a t a h ig h l e v e l . Spiegelm an and Kamen (1946) in a b r i e f p ap er show t h a t enzyme s y n th e s is i n y e a s t i s a s s o c ia te d w ith a marked lo s s of phosphate from n u c le o p ro te in a . D rab- k in (1947) showed th a t i n anim als w ith p a r t i a l hepatectom y more r ib o n u c le ic a c id accum ulated i n th e l i v e r s o f r a t s on a p r o te in - f r e e d ie t th a n on a h ig h p r o te in d i e t , and concluded th a t rib o n u c le ic a c id may be in v o lv ed i n th e p ro c e ss o f p r o te in s y n th e s is . From th e se d a ta i t has n o t seemed u n lik e ly th a t th e work u n d er ta k e n i n t h i s th e s i s would p o in t to an in c re a s e d n u c le o p ro te in m etab o lism a s s o c ia te d w ith th y ro id a c tiv ity * On th e o th e r hand, th e re e x is t s some ex p erim en tal evidence p u r p o rtin g to prove th a t th e th y r o id hormone a n d /o r th y ro x in e can cause an in c re a s e d §Qg i n v i t r o . A dler and L ip s c h itz (1922) re p o rte d th a t th y ro x in e in v i t r o caused an in c re a s e in th e "N itroatm ung* o f su rv iv in g t i s s u e . A bderhal- den (1922) re p o rte d a s tim u la tio n o f y e a s t fe rm e n ta tio n by th e a c tio n o f one p e rc e n t th y ro x in e . A hlgren (1925) found t h a t th y ro x in e a t a concen t r a t i o n o f 1 0 1 4 to 1 0 g ./m l. caused an a c c e le r a tio n o f m ethylene b lu e d e c o lo r iz a tio n by hom ogenates, w hile h ig h e r co n cen tratio n s, were i n h ib ito r y . Haarmann (1936) lik e w is e showed th a t th y ro x in e in th e range 1 0 to 1 0 g ./m l. cau sed an in c re a s e in oxygen consum ption o f s u r v iv in g t i s s u e m ince, w h ile h ig h e r c o n c e n tra tio n s were in h ib ito r y . G a n za n elli (1937, 1939) found t h a t th y ro g lo b u lin could in c re a s e th e oxygen consum ption o f l i v e r , k id n ey , t e s t i s , and h e a r t, b u t th a t th y ro x in e was u s u a lly in e f f e c tiv e ( in two in s ta n c e s an in c re a s e was o b se rv e d ), S a lte r and C raig (1938) and C raig and S a lte r (1945) re p o rte d t h a t h y p e rth y ro id plasm a caused a r e ta r d a tio n o f th e norm al d ecrease in Q q o f su rv iv in g l i v e r t i s s u e , w h ile norm al plasm a, plasm a p lu s th y ro x in e and b u ff e r p lu s th y ro x in e had no e ffe c t* I f we a re to a cc e p t th e s e d a ta , i t i s d i f f i c u l t t o b e lie v e th a t 6 such e f f e c t s co u ld he caused hy in c re a se d enzyme s y n th e s is , sin c e th e tim e n e c e ssa ry f o r th e e f f e c t s to he m a n ife ste d i s v ery s h o r t, w h ile in v iv o , IS t o 24 hours a re n e c e ssa ry in th e r a t b e fo re in c re a s e d BMRs and QOgS a re observed* I t i s th e r e f o r e e v id e n t th a t i n t h i s stu d y two e n t i r e l y d i f f e r e n t e f f e c t s may be found. I n in c re a se d tu rn o v e r o f n u c le ic a c id phosphorus would perh ap s in d ic a te an e f f e c t on p r o te in s y n th e s is , w hile an in c re a s e d tu rn o v e r o f th e a c id - s o lu b le phosphorus f r a c tio n s would re p re s e n t in c re a se d r e s p ir a tio n a n d /o r g ly c o ly s is . CHAPTER I I I PROCEDURE AN D M ETHODS A. TREATM ENT OF ANIMALS I ; Normal r a t s . A dult: fem ale r a t s ; o f th e U n iv e rs ity o f S outhern C a lif o r n ia s t r a i n were u sed th ro u g h o u t th e in v e s tig a tio n . They were m a in ta in ed on th e s to c k d ie t g iv en in T able I . A ll anim als re c e iv e d food and w ater ad lib itu m . 2* T h io u r a e il- tr e a te d r a t s . These r a t s were m a in ta in ed on th e b a s ic s to c k d ie t to w hich had been added 3 grams o f 2 - th i o u r a c il p e r k i l o gram o f d i e t . They rem ained on t h i s d ie t from f iv e t o seven months b e fo re b e in g s a c r i f i c e d . . 3. Hyper th y ro id r a t s . Animals were made h y p e rth y ro id by th e d a ily in tr a p e r ito n e a l in je c tio n o f 1 0 0 mg. o f t r i p l e s tr e n g th d e sic c a te d hog th y ro id .. The anim als were s a c r if ic e d a f t e r th e f o u r th in je c tio n . 32 32 4 . p - t r e a t e d r a t s . P , when in je c te d in to th e an im al, was a d m in iste re d in a s in g le in tr a p e r ito n e a l dose o f about 1 0 0 m ie ro c u rie s th re e days b e fo re th e anim al was to be s a c r if ic e d . B. INCUBATION MEDIA The in c u b a tio n m edia were designed t o approxim ate as c lo s e ly as p o s s ib le th e io n ic co m position o f a plasm a u l t r a f i l t r a t e (W arren and Ebaugh, 1946; H a stin g s e t a l , 1939; H a stin g s , 1940-41). S e v e ra l v a r ia tio n s were n e c e ssa ry f o r s p e c ia l p u rp o se s. D iff e re n t b u ff e rs were 32 needed to s l i c e th e t i s s u e i n , f o r in c u b a tio n , f o r w ashing o u t P , 8 TJBEE I COMPOSITION OF STOCK DIET In g re d ie n t Amount I h o le Wheat F lo u r ► 340 grams Ground O ats 340 w Skim M ilk Powder . . . . . . . . . . . . . . . . . . . 150 w A lf a lf a M eal 40 n D ried Y east powder . . . . . . . . . . . . . . . . . . 20 ’* C o tto n seed O i i ^ . . . . . . . . . . . . . . . . . . . 100 w CaC0„ * * . . . ............................................................... . * . . 5 " o - • • - NaCl ........................................................ . . . . . . 5 » (a) F o r tif ie d w ith : Y itam in A . . . . . . . 32*000 u n its Y itam in D . . . . . . . 3,200 u n its 9 and f o r rem oving in o rg a n ic p h o sp h ate. B u ffe r #1 was d esig n ed to have a pH o f 7 .4 a t 57° C. in e q u ilib riu m w ith 5 % C0g gas m ix tu re. B u ffe r #2 was designed to have a pH o f 7 .4 a t 23° C* in e q u ilib riu m with;. 5$ COg gas m ix tu re . B u ffer § 5 was desig n ed t o have a pH o f 7 .4 a t 23° G. in e q u ilib riu m w ith a i r . B u ffer #4 was d esigned t o have a pH o f 7 .4 a t 23° C. in e q u ilib riu m w ith a ir* and to be p h o s p h a te -fre e . The com position o f th e s e v e ra l b u f f e r s in m illim o le s p e r l i t e r i s g iv en in T able I I . n O P , when added to th e in c u b a tio n medium, was u sed a t a con c e n tr a tio n o f about 25 m ic ro c u rie s p e r tu b e (20 m l.) . T his re p re s e n ts a t r a c e r dose c o n ta in in g a n e g lig ib le amount o f phosphorus. T hyroxine, when added to th e in c u b a tio n medium, was u sed a t a c o n c e n tra tio n o f 2*5 m icrogram s p e r m l. C. REM O V A L OF TISSUE AND INCUBATION The anim als were k i l l e d by a blow on th e head and th e diaphragm r a p id ly d is s e c te d o u t, care b ein g ta k en to han d le th e t is s u e as l i t t l e as p o s s ib le . The t i s s u e was im m ediately p la ce d in a P e t r i d ish on a p ie c e o f f i l t e r pap er m oistened w ith an a p p ro p ria te b u ffe re d s a l t s o lu tio n (B u ffe r # 3 ) , and cu t in to two o r fo u r p o rtio n s w ith a r a z o r . The seg m ents were th e n p la c e d in b eak ers c o n ta in in g B u ffe r #2, and g a ssin g w ith 95$ Og-5$ GOg gas m ix tu re begun im m ediately. The segm ents were d iv id e d 10 TABLE I I . COMPOSITION OP INCUBATION MEDIA (IN MILLIMOLES PER LITER) C O M PO U N D M EDIUM #1 M EDIUM #2 M ED IU M ' #3 M EDIUM #4 NaCl 126*1 108.5 151,0 154.0 EC1 5.50 5,50 5.50 5.50 CaClg 1.40 1.40 1.40 1*40 MgClg 0.75 0 .75 0.75 0 .7 5 NaHCOg 2 5 .4 4 3 .0 0.17 0.17 NagHP0 4 1,08 1.08 1.08 0 . 0 0 NaH2 P04 0 .2 7 0 .2 7 0.27 0 . 0 0 NagSO^ 0.25 0.25 0*25 0.25 Glucose 5.55 5.55 5.55 5.55 11 so th a t each o f a p a ir (o r t r i o ) o f b eak ers c o n tain ed one segment from each diaphragm -so th a t v a r ia tio n from anim al to anim al would be e q u a l iz e d . ' Each beak er f i n a l l y c o n tain ed one segment from each of s ix d ia - N . phragm s. Ti'hen a l l 't i s s u e s were re a d y , th e b u ff e r was poured o f f and th e segm ents'w ere tr a n s f e r r e d to in c u b a tio n tu b es ( 6 x 1 inch Pyrex t e s t tu b e s) w ith 2 0 m l. o f b u f f e r # 1 c o n ta in in g th e d e s ire d a d d itio n a l components (P and th y ro x in e ). In c u b a tio n was c a r r ie d out a t 57° C. f o r fo u r h ours w ith c o n tin u a l g a ssin g w ith 95$ 0 2 - 5 $ COg. D. FRACTIONATION OF TISSUES 1 . R eag en ts. ( 1 ) 5 .0 $ T r ic h lo r a c e tic A cid ( 2 ) 5 .0 N H S L 4 0H (5) 2 .5 $ CaClg (4) 0 .5 $ S uspension o f “l i g h t ” MgCO^ {5) 0 .3 M jSSS^O S ( 6 ) 6.0 N HNO^ (7) “Ammonium m olybdate re a g e n t” 90 g . ammonium m olybdate d is so lv e d in 1 0 0 m l. o f 5 N NH^OH, 240 g. M 4 M>3 added, and th e whole made up to 1 l i t e r ( 8 ) 12.0 N HgS04 —C. P. S p e c ia l—low in N and As (9) 6 .0 N H C 1 0 4 ( 1 0 ) 1 *2 . N HNOg (11) A lc o h o 1 -e th e r, 3 :1 .(12) C hloroform -m ethanol, 1 :1 (13) 1 .0 N NaOH— "Phosphate»f- f r e e 30 g. o f m e ta llic Na c a u tio u s ly r e a c te d w ith 1 l i t e r o f w a ter c o n ta in e d in a s t a i n l e s s s t e e l b eak er and d ilu te d to th e p ro p er c o n c e n tra tio n a f t e r a p re lim in a ry t i t r a t i o n . The s o lu tio n i s s to re d in a p a ra f f i n - l i n e d b o t t l e (14) C o n cen trated HCl (15) 50$ T r ic h lo r a c e tic A cid (16) 1$ A scorbic A cid—f r e s h ly p re p a red and f i l t e r e d on th e day i t i s t o be used (1?) 2$ Ammonium m olybdate—f r e s h ly p re p a re d on th e day i t i s to be u sed (18) 0 .5 $ P h e n o lp h th a le in in 95$ e th a n o l (19) 1$ s o lu tio n o f ,,0 rv u sn d e te rg e n t in w ater (20) 10 N NaOE—Approxim ate 2* p re lim in a ry tre a tm e n t. At th e c lo se o f th e in c u b a tio n p e rio d , th e b u ff e r was poured o f f and re s e rv e d (sam ple S ). T w enty-five m l. o f buf f e r § 5 were added, allow ed to s ta n d 3 m in u te s, and poured o f f . T his was re p e a te d once. Then th e same p ro cedure was. re p e a te d tw ice w ith 25 m l. p o rtio n s o f b u ff e r # 4 . T his s e r ie s o f w ashings se rv e s f i r s t to e lu te from th e t is s u e any e x t r a c e l l u l a r phosphorus o f h ig h s p e c if ic a c t i v i t y and second to r in s e th e t i s s u e o f m ost o f th e chem ical phosphorus o u r- sld e o f th e c e l l s (F u rc h g o tt and S c h o rr, J.942; K alck er e t a l , 1944). 13 A fte r th e l a s t p o rtio n o f b u f f e r had co m p letely d ra in e d o f f , 15 ml* o f 5$ t r i c h l o r a c e t i c a c id a t 0° C. was added. The tu b e s th e n sto o d 15 m inutes a t 0° C* w ith o c c a sio n a l sh ak in g , and th e e x tr a c t was th e n c a r e f u lly d ecanted th ro u g h a f i l t e r p aper in to a c h ille d g rad u ated c y l in d e r. The re s id u e was tw ice washed w ith 3 m l. o f 5$ t r i c h l o r a c e t i c a c id and th e e x tr a c t and w ashings made up to 2 0 . 0 m l. 3. E r a c tio n a tjo n o f A c id -so lu b le Phosphorus. T his f r a c tio n a tio n employs th e method o f D elory (1938) f o r p r e c ip ita tio n o f in o rg a n ic phos p h o ru s, fo llo w ed by a m o d ific a tio n o f th e s e p a ra tio n p ro ced u re o f Sacks (1942) f o r p h o sp h o crea tin e and adenosine trip h o s p h a te . A 2 m l. sample o f th e t r i c h l o r a c e t i c a c id f i l t r a t e was ta k en f o r d e te rm in a tio n o f t o t a l a c id - s o lu b le phosphorus (TAS). The rem ainder was tr a n s f e r r e d to a 6 x 1 in ch t e s t tu b e w ith two 2 m l. p o rtio n s o f w a te r, and th e s o lu tio n was n e u tr a liz e d to p h e n o lp h th a le in w ith 5 N NE^OH and an ex cess o f one m l. added. Two m l. o f 2 .5 $ CaClg, and 2 m l. o f 0 .5 $ MgCOg were added and th e tu b e s allow ed to s ta n d 30 m inutes in th e r e f r i g e r a t o r . The s o lu tio n was th e n f i l t e r e d th r u a 20 m l. s in te r e d g la s s fu n n e l and th e re s id u e washed w ith two 2 m l. p o rtio n s o f co ld 0 .3 N NE^CH. The p r e c i p i t a t e ( in th e tube and on th e fu n n e l) was th e n d is so lv e d w ith two 2 m l. p o rtio n s o f 1 . 2 N HNOg and r in s e d th ru th e fu n n e l in to a 15 m l. g ra d u a ted c e n tr ifu g e tu b e . The s o lu tio n was th e n made up to 15 m l. aiid saved in a sto p p e re d b o t t l e f o r d e term in a tio n o f in o rg a n ic phosphorus ( I P ) . To th e f i l t r a t e and w ashings was added 1 .4 m l. ex cess o f 6 N HN03 ( to a f i n a l a c id c o n c e n tra tio n o f 0 .2 N) and 3 .3 m l. rtammonium m olybdate 14 re a g e n t* . The m ix tu re was th e n allow ed to sta n d one and o n e -h a lf hours a t room te m p e ra tu re , th e n f i l t e r e d th r u a s in te r e d g la s s fu n n e l a s b e fo re and washed tw ice w ith 2 m l. p o rtio n s o f w a te r. The p r e c i p i t a t e was th e n d is so lv e d in two 2 ml* p o rtio n s o f 5 N Nff^OH and washed th r u th e f i l t e r a s b e fo re , made up to 15 m l. and saved f o r th e d e te rm in a tio n o f phosphe- c r e a tin e phosphorus (CP). The f i l t r a t e was tr a n s f e r r e d to a 125 m l. Erlenm eyer f l a s k , 2 .7 m l. o f 6 N HN03 added ( to 0 .5 N ), and th e f la s k h e a te d i n a b o ilin g w ater b a th f o r 20 m in u te s. I t was th e n c o o led , allow ed to sta n d f o r one hour to com plete th e p r e c ip ita tio n o f ammonium phosphom olybdate. I t was th e n f i l t e r e d , w ashed, and th e p r e c i p i t a t e d isso lv e d as above. The sample was made up to 15 m l. and re s e rv e d f o r d e te rm in a tio n o f adenosine t r i phosphate phosphorus (1TP). As h e re employed, th e f r a c tio n c a lle d ATP C o n tain s a l l o f th e g lu c o s e -l-p h o s p h a te , and 25 f a o f th e f r u c to s e d i phosphate i n th e sample (U m breit e t a l , 1945). However th e s e compounds re p re s e n t le s s th a n 5f0 o f th e ph o sp h ate determ ined as h e a t - l a b i l e phos p h o ru s, th e r e s t re p re s e n tin g th e l a b i l e phosphate groups o f adenosine d i- and tri-p h o sp h a te * The f i l t r a t e was made up to a volume o f 75 m l. and a 15 m l. sample ta k e n f o r r e s id u a l a c id - s o lu b le phosphorus(R P ). To t h i s sample was added one m l. o f 1 2 H HgSO^, and i t was th e n d ig e ste d over an open flam §, con tin u in g th e h e a tin g f o r one m inute a f t e r SOg fumes ap p eared . A fte r c o o l in g , 5 m l. o f w ater were added and i t was h e a te d in a b o ilin g w ater b a th f o r 10 m inutes to hy d ro ly ze p y ro p h o sp h ate. I t was th e n cooled and made up to 15 m l. f o r a n a ly s is . 15 The sample f o r t o t a l a c id -s o lu b le phosphorus (TAS) was d ig e s te d w ith 1 ml* o f 12 IT EgSO^, and 0 .5 ml* o f 6 N HNOg, and tr e a te d a s above. 4 . E x tra c tio n o f L ip id Phosphorus* The e x tr a c tio n o f l i p i d s i and f r a c tio n a tio n o f th e p ro te in s was perform ed by th e method o f Schmidt and T annhauser (1945). The a c id - in s o lu b le re s id u e was e x tra c te d w ith 20 ml* o f 5 % t r i c h lo r a c e tic a c id f o r 10 m inutes and th e e x tr a c t was d is c a rd e d . The re s id u e was th e n e x tr a c te d f o r 2 hours w ith 2 0 m l. o f a lc o h o l-e th e r m ix tu re w ith co n tin u o u s sh ak in g . A fte r rem oval o f th e e x tr a c t by d eean t- a tio n , th e re s id u e was f u r th e r e x tra c te d by r e flu x in g w ith 2 0 ml* o f chloroform -m ethanol m ix tu re . T h is e x tr a c t was lik e w is e removed by d e- c a n ta tio n , and th e combined l i p i d e x tr a c ts were e v ap o rate d to d ry n ess. The l i p i d re s id u e was th e n d ig e s te d w ith 1 m l. o f 12 N HgSO^ and 2 m l. o f 6 N HC1G4 w ith a d d itio n s o f 6 IT HNOg to e f f e c t f i n a l d e c o lo n iz a tio n o f th e d ig e st* The d ig e s t was th e n d ilu te d to 5 m l*, h y d ro ly zed f o r 15 min u te s a t 100° C ., and d ilu te d to ’15 m l. f o r a n a ly s is (L P ). 5. E ra c tio n a tlo n o f p r o te in P hosphorus. The p r o te in re s id u e was washed tw ice w ith 1 0 ml* p o rtio n s o f e th e r and th e n d rie d in vacuo f o r a few m in u te s. (The w eight o f t h i s re s id u e , c o n s is tin g alm ost e x c lu s iv e ly o f p r o te in , i s p ro b ab ly th e b e s t m easure o f th e amount o f tis s u e u sed in th e ex p erim en t.) To t h i s dry p r o te in was added 5 m l. o f 1 .0 N ITaO H ( ’ ’P hosphate”- f r e e ) and th e m ix tu re was in c u b a te d fo r 15 h ours a t 37° C ., during which tim e com plete s o lu tio n o f th e p r o te in was e f f e c te d to g e th e r w ith h y d ro ly s is o f rib o n u c le ic a c id to m o n o n u cleo tid es. The s o lu tio n was 16 d ilu te d to 1 0 m l. and a 2 m l. sample ta k e n f o r t o t a l p r o te in phosphorus (TPP). To t h i s sample was added 0 .5 m l. o f 12 N H2 SO 4 and 1 m l. o f 6 M HCIO4 , and i t was th e n d ig e s te d in th e same manner as was th e l i p i d sam ple. The rem ain in g 8 m l. of s o lu tio n \m s tr a n s f e r r e d to a 6 x 1 in ch t e s t tu b e w ith two 2 m l. p o rtio n s o f w a te r* and 1 * 2 m l. each o f concen t r a t e d HCl and 50$ t r i c h l o r a c e t i c a c id (0 .1 volumes) were added. A fte r thorough m ixing and 1 0 m in u te s' s ta n d in g , th e tu b e was c e n trifu g e d and th e su p e rn a ta n t decan ted th r u f i l t e r p a p e r. The re s id u e was washed tw ice w ith 5 m l. p o rtio n s o f 5$ t r i c h l o r a c e t i c a c id w ith c e n tr ifu g in g and f i l t r a t i o n as above. The re s id u e was th e n washed w ith 15 m l. o f 5$ t r i c h l o r a c e t i c a c id and t h i s w ashing was d isc a rd e d . The re s id u e was di>- g e s te d in th e same manner as sample TPP and was re s e rv e d f o r th e d e te r m in a tio n o f d e so x y rib o n u cle ic a c id phosphorus (DNA). To th e f i l t r a t e , c o n tain ed in a 125 m l. Erlenm eyer f l a s k , was added 2 m l. o f 12 N HgSO^ and 0 .5 m l. o f 6 N H N O 3 , and th e s o lu tio n was b o ile d down to 2 o r 3 m l. on th e h o t p l a t e . D ig estio n , h y d ro ly s is and d ilu tio n were th e n c a r r ie d out e x a c tly as f o r sample TPP and th e s o l u tio n was re s e rv e d f o r th e d e te rm in a tio n o f rib o n u c le ic a c id phosphorus (UNA). E. ANALYSIS FOR P3 2 AN D P3 1 C hem ical a n a ly s is f o r P*^ was perform ed by th e method o f Shannon and W erkheiser (1946) b ased on th e work o f Ammon and H insberg (1936). One m l. p o rtio n s o f a l l sam ples were t i t r a t e d to determ ine t h e i r a c id o r base c o n te n t. The a c id sam ples were t i t r a t e d a g a in s t 1 .0 N NaOH 17 u sin g p h e n o lp h th a le in as in d ic a to r . The ammoniacal sam ples were t i t r a t e d a g a in s t 1 * 0 N H2 SO 4 . u sin g m eth y l re d as in d ic a to r . Samples o f such a s iz e as to g iv e 5 to 15 m ierogram s o f phosphorus w ere p la c e d in 5 in ch t e s t tu b e s and 12 N H2 SO 4 . added to g iv e a t o t a l a c id c o n ten t o f 6 m illie q u iv a le n ts , c a lc u la te d from th e above t i t r a t i o n ) . W ater was th e n added to g iv e a f i n a l volume o f 10 m l. One m l. each o f 1 ; % a sc o rb ic a c id and ammonium m olybdate were added in o rd e r, th e tu b e s were mixed by in v e rs io n , and th e n h e a te d f o r e x a c tly two m inutes in a b o ilid g w ater b a th . S ince th e c o lo r i s v ery s e n s itiv e to th e le n g th o f th e h e a tin g p e rio d , th e same number o f tu b es and th e same s iz e w a te r b a th were used in a l l cases and a d u p lic a te b la n k and sta n d a rd sample were c a r r ie d th r u w ith each b a tc h . The tu b es were th e n q u ic k ly cooled in c o ld w a te r. The s o lu tio n s were th e n re a d in a K lett-S um erson photo e l e c t r i c c o lo rim e te r u s in g a No. 62 f i l t e r a g a in s t a b la n k and a sta n d a rd c a r r ie d th r u th e d e te rm in a tio n . The curve i s l i n e a r up to a re a d in g o f 350. A m o d ific a tio n was n e c e ssa ry f o r th e d e te rm in a tio n o f r e s id u a l phos p h o ru s, sin c e t h i s sample a lre a d y c o n tain ed ammonium m olybdate. F ive m l. sam ples o f th e d ig e s t were u sed , c o n ta in in g 2 0 m l. o f ammonium m olybdate (.5. x i x 3 .3 x 9 0 ), th e e x a c t amount u sed in th e m ethod, hence one m l. o f 15 5 w ater was s u b s titu te d f o r th e ammonium m olybdate a d d itio n . For th e d e te rm in a tio n o f r a d io a c ti v ity , sam ples o f 3 m l. o r le s s (c o n ta in in g le s s th an 10,000 and more th a n 30 counts p e r m in u te) were m easured in to 1 /4 o z. t i n ointm ent d is h e s , 1 drop o f p h e n o lp h th a le in s o l u tio n and 1 drop o f a d ilu te s o lu tio n o f "O rvus" d e te rg e n t were added, and 18 th e sam ples re n d e red a lk a lin e by th e dropw ise a d d itio n o f 10 N NaOH* The sam ples were th e n d rie d a t 110° C. and th e r a d io a c ti v ity d eterm ined in a G eig er-M u ller c o u n te r, m anufactured by T e c h n ica l A s s o c ia te s , I n c ., G len d ale, C a l if . The co u n tin g tu b e was o f th e t h i n g la s s w a ll ty p e (TA -B l). The n e t co u n tin g e f f ic ie n c y , b ased on a n a ly se s o b ta in e d from th e C lin to n la b o ra t o r i e s , Oak R id g e, T e n n ., was 1 5 .4 $ . The c o rre c tio n f a c to r a p p lie d f o r co in cid en ce e r r o r amounted to about 1 $ p e r tho u san d counts p e r m in u te. N e g lig ib le e r r o r s were caused by s e lf - a b s o r p tio n up to a t o t a l s o lid con te n t o f 500 mg. p e r d is h , w hich was c o n sid e ra b ly h ig h e r th a n th e s o lid s en co u n tered i n t h i s work* A ll sam ples o f a g iv en s e r ie s were re a d on th e co u n ter a t th e same tim e , th u s making i t u n n ecessary to a p p ly decay o r c o u n ter c o r r e c tio n s . F. HANDLING OF RESULTS r e s u l t s a re re p o rte d a s p e rc e n t o f t o t a l p r o te in phosphorus, s in c e Berenblum e t a l (1939) and o th e rs c o n sid e r t h i s to be an in d ex o f th e w c e l l u l a r i t y w o f th e t is s u e and hence more s ig n if ic a n t th a n v a lu e s based on dry w eig h t. 3? P r e s u lts , a r e re p o rte d i a term s o f r e l a t i v e s p e c if ic a c tiv ity * th a t i s th e s p e c if ic a c t i v i t y ( i n cotints p er m icrogram o f P3^) o f each f r a c t i o n a s p e rc e n t o f th e s p e c if ic a c t i v i t y o f th e in o rg a n ic phosphorus » f r a c tio n . G. CONSIDERATION OF ERRORS A lthough i t w i l l be n o ted th a t re c o v e ry o f phosphorus in th e 19 v a rio u s f r a c tio n s i s u s u a lly q u ite high., no a tte m p t was made to malce th e s e p a ra tio n s co m p letely q u a n tita tiv e , th e aim b ein g r a th e r to en su re th a t each f r a c t i o n was co m p letely f r e e o f any o th e r f r a c tio n , so th a t the. sp e c i f i c a c t i v i t y re p o rte d would be an a c c u ra te m easure o f t h a t e x is tin g in 51 th e t i s s u e . N o n e th ele ss, th e v a lu e s re p o rte d f o r P co n ce n tra tio n s, a re s u f f i c i e n t l y a c c u ra te to be r e p r e s e n ta tiv e o f th e amounts e x is tin g in th e tis s u e s * CHAPTER IV EZPERBiENTAL BATA The i n i t i a l experim ent (S e r ie s I ) was c a r r ie d out on s i x t h i o u r a c i l - t r e a t e d r a t s . One hem i-diaphragm from each r a t was p la c e d in Tube 1 and th e o th e r p la c e d in Tube 2. The in c u b a tio n , f r a c tio n a tio n , and a n a ly s is was c a r r ie d out as d e sc rib e d in C hapter I I I , 25 m ic ro c u rie s o f P 3 2 b ein g added to each tu b e j u s t p r io r to in c u b a tio n . In S e rie s I I , two groups o f s i x t h i o u r a c i l - t r e a t e d r a t s were u sed . Tubes 1 and 3 , and Tubes 2 and 4 were, d u p lic a te s made up as above. To rzp Tubes 3 and 4 th y ro x in e was added, p was added to a l l tu b e s im m ediately b e fo re in c u b a tio n . T his s e r ie s i s s u b je c t to c r itic is m , s in c e th e t i s s u e s rem ained u n a g lta te d in b eak ers f o r a t l e a s t one h o u r. T his p ro b ab ly r e s u lte d in a t l e a s t p a r t i a l l y a n ae ro b ic c o n d itio n s , from which th e tis s u e s may not have co m p letely re c o v e re d . S e rie s I I I was id e n tic a l to S e rie s I I , ex cep t t h a t COg-Og gas m ix tu r e was b ubbled th ro u g h th e b e ak e rs in which th e t i s s u e s were p la c e d . In S e rie s V, th e diaphragm s from s ix h y p e r th y r o id 'r a ts were p la c e d in Tubes 1 and 2 and th e diaphragm s from s i x norm al r a t s p la c e d in Tubes 3 and 4 . P * 5 2 was added j u s t p r io r to in c u b a tio n . In S e rie s IV two groups o f s ix r a t s , in je c te d w ith P^ 2 th re e days p re v io u s ly , were used* The diaphragm s from each gfoup were cut. in to th r e e e q u a l segm ents and d is tr ib u te d in th re e tu b e s so t h a t each tu b e was e s s e n t i a l l y a r e p lic a t e o f th e o th e r s . Tubes 1, 2, and 3 co n tain ed th e diaphragm s from th e f i r s t group o f r a t s and Tubes 4 , 5, and 6 con- 2 1 ta in e d th o se from th e second. Tubes 1 and 4 were im m ediately tr e a te d with, t r i c h l o r a c e t i c a c id t o serv e as i n i t i a l c o n tro ls w h ile th e rem ain ing tu b e s were in c u b ated as u s u a l. Thyroxine was added to Tubes 5 and 6 . The d a ta from th e se experim ents i s ta b u la te d in T ables I I I to ¥111, and d isc u sse d in C hapter ¥ . CHAPTER 7 DISCUSSION In Table I I I a re ta b u la te d th e d a ta f o r tis s u e s from norm al, h y p e rth y ro id , and h y p o th y ro id ( th i o u r a c i l - t r e a t e d ) r a t s . S ince th e d ata a re in term s o f s p e c if ic a c t i v i t y r e l a t i v e to th e in o rg a n ic f r a c tio n , th e h ig h e r th e v alu e f o r any p a r t i c u l a r f r a c tio n , th e g r e a te r has been th e u p tak e o f P32; From a stu d y o f th e t a b l e , i t may be n o te d th a t th e re a re s e v e ra l v e ry d e f in ite tre n d s . F i r s t , th e re i s an in d ic a tio n th a t h y p erth y ro id ism cau ses an in c re a s e d u p tak e o f P 3 2 in to phosphocrea- ti n e (GP) and adenosine trip h o s p h a te (ATP). T h is may p erh ap s be i n t e r p re te d as a r e f l e c t i o n o f th e in c re a s e d m etab o lic r a t e o f th e h y p erth y r o id t i s s u e . C oncom itantly th e re ap p ears to be a d e crea se in c o n cen tra tio n o f in o rg a n ic phosphate ( I P ) , CP, ATP, and t o t a l a c id - s o lu b le phosph o ru s (TAS), w ith re s p e c t to n u c le o p ro te in phosphorus (TPP) (T able 1 7 ), a s s o c ia te d w ith in c re a s e d m e ta b o lic a c t i v i t y . A lthough th e s e e f f e c t s have n o t been p re v io u s ly re p o rte d , B ark er, F u rc h g o tt, and S ch o rr (1941) 52 have observ ed th a t th e u p tak e o f P in to ATP and CP by s u rv iv in g e a r - d ia e s l i c e s i s in c re a s e d w ith in c re a s e d • These e f f e c t s a re com pat- 2 ib le w ith c u rre n t co n cep ts o f carb o h y d rate m etabolism , s in c e in c re a s e d a c t i v i t y would be ex p ected to r e s u l t in in c re a s e d tu rn o v e r and breakdown o f h ig h energy phosphate compounds, p a r t i c u l a r l y in s u rv iv in g t i s s u e , where r e s y n th e s is i s a t l e a s t in p a r t subnorm al. R e fe rrin g a g a in to T able I I I , i t may be n o ted th a t th e v a lu e s f o r th e u p tak e o f phosphorus by l i p i d (IP ) a re much low er f o r th e th i o u r a c i l - TABLE I I I PHOSPHORUS METABOLISM IN DIAPHRAGM S PRO M NORM AL, HYPOTHYROID, AND HYPERTHYROID RATS P3^ U ptake by P h o sp h o rus-C ontaining Compounds F ra c tio n T h io u r a c il- tr e a te d S e rie s I S e rie s I I I Tube 1 Tube 2 Tube 1 Tube 2 Normal S e rie s V Tube 3 Tube 4 H y p erth y ro id S e rie s V Tube 1 Tube 2 I P i o o ( a ) 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 CP 73 77 76 82 75(fc) 75(b ) 85(*») 92(b ) ATP 59 6 6 59 65 63 62 75 8 8 RP 16^e ) 1 9 (C> 43 54 54 41 55 52 TAS 81 80 74. 73. 55. 58. 59 49 . I P 2 .3 1 .9 3 .1 2 . 8 6 .6 6 .3 8 .3 6 .4 DM 0 .2 5 0 .3 4 0 .4 2 0 .4 4 0 .9 1 . 1 1 . 6 1 . 2 E M 5 .0 5 .8 8 . 0 8 . 0 16 17 13 1 1 TPP 3 .8 4 .5 6 . 1 6 . 2 13 14 1 1 9 ( q)a i i v a lu e s i n t h i s ta b le a re re p o rte d in term s o f th e s p e c if ic a c t i v i t y o f th e f r a c tio n as p e rc e n t o f th e s p e c if ic a c t i v i t y o f th e in o rg a n ic f r a c t i o n . (b) Owing to an e r r o r i n te c h n ic , th e se v a lu e s had to be e stim a te d by d if f e r e n c e . f c ) ' 'T hese v a lu e s a re low p ro b ab ly because o f th e a n ae ro b ic s ta n d in g p e rio d d e sc rib e d in C hapter IT . TABLE 3nr PHOSPHORUS METABOLISM IN DIAPBRAGM S FROM NORM AL, HYPOTHYROID, AND HYPERTHYROID RATS Phosphorus D is tr ib u tio n in P h o sp h o ru s-C o n tain in g Compounds F ra c tio n T h io u r a c il- tr e a te d S e rie s I S e rie s I I I Tube 1 Tube 2 Tube 1 Tube 2 Normal S e r ie s V Tube 3 Tube 4 H y p erth y ro id S e r ie s V Tube 1 Tube 2 IP 78^a ) 81 81 72 6 6 80 41 32 CP 39(c ) 3 3 (c ) 83 51 2 8 ^ 2 ? ( b ) 7 (b ) 7 (b) ATP 23(c ) 19(c) 39 30 8 1 2 17 9 RP 4 2 (c ) 40 ( c ) 23 . 17 25 37 28 23 TAS 170 167 233 205 140 151 91 79 LP 60* 55<d> 182 173 146 178 8 3 ^ 136 D N A 26 24 31 30 30 29 27 29 R N A 77 74 6 8 65 67 73 6 6 61 TPP 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 (c ^ A ll v a lu e s in t h i s ta b le a re re p o rte d a s p e rc e n t o f t o t a l p ro te in phosphorus. ^ O w in g t o an e r r o r ' in te c h n ic , th e se v a lu e s had to be e stim a te d by d if f e r e n c e . ( c )These v a lu e s a re low p ro b ab ly because o f th e an aero b ic p e rio d d e sc rib e d in C hapter IV. ( d ^A p o rtio n o f th e s e sam ples was l o s t d u rin g e v a p o ra tio n . 2 5 tr e a te d anim als th a n f o r th e o th e r g ro u p s. T his i s und o u b ted ly in d ic a tiv e o f a low ered p h o sp h o lip id m etabolism in th e s e an im als. The v a lu es f o r 31 P in t h i s f r a c tio n (T able IV ), a f t e r d isc o u n tin g th o se known to be low, show no g e n e ra liz e d e ffe c t* C o n sid e rin g now th e n u c le o p ro te in v a lu e s in T able I I I i t i s imme d ia te ly ap p aren t th a t t h io u r a c il ag ain causes a v e ry m arked re d u c tio n in up tak e f o r b o th n u c le o p ro te in f r a c tio n s , w ith perh ap s a somewhat g r e a te r e f f e c t on BM. th a n on RN A .. Comparing th e s e ‘r e s u l t s w ith th e c o n c e n tra tio n s re p o rte d in T able IV, we see t h a t ,a s w ith t h e . l i p i d f r a c tio n , in c re a s e d tu rn o v e r i s not a s s o c ia te d w ith d ecreased co n ce n tra tio n * However, we must remember th a t th e sm all uptake in v o lv ed in d ic a te s th a t o n ly a sm all p a r t o f th e f r a c tio n has been p a r t ic ip a tin g in th e m etabolism and henee la rg e changes in c o n c e n tra tio n a re n o t to be expected* I t i s im p o ssib le a t t h i s p o in t to decid e w hether th e e f f e c t s on l i p i d and p r o te in phosphorus u p tak e a re d ir e c t r e s u l t s o f a change in o v e r a ll m e ta b o lic a c t i v i t y a s s o c ia te d w ith changes in th y ro id fu n c tio n , o r a re an independent e f f e c t , perhaps o r ig in a tin g e a r l i e r th a n th e change in m etabolism and b e in g i n d i r e c t l y re s p o n s ib le f o r i t . The ex p erim en ts re c o rd ed in T ables V and VI were p a r t o f a stu d y on th e e f f e c t o f th y ro x in e in v i t r o on tis s u e s from t h i o u r a c i l - t r e a t e d a n im a ls. The d a ta ta k e n from S e rie s I and I I do n o t show th e same ran g es o f v a lu e s as do th o se from S e rie s I I I , f o r th e re a so n e x p la in e d i n C hapter IV, and m entioned i n th e fo o tn o te to th e ta b le s* However, s in c e th e in d i v id u a l experim ents were a d eq u a te ly c o n tr o lle d , we may s t i l l draw co n clu sio n s co ncerning th e e f f e c t o f th y ro x in e . The d a ta from S e rie s I was in c lu d e d TABLE V THE EFFECT OF THYEOXTEE in v i t r o O N PHOSPHORUS METABOLISM OF DIAPHRAGM S FR O M RATS TREATED WITH THIOURACIL P^2 U ptake by Phospb.orus-Contain.ing Compounds F ra c tio n S e r ie s l ( a ) Tube 1 Tube 2 S e rie s I l ( a ) Tube 1 Tube 3 S e rie s I l ( a ) Tube 2 Tube 4 S e rie s I I I Tube 1 Tube 3 S e rie s I I I Tube 2 Tube 4 T h io - U r a c il C o n tro l T h io - u r a c i l C o n tro l T hio- u r a c i l C o n tro l Thyroxine added T h io - u r a c i l C o n tro l Thyroxine added T h io - u r a c i l C o n tro l Thyroxine added T h io - u r a c i l C o n tro l Thyroxine added IP i o o 'B) 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 CP 73 77 57 76 73 92 76 79 82 67 ATP 59 6 6 32 45 38 60 59 63 65 64 RP 16 19 1 2 42 13 41 43 72 54 70 TAS 81. 80. 60 74 73 79. 74 7 7 73 53 LP 2 .3 1 .9 5 .7 2 . 6 3 .8 4 .1 3 .1 2 . 8 2 . 8 6 . 6 D M A 0 .2 5 0 .3 4 0 .3 4 0 . 2 0 0.32 0.34 0.42 0 .4 8 0 .4 4 0 .5 4 RNA 5 .0 5 .8 1 1 * 1 8 .9 1 2 . 2 10.9 8 . 0 8 .5 8 . 0 1 1 .4 TPP 3 .8 4 .5 6 . 1 5 .4 7 .8 - 7 .3 6 . 1 5 .9 ' 6 . 2 7 .6 ----------------- ( a )These tu b e s ex p erien c e d an a n a e ro b ic stan d in g p e rio d as d e sc rib e d in C hapter IV. {k^AU v a lu e s in t h i s ta b le a re re p o rte d i n term s o f th e s p e c if ic a c t i v i t y o f th e f r a c t i o n a s p e rc e n t o f th e s p e c if ic a c t i v i t y o f th e in o rg a n ic f r a c tio n . w cn T A B L E T FT THE EFFECT OF TEEROXEHE in v i t r o O N PHOSPHORUS METABOLISM OF DIAPHRAGM S FRO M RATS TREATED WITH THIOURACIL Phosphorus D is tr ib u tio n i n P h o sp h o rus-C ontaining Compounds F r a c tio n S e rie s l ( a ) Tube 1 Tube 2 S e rie s I l ( a ) Tube 1 Tube 3 S e rie s I l ( a ) Tube 2 Tube 4 S e rie s I I I Tube 1 Tube 3 S e rie s I I I Tube 2 Tube 4 T h io - T h io - u r a c i l u r a c i l C o n tro l C o n tro l T hio- u r a c i l C o n tro l Thyroxine added T hio- u r a c i l C o n tro l Thyroxine added T hio- u r a c i l C o n tro l Thyroxine added T h io - u r a c i l C o n tro l Thyroxine added IP 78 81 19 33 42 48 81 76 72 79 CP 39 33 2 1 30 48 41 83 65 51 63 ATP 23 19 2 .4 1 2 24 17 39 26 30 31 RP 42 40 33 1 1 38 1 2 23 13 17 13 TAS 170 167 63 92 128 138 233 205 205 132 LP 60 55 78 143 131 116 182 157 173 165 DN A 26 24 25 31 30 29 31 24 30 29 RNA 77 74 51 52 55 55 6 8 53 65 59 TPP 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 1 0 0 ( a )These tu b e s e x p erien c e d an an aero b ic sta n d in g p e rio d a s d e sc rib e d in C hapter IF . (b )A ll v a lu es i n t h i s ta b le a re re p o rte d in term s o f p e rc e n t o f t o t a l p ro te in phosphorus. ro N J 28 in th e s e ta b le s m erely to in d ic a te th e degree o f r e p r o d u c ib ility to be ex p e c te d . A c o n s id e ra tio n o f T able V shows a u n ifo rm s tim u la tio n by th y ro x in e o f phosphorus up tak e in to th e r e s id u a l phosphorus f r a c t i o n KP). (T h is ♦ f r a c tio n i s a complex m ix tu re c o n ta in in g , among o th e r th in g s , hexose and t r i o s e phosphates and o th e r in te rm e d ia te s o f carb o h y d rate m etabolism , as w e ll as sm a lle r amounts o f n u c le o tid e s and p en to se p h o sp h a te s). In T able VI i t is a ls o n o tic e d th a t in th e th y r o x in e - tr e a te d tis s u e th e r e i s a mar ked d ecrease in c o n c e n tra tio n o f phosphorus in t h i s f r a c tio n as compared to th e c o n tr o ls . A second e f f e c t o b serv ab le in two o f th e fo u r e x p e ri- 32 m ents i s found in th e P u p tak e by p h o sp h o crea tin e and adenosine t r i p h o sp h ate. However, s in c e t h i s was n o t u n ifo rm ly o b serv ed , i t may be an a r t i f a c t . C a re fu l stu d y o f th e rem ainder o f th e s e ta b le s f a i l s to show any o th e r even p a r t i a l l y re p ro d u c ib le tr e n d s . I t was a ls o o f i n t e r e s t to stu d y th e in v i t r o e f f e c t o f th y ro x in e on th e change in tu rn o v e r o f in tis s u e s a lre a d y c o n ta in in g p * ^ . T his approach appeared p a r t i c u l a r l y d e s ir a b le sin c e Spiegelm an and Kamen (1946) in t h e i r work on y e a s t found n o t an in c re a s e d up tak e b u t an in c re a se d lo s s of from n u c le o p ro te in s a s s o c ia te d w ith enzyme sy n th e sis* In T able V II, th e s p e c if ic a c t i v i t i e s o f a l l f r a c tio n s in each o f th e two experim ents a re r e f e r r e d to th e s p e c if ic a c t i v i t y o f th e i n i t i a l in o rg an ic phosphate o f th e ex p erim en t, sin c e t h i s r e p re s e n ts b e s t th e changes which ta k e p la c e d u rin g in c u b a tio n . In Table V III, how ever, th e concen t r a t i o n v a lu e s a re re p o rte d as b e fo re , s in c e th e a b so lu te v a lu e s a re a fu n c tio n of th e t o t a l amount o f tis s u e u sed , and th e t o t a l p ro te in does TABLE H I THE EFFECT OF THYROXINE in v i t r o OH PHOSPHATE METABOLISM IN DIAPHRAGM S OF THIOORAiCIL-TREATED RATS PREVIOUSLY TREATED W EEK P3 2 P 3 2 Turnover i n P h o sp h o rus-C ontaining Compounds O ccurring du rin g In c u b a tio n F r a c tio n I n i t i a l RSA*a ) In cu b ated C o n tro l RSA Change In cu b ated w ith T hyroxine RSA Change I n i t i a l RSA In cu b ated C o n tro l RSA Change In c u b a te d w ith Thyroxine RSA Change IP 1 0 0 85 -15 81 ^19 1 0 0 87 -1 3 95 - 5 CP 69 55 -14 37 -32 97 70 -27 54 to i ATP 80 25 -55 37 -43 67 6 8 * 1 47 i to o RP 6 8 60 - 8 60 - 8 51 52 f 1 70 *19 TAS 79 63 i H < P 54 -25 87 74 -1 3 72 -15 IP 6 ? 60 - 7 58 - 9 52 45 - 7 38 -14 DN A 5 .7 4 .9 - 0 * 8 4*5 * • 1 * 2 6 .7 3 .7 - 3*0 2 .9 - 3 .8 REA 44 47 * 3 40 - 4 53 45 - 8 40 -1 3 TPP 38 38 0 34 - 4 35 36 f 1 32 - 3 ( a )R e la tiv e S p e c ific A c tiv ity —S p e c ific a c t i v i t y o f f r a c tio n as p e rc e n t o f i n i t i a l s p e c if ic a c t i v i t y o f in o rg a n ic f r a c tio n . to t o T A B L E T T i l l THE EFFECT OF THYROXINE i n V itro ON PHOSPHATE METABOLISM IN DIAPHRAGM S OF THIOORACIL-TREATED RATS PREVIOUSLY TREATED UITH P3 2 Change i n Phosphorus D is tr ib u tio n in P h o sp h o ru s-C o n tain in g Compounds O ccurring during In c u b a tio n F ra c tio n I n i t i a l In c u b a ted C o n tro l Change In cu b ated w ith Thyroxine Change I n i t i a l In cu b ated C o n tro l Change In c u b a ted . w ith Thyroxine Change IP 108( a ) 89 - 19 95 -13 90 94 4 4 95 t 5 CP 189 69 - 1 2 0 127 -62 115 80 -35 95 - 2 0 ATP 28 34 4 6 17 - 1 1 38 2 2 -16 19 -19 RP 35 32 - 3 27 “ . 8 41 39 - 2 31 - 1 0 TAS 285 176 -109 186 -99 266 218 -48 193 -73 LP 134 2 0 1 f 67 179 445 125 158 433 178 453 U N A 17 30 4 13 30 *13 16 19 f 3 27 411 RNA 72 6 8 - 4 71 - 1 44 46 4 2 6 6 422 TPP 1 0 0 1 0 0 ----- 1 0 0 ----- 1 0 0 1 0 0 ----- 1 0 0 ----- ( a )A ll v a lu e s a re re p o rte d as p e rc e n t o f t o t a l p r o te in phosphorus. 51 n ot change d u rin g th e course o f th e in c u b a tio n . In th e se ta b le s th e n e t change in a c t i v i t y and c o n c e n tra tio n d u rin g th e in c u b a tio n a re a ls o r e corded, and th e s e a re th e v a lu e s th a t a re to be d isc u sse d . The d a ta re p o rte d h ere p re s e n t p erhaps th e g r e a te s t d i f f i c u l t i e s in in te r p r e ta tio n sin c e th e re i s c o n sid e ra b le la c k o f c o n siste n c y betw een, ex p erim en ts. N o n eth eless i t may be n o ted th a t once more th y ro x in e cau se s a d ecrease i n c o n c e n tra tio n o f r e s id u a l phosphorus (T able V I I I ) , alth o u g h th e p re v io u s ly m entioned s tim u la tio n o f uptak e by t h i s f r a c tio n ap p ears in o n ly one o f th e two ex p erim en ts. T his does r e p r e s e n t, how ever, p o s i t i v e in v i t r o e f f e c t s o f th y ro x in e on r e s id u a l phosphorus in e le v e n out o f th e p o s s ib le tw elve c ase s in c lu d e d in T ab les V t o V III. 32 In T able V II, one may a ls o n o te an in c re a s e d lo s s o f P from p h o s- p h o c re a tin e , unaccom panied by any e f f e c t o f phosphorus c o n c e n tra tio n (T able V I I I ) . llth o u g h no e f f e c t i s o b serv ab le in th e l i p i d f r a c tio n in e i t h e r ta b l e , i t may be n o ted th a t th e n u c le o p ro te in f r a c t i o n s , p a r t i c u l a r l y E M (T able V II) , show a sm all e f f e c t t h a t m ight p erh ap s be s ig n if ic a n t. No o th e r e f f e c t s on phosphorus c o n c e n tra tio n a re o b served. A f i n a l p o in t o f i n t e r e s t may be n o te d in T able V III where i t i s seen th a t th e re i s d e f i n i t e in d ic a tio n th a t during th e in c u b a tio n p e rio d th e re occu rs s y n th e s is o f p h o sp h o lip id , th e v a lu es s ifte r in c u b a tio n b e in g in ex cess o f 25fo over th e c o n tr o ls . No in te r p r e ta tio n o f t h i s e f f e c t may 'be made a t p re s e n t, alth o u g h i t i s o b v io u sly u n re la te d to th y ro x in e . I n c o n c lu sio n , i t may be t e n t a t i v e l y s ta t e d t h a t , in th e e x p e ri m ents o f T ables V and VI and in T ables V II and V III , th y ro x in e i n v i t r o caused an in c re a s e d tu rn o v e r o f p h o sp h o creatin e and r e s id u a l phosphorus 32 as w e ll a s a d e crea se i n c o n c e n tra tio n o f r e s id u a l phosphorus. F u rth e r more , th e re i s a ls o an in d ic a tio n th a t th y ro x in e may s tim u la te th e lo s s o f phosphorus from , h u t n o t th e accum ulation o f phosphorus i n , n u c le o p ro te in s . C H & P T E R 71 SDMMRY A stu d y h as b een made o f th e tu rn o v e r r a t e s and c o n c e n tra tio n o f v a rio u s phosphate f r a c tio n s o f r a t diaphragm m uscle a f t e r in c u b a tio n w ith P32. I t has been shown th a t th e tu rn o v e r r a t e s and c o n c e n tra tio n o f a c id -s o lu b le phosphorus compounds o f t h i s t is s u e a re changed i n an im als tr e a te d w ith th y ro id o r t h i o u r a c i l . S im ila r changes in tu rn o v e r r a t e s a re a ls o observed in th e p h o sp h o lip id and n u c le o p ro te in f r a c tio n s . In d ic a tio n s have been shown o f an in v i t r o e f f e c t o f th y ro x in e on th e tu rn o v e r o f a c id - s o lu b le phosphorus compounds o f r a t diaphragm m uscle, p a r t i c u l a r l y in th e r e s id u a l phosphorus f r a c t i o n . I t has been observed th a t d u rin g in c u b a tio n o f diaphragm segm ents, a s y n th e s is o f p h o sp h o lip id o c c u rs. BIBLIOG RA PH Y A bderhalden, E ., "A lco h o lic F erm en tatio n by Means o f Y east C e lls u n d er D iff e re n t C o n d itio n s .V I." Ferm entforschung, 6 : 149, 1922* A d le r, L*, and W. L ip s c h itz , "Die W irkung von Hormonen au f d ie Z e l l - o xydationen und den W aerm ehaushalt des O rg a n is m s ." Arch* f . ex p er. P a t h ,, 95: 181, 1922. A hlgren, G ., "T hyroxin und Gewebsatmiing zu r K enntnis der t i e r i s c h e n G ew ebsoxydation." Skand. A rc h ., 47: 1925 Suppl. A lth au sen , T. L . , and M, Stockholm , " In flu e n c e o f th e T hyroid Gland on A b so rp tio n in th e D ig e stiv e T r a c t." Am. J . p h y s io l., 123: 57?, 1938* Ammon, R ., and K » H in sb erg , "C o lo rim e trisc h e Phosphor- und A rsen sa u re - bestimmung m it A se o rb e n sau e re ." 2 . P h y s io l, Chem., 239: 207, 1936 Astwood, E. B.-, J . S u lliv a n , A dele B i s s e l l and R* T y slo w itz , "A ction o f C e rta in Sulfonam ides and o f T hiourea upon th e F u n ctio n o f th e T h y ro id Gland o f th e R a t." E n d o c rin ., 32: 210, 1943. B ark er, S. B ., " E ffe c ts o f T h y ro tro p h in On M etabolism o f T h io u ra c il- t r e a t e d R a ts ." E n d o c rin ., 57: 230, 1945. B ark er, S. B ., R. F. F u rc h g o tt, and E. S h o rr, "O rganic phosphate Exchanges in R e stin g C ard iac M uscle as In d ic a te d by R adio- A c tiv ity S tu d ie s ." Am. J . P h y s io l. , 133: P202, 1941. B erenblun, I . , E, C hain, and N. G. H e a tle y , "IX . The Study o f Metab o l i c A c tiv itie s o f Sm all Amounts o f S u rv iv in g T is s u e s ." Biochem. J . , 33: 6 8 , 1939. B ru es, A. M ., M. M. T racy, and W. E. Cohn, "The R e la tio n betw een N ucleic A cids and Growth. T urnover R ates of N u cleic A c id s." S c ie n c e , 95: 558, 1942. B ru es, A. M ., H. M . T racy , and W. E. Cohn, "N ucleic A cids of R at L iv er and H epatom a--T heir M etab o lic Turnover R ate in R e la tio n to Growth. J . B io l* Chem., 155: 619, 1944. C a n z a n e lli, A. R ., G u ild , and D. RA pport, "F u rth e r O b serv atio n s on th e E f fe c t o f T hyroglobU lin and Thyroxine oh th e Oxygen Consumption o f T issu e s in v i t r o . " E n d o c rin ., 25: 707, 1939*, 35 C a n z a n e lli, A ., and D. R ap p o rt, "The E ffe c t o f T h y ro g lo b u lin a n d 'R e la te d S ub stan ces on t h e ' Oxygen Consumption o f R at L iv er in v i t r o . ’* E n d o c rin ., 21: 779, 1937. Cohen, R, A ., and R. ty. G erard, ’’H yperthyroidism ' and B ra in O x id a tio n s” , jr. C e ll, and Comp. P h y s io l., 10: 223, 1937. C ra ig , F. N ., A. M. B a s s e t, and W . T. S a l t e r , * * A rtific ia l Benignancy o f Neoplasm. 171. O b serv atio n s on th e O x id ativ e B ehavior o f Tumors, A r t i f i c i a l l y B enign Tumors, and Homologous Normal T iss u e s ’*. C ancer R ese arch , 1: 859, 1941. C ra ig , F. N ., and W. T. S a l t e r ,; "V icario u s M etabolic R esponse—The I n f lu ence o f Blood from T h y ro xinized'A nim als on th e Oxygen Consumption o f S u rv iv in g Tissue**. E n d o c rin ., 37: 117, 1945. D elo ry , G. E . , "A Note on th e D eterm in atio n o f ’P h o sp h a te ' i n ' th e P resen ce o f I n te r f e r in g S u b sta n ce s’*. Biochem. J . , 32: 1161, 1938. B rab k in , D. L. "L iv er R eg en eratio n and Cytochrome C M etabolism .’ In flu en c e o f A noxia'and o f I n je c tio n o f Cytochrome C ." J . B io l. Chem., 171: 409, 1947. F u rc h g o tt, R. F . , and E . S h o rr, ’’Phosphate Exchange in R e s tin g C ard iac M uscle as In d ic a te d by R a d io a c tiv ity S tu d ie s . II.**. Fed. P r o c ., 1: 27, 1942. Gaddum, j ; H ., ‘ ’’Q u a n tita tiv e O b serv atio n s on Thyroxine and A llie d Sub s ta n c e s . I I . E f f e c ts on Oxygen Consumption o f R a t s .” J . p h y s i o l ., 6 8 : 383, 1930. G erard, R. W ., and M. M cIn ty re, " E f f e c t.o f T hyroid F eeding on T issu e R e s p ira tio n ”. Am. J . p h y s i o l ., 103: 225, 1933* Gordon* E. S ., and A. E . Heming, " E ffe c t o f T hyroid T reatm ent on R e sp ir a tio n o f V arious R at T is s u e s ’ *. E n d o c rin ., 34: 353, 1944. G reenberg, D. M ., J . F ra e n k e l-C o n ra t, and M. B. G lendening, " S tu d ie s w ith R ad io ac tiv e P h o sp h o ru s'o f A cid -S o lu b le phosphate Changes in H y p erthyroidism ” . Fed. P r o c .* 6 : 256, 1947. Haarmann, W ., "Ueber den E in f lu s s von Thyroxin au f d e n 'S a u e rs to ffv e r- b rau ch u e b erleb e n d e r Gewebe” . A rch, e x p tl. P a th , u . P h a m ., 180: 167, 1936. H a stin g s , A. B .' " E le c tr o ly te s 'O f T issu e s and Body F lu id s ” . H arvey L e c tu re s, 36: 91, 19S0-S1. 5 6 H a stin g s , A. B ., J . Mans, and 0. A. B essey, ’ ’T issu e M etabolism in V itam in D e fic ie n c ie s* I . The E f fe c t o f D e fic ie n c ie s in R ib o fla v in a n d 'th e H eat S ta b le V itam in B. Components” . J . B iol* Chem., 129: 295, 1939. J a n d o rf, B; J . , and B. H. W illiam s, "The E f f e c ts o f O ral A d m in istra tio n o f T h io u ra c il on th e M etabolism of I s o la te d T issu e s from norm al and H y p erth y ro id H a ts” . Am. J . P h y s io l., 141: 91, 1944. K alck ar, H. M ., J . D e h lin g e r, and A. M ehler, ”The R ate o f R eju v en atio n o f L a b ile ‘Phosphorus Compounds in M uscle and L iv e r” . T. B io l. Chem., 1 5 4 :, 275, 1944.. MacEachen, D ., ”The M etabolism o f I s o la te d S u rv iv in g T issu e s 'from Animals Rendered H y p erth y ro id w ith T hyroxine” . B u ll. Johns Hopkins Hosp. , 56: 145, 1935. Magnus-Levy, A ., "Ueber den R e s p ira to ris c h e n G asw echsel i n t e r den E in flu s s der T hy ro id en , sowie u n te r'V e rsc h ie d e n e h P a th o lo g isc h en Z ustaenden” . B e rl. k i l n . W oehschr., 32: 650, 1895. Palm er, W. W ., and J . P . L elan d , "Com parative C a lo rig e n ic A ctio n o f Normal and p a th o lo g ic a l T hyroid Glands A d m in istered in E qui- T hyroxine Doses” . _J. C lin . I n v e s t ., 14: 619, 1935. R ubino, A ., "Endogenous P u rin e M etabolism and th e v e g e ta tiv e Nervous System o f th e E ndocrine G lands. I . In flu e n c e o f th e T hyroid Gland on t h e 'Endogenous P u rin e M etabolism ” , p o lic lin ic o (Rome) M ed., 48: 349, 1941. S ack s, J . , and C. H. A lts h u le r , "R ad io activ e Phosphorus S tu d ie s on S tr ia te d 'a n d C ard iac Muscle M etabolism ” . Am. J . P h y s io l., 137: 750, 1942. S a l t e r , W. T , , and P. N. C ra ig , "V icario u s M etab o lic Response— Oxygen Consumption o f S u rv iv in g T issu e in Plasm a from H y p erth y ro id A nim als” . J . C lin . I n v e s t . , 17: 502, 1938. Schm idt, G ., and S. J . T annhauser, ”A Method f o r th e D eterm in atio n o f D eso x y rib o n u cleic A cid, R ib o n u c le ic 'A c id , and p h o sp h o p ro tein in Anim al T iss u e s ” . J . B io l. Chem., 161: 83, 1945. Shannon, A ., and W. C. W erk h eiser, "A M icro-M ethod f o r th e D eterm in atio n o f Phosphorus” . U npublished D ata, S m elser, G. K ., "Oxygen Consumption o f ly e M u sc le 'o f T hyroidectom ized and T hyroxine I n je c te d Guinea P ig s ” . Am. J* P h y s io l. , 142: 396, 1944. ' Spiegelm an, S .,'a n d Kamen, M. D ., "G enes'and N u c leo p ro tein s in th e S y n th e sis o f Enzymes". S c ie n c e , 104: 581, 1946. 37 S te rn h e im e r, R . ' ”l f f e e t o f a S in g le I n je c tio n o f T hyroxine on C arb o -' h y d ra te s , P r o te in , and Growth in R at L iv e r” . E n d o c rin ., 25: 899, 1939. T ip to n , S. R ., and N ixon, W. L . , ”The E f fe c t o f T h io u ra c il on S uecin o x id ase and Cytochrome O xidase o f R at L iv e r” . E n d o crin . , 39: 300, 1946. T i s s ie r e s , A ., ”T hyroid an d .A d ren al Glands and M uscular Cytochrome C o f th e R at”. A rch. I n te r n a tio n a le s P h y s io l., 54: 305, 1946. Tweedy, W. R ,, M. E. C h ilc o te , and M. C. P a tr a s , ”D is tr ib u tio n , R e te n t io n , and E x c re tio n o f R ad io ac tiv e Phosphorus fo llo w in g T hyropara- th y ro id ecto m y and th e I n je c tio n o f P a ra th y ro id E x tr a c t”. Fed. P r o c ., 5 : 159, 1946. U m breit, W . W., R.H. B u r r is , and J , F. S ta u f f e r , ”M anom etric Techniques and R e la te d Methods f o r th e 'S tu d y o f T issu e M etabolism ”. B urgess P u b lish in g C o ., M in n eap o lis, 1945. W arren, C. 0 . , and F. G. Ebaugh, ”The E f fe c t o f V arious Ib n s on th e ' A naerobic G ly c o ly sis o f R at L iv e r in v i t r o ”. Am. J . P h y s io l., 147: 509, 1946* In iu u /si'y cf S ^ u Vin m i- . * 1 1 - rn Caifvom ja Llktm

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Creator Werkheiser, William Clark (author)

Core Title The effect of thyroxine on the rate of incorporation of P32 into the phosphorus-compounds of rat diaphragm in vitro

Contributor Digitized by ProQuest (provenance)

Degree Master of Science

Degree Program Biochemistry

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-775872

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Rights Werkheiser, William Clark

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