University of Southern California Dissertations and Theses

Studies on metabolism of tissues. I. The polarographic measurement of cytochrome oxidase in liver homogenates. II. Studies on the effect of thyroxine on respiration of isolated rat diaphragm

(USC Thesis Other)

Studies on metabolism of tissues. I. The polarographic measurement of cytochrome oxidase in liver homogenates. II. Studies on the effect of thyroxine on respiration of isolated rat diaphragm

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content STUDIES ON METABOLISM OF TISSUES I . THE POLAROGRAPHIC MEASUREMENT OF CYTOCHROM E OXIDASE IN LIVER HOMOGENATES I I . STUDIES ON THE EFFECT OF THYROXINE ON RESPIRATION OF ISOLATED RAT DIAPHRAGM A T h e s is P r e s e n t e d t o th e F a c u lt y o f th e D ep artm en t o f B io c h e m is tr y The U n iv e r s i t y o f S o u th e r n C a l i f o r n ia I n P a r t i a l F u l f il l m e n t o f t h e R eq u irem en ts f o r t h e D e g re e M aster o f S c ie n c e b y Jam es E ugene F a s o la s C a p t. U .S .A .F . A u g u st 1 9 4 9 UMI Number: EP41303 All rights reserved IN FO R M A TIO N 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 EP41303 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 4 8 1 0 6 -1 3 4 6 Bio p ^ D a ^ ..A l2g L L S .t...l9.^ $- Faculty Committee / Chairnmn KO^jJkS L -e !FAu thesis, written by .... p JA.^.S..E.,...PASOLAS t............. y * * under the guidance of h..%.P.. Faculty Committee, and approved by all its members, has been presented to and accepted by the Council on \T \ Graduate Study and Research in partial fulfill ment of the requirements for the degree of MASTER OP SCIENCE ..E ^ .S ^ .B a g .a r.d u .a.... Sean TABLE OP CONTENTS CHAPTER PAGE THE POLAROGRAPHTC MEASUREMENT OF CYTOCHROM E OXIDASE IN LIVER HOMOGENATES . . . . . . . . . 1 I . INTRODUCTION........................................ 1 I I . LITERATURE....................................................................................... 2 I I I . M ETHODS............................................................................................ 3 IV . R E S U L T S ........................................................... 7 R e l a t i o n b e tw e en r e s p i r a t i o n r a t e and am ount o f h om ogen ate • •• •• • • • • • • 7 C om p arison o f p o la r o g r a p h ic and Warburg m e th o d s. • • • . • • • • • • • • • • • • • 12 V . DISCUSSION AND SUMMARY........................................................ 14 STUDIES ON THE EFFECT OF THYROXINE ON RESPIRA TION OF ISOLATED RAT DIAPHRAGM......................... 15 V I . INTRODUCTION .............................................. 1 5 V I I . LITERATURE ................................................... 1 6 V I I I . METHODS . 1 9 IX . RESULTS..................................... . . . 23 X . DISCUSSIO N................................................. 39 BIBLIOGRAPHY............................• • • • ................................................. 39 LIST OP TABLES TABLE PAGE I . T a b u la tio n o f S o lu t io n s U sed i n C ytochrom e O x id a se Enzyme S y stem . . • • • • • • • • * 9 I I . T a b u la tio n o f G alvan om eter D e f l e c t i o n s v s . Time i n M in u tes • • • • • • • • • • • • • • 1 0 I I I . S o lu t io n s U sed i n Warburg E x p erim en t . . . . 1 5 IV . The I n V it r o E f f e c t o f T h y r o x in e i n R in g e r P h o sp h a te G lu c o s e (R .P .G .) and Normal Serum ( N . S . ) ................................................................................... 24 V* S t u d ie s on t h e I n V itr o E f f e c t o f T h y ro x in e i n R in g e r P h o sp h a te G lu c o s e O ver a n E ig h t - Hour P e r io d • • • • • . • • • • • • • • • • 26 V I. The I n V it r o E f f e c t o f T h y r o x in e i n Norm al Serum O ver a N in e-H ou r P e r io d • • • • • • • 29 V I I . The I n V it r o E f f e c t o f H y p e r th y r o id Serum • • 34 V I I I . The I n V it r o E f f e c t o f A dding H y p e r th y r o id Serum t o R .P .G . 35 LIST OP FIGURES FIGURE PAGE 1 , The P r i n c i p l e o f th e H e y r o v sk y -S h ik a ta P o la r o g r a p h • • « • • • • • • • • 5 2 . The E f f e c t o f C o n c e n tr a tio n o n th e R ate o f 11 R e s p ir a t io n • • • • • • • • • • . » • • • • • 3* S c h e m a tic D iagram o f R at D iaphragm Show ing th e M ethod o f R em oval o f T is s u e • • » • • • • 21 4« The I n V it r o E f f e c t o f T h y ro x in e i n R in g e r P h o sp h a te G lu c o se and Normal Serum • • . . . 2 5 5 . The I n V it r o E f f e c t o f T h y ro x in e i n R in g e r P h o sp h a te G lu c o se • • • • •• • « • •• • •* 2 7 6* The I n V it r o E f f e c t o f H y p e r th y r o id Seram • * • 3 0 7* The I n V it r o E f f e c t o f T h y r o x in e i n Normal S e r u m .........................♦ . . .................................................... . 36' 8* The I n V it r o E f f e c t o f H y p e r th y r o id Serum and Norm al Serum • • • • • • • • • • • • • • 333 PART I THE POLARGGRAPHIC MEASUREMENT OF CYTOCHROM E OXIDASE IN LIVER HOMOGENATES CHAPTER I INTRODUCTION I t i s th e p u rp o se o f t h i s t h e s i s t o p r e s e n t p r e lim in a r y w ork w h ic h i n d i c a t e s t h a t th e cy to ch ro m e o x id a s e c o n te n t o f h om ogen ized t i s s u e may h e d e te r m in e d u s in g th e p o la r o g r a p h ic m eth od o f a n a l y s i s . The d e v e lo p m en t o f t h i s te c h n iq u e sh o u ld a f f o r d a m eans o f f a c i l i t a t i n g s t u d i e s on th e r e s p i r a t o r y a c t i v i t i e s o f c e l l s . The sta n d a r d p r e s e n t d ay t e c h n iq u e s in v o lv e th e r a th e r t e d io u s in s t r u m e n t a t io n o f th e W arburg te c h n iq u e and a l s o p e r m it o f enzym e d e c o m p o s itio n due t o th e lo n g p e r io d s n e c e s s a r y to s e t up th e a p p a r a tu s . The p r e s e n t work w as i n i t i a t e d a s a r e s u l t o f a d e s i r e to s tu d y th e e f f e c t s o f ca rb o n m onoxid e on th e c y t o chrom e o x id a s e sy ste m o f a n im a l t i s s u e s . The program was d is c o n t in u e d a s a r e s u l t o f c e r t a i n t e c h n i c a l d i f f i c u l t i e s p e r t a in in g t o th e o p t i c a l s y s te m . The r e s p i r a t i o n s t u d i e s , h o w ev er, had p r o g r e s s e d s u f f i c i e n t l y f a r to m e r it t h e i r d e s c r i p t i o n a s p a r t o f t h i s t h e s i s . CHAPTER I I LITERATURE * * ' S in c e i t s d e v e lo p m en t b y H ev ro v sk y and S h ik a ta (1 9 2 5 ) th e p o la r o g r a p h h a s had many and v a r ie d a p p l i c a t i o n s i n th e r e s e a r c h la b o r a t o r y . O f p a r t i c u l a r i n t e r e s t to th e p r e s e n t p rob lem i s t h e te c h n iq u e f o r s t u d y in g th e r e s p i r a t i o n o f y e a s t s u s p e n s io n s a s d e v e lo p e d b y M il l e r and Baum berger ( 1 9 3 5 ) , and e la b o r a t e d upon b y M il l e r (1 9 3 9 ) and B aum berger ( 1 9 3 9 ) . The Qog v a lu e s o b ta in e d b y d u p lic a t e r u n s on th e W arburg a p p a r a tu s . The te c h n iq u e w as f u r t h e r a p p lie d to th e s tu d y o f th e m e ta b o lis m o f y e a s t u n d er v a r io u s c o n d it io n s (W inaL er, 1 9 4 1 , 1 9 4 3 ) . The a p p l i c a t i o n o f t h i s m eth od to s t u d i e s o f th e r e s p i r a t i o n o f a n im a l t i s s u e s h a s th u s fa r b e e n a p p lie d o n ly b y W arren (1 9 4 2 ) to th e m e ta b o lism o f b on e marrov/ c e l l s u s p e n s io n s . The a p p l i c a t i o n o f th e te c h n iq u e to o t h e r t i s s u e s h a s n o t b e e n d e v e lo p e d . The im m ed ia te p u rp o se o f t h i s work w as to d e v e lo p r a p id m eth od s f o r m e a su r in g th e a c t i v i t y o f th e cytoch rom e i o x id a s e sy stem o f a n im a l t i s s u e s . The p r o c ed u r e w as b a se d on th e u s e o f h om ogen ized t i s s u e s f o r t i f i e d w ith cytoch rom e C a s d e v e lo p e d b y P o t t e r (1 9 3 6 , 1 9 4 0 , 1 9 4 2 ) , S c h n e id e r and P o t te r ( 1 9 4 3 ) , D u B ois and P o t t e r (1 9 4 3 ) and T ip to n and N ix o n ( 1 9 4 6 ) . CHAPTER I I I METHODS The p o la r o g r a p h ic m ethod o f a n a ly s e s i n v o l v e s th e d e te r m in a tio n o f s u b s ta n c e s w h ich a r e r e d u c ib le a t th e d r o p p in g m ercu ry e l e c t r o d e . The v o lt a g e a c r o s s th e e le c t r o d e i s ch a n g ed and th e c u r r e n t m easu red b y m eans o f a c i r c u i t shown s c h e m a t ic a lly i n F ig u r e 1 . S in c e o x y g e n i s r e d u c ib le a t th e d r o p p in g m ercu ry e l e c t r o d e and a t any f i x e d v o l t a g e th e c u r r e n t i s p r o p o r t io n a l t o th e c o n c e n t r a t io n o f o x y g e n i n th e s o l u t i o n i n w h ic h th e d r o p p in g m ercu ry e l e c t r o d e i s im m ersed , i t i s p o s s i b l e t o m easu re c h a n g e s , i n o x y g en t e n s i o n i n a s o l u t i o n a s a f u n c t io n o f tim e* From su c h c h a n g e s and t h e B unsen s o l u b i l i t y c o e f f i c i e n t , th e f l u i d volum e and th e t i s s u e w e ig h t th e Q .0 2 o f th e c e l l s ca n b e c a lc u la t e d * T h is c a l c u l a t i o n w as made a s f o l l o w s : ^ ° 2 = J = N x ° * 23l x x w h ere A - A ir v a l u e , g a lv a n o m e tr ic r e a d in g w ith s o l u t i o n i n e q u ilib r iu m w it h a i r ( 2 1 #(>2 ) B - N itr o g e n v a l u e , g a lv a n o m e tr ic r e a d in g w it h a l l o x y g en rem oved S - S lo p e , ch an ge i n g a lv a n o m e tr ic r e a d in g p e r m in u te - B unsen s o l u b i l i t y c o e f f i c i e n t (0 * 0 2 3 7 a t 37°C *) W - D ry w e ig h t o f t i s s u e / m l * o f s u s p e n s io n i n m i l l i gram s. The a p p a r a tu s u s e d w as a H evrovsk y P o la r o g r a p h manu f a c t u r e d b y E . H* S a r g e n t and Company, C h ic a g o , I l l i n o i s * F ig u r e 1 i s a s c h e m a tic d ia g ra m o f th e p o la r o g r a p h . S in c e th e a p p lie d v o l t a g e w as h e ld c o n s t a n t th r o u g h o u t th e e x p e r i m en ts th e m o to r ( A ) , w h ic h d r i v e s th e a p p lie d v o l t a g e drum ( B ) , w as n o t tu r n e d on* The s e n s i t i v i t y (F ) was s e t a t 1 0 , th e a p p lie d v o lt a g e (H) a t -0 * 5 Z and t h e g a lv a n o m e te r d e f l e c t i o n s r e a d and r e c o r d e d a t d e f i n i t e tim e i n t e r v a l s d e p e n d in g upon th e r a t e a t w h ic h th e r e a d in g s d e c r e a se d * The i n t e r v a l b e tw e e n r e a d in g w as u s u a l l y 1 5 s e c o n d s t o on e m in u te* The r e s p i r a t i o n c e l l w as c o n s t r u c t e d w it h a w a te r j a c k e t s o t h a t w a te r a t 3 7 °C . c o u ld b e c i r c u l a t e d th r o u g h i t * By m a in t a in in g a l l s o l u t i o n s e x c e p t th e t i s s u e h o m o g en a te, a t 37°C* p r i o r t o a d d in g t o th e t e s t c e l l , a tim e i n t e r v a l o f a b o u t one m in u te w as n e c e s s a r y b e f o r e th e c e l l r e a c h e d a n e q u ilib r iu m te m p e r a tu r e w ith th e c i r c u l a t i n g w a ter* T h is i n t e r v a l w as u t i l i z e d i n sh a k in g th e s o l u t i o n to s a t u r a t e i t w it h a i r p r io r t o e a c h run* The h o m o g e n iz in g a p p a r a tu s and s o l u t i o n s u s e d a r e d e s c r ib e d i n TJmbreit e t a l . (1 9 4 5 ) b y P o t t e r i n C h ap ter N in e o f "M anom etrlc T e c h n iq u e s " . I t w as fo u n d t h a t th e sodium a s c o r b ic s o l u t i o n ( 0 .1 1 4 M ) had t o b e made f r e s h d a l l y and u s e d a s so o n a f t e r i t s p r e p a r a tio n a s p o s s i b l e b e c a u s e a u t o o x i d a t i o n te n d s to d e s t r o y i t s u s e f u l n e s s I n th e enzym e s y s te m . I A '0 H The principle of the Hevrovsky-Shikata Polarograph. j 5 The cy to ch ro m e C w as o b t a in e d c o m m e r c ia lly and c o n ta in e d 5 m g /c c . The maximum g a lv a n o m e te r r e a d in g (m ax, o x y g en ) w as o b t a in e d on e a c h sam p le a f t e r th e ru n w as c o m p le te d b y a d d in g sodium a z id e and s h a k in g w it h a i r u n t i l a c o n s ta n t r e a d in g w as o b t a in e d . Z ero o x y g e n w as o b ta in e d when th e g a lv a n o m e te r c e a s e d t o d e f l e c t a t t h e en d o f e a c h r u n . The r a t s w ere s a c r i f i c e d e i t h e r b y d e c a p i t a t io n o r a b lo w on th e h e a d . The t i s s u e rem oved i n a l l c a s e s w as th e l i v e r . A f t e r a p r e lim in a r y w e ig h in g t h e t i s s u e w as h o m ogen ized a c c o r d in g to P o t t e r (1 9 5 6 ) and ‘ D m b reit e t a l , ( 1 9 4 5 ) , Mid th e n e c e s s a r y volum e add ed t o th e s o l u t i o n fo r s t u d y . An a l i q u o t p o r t io n o f th e c e l l u l a r s u s p e n s io n w as rem oved , d r ie d and w e ig h e d t o d e te r m in e th e a c t u a l d r y w e ig h t o f t i s s u e p e r c c . T h is s e r v e d t o c h e c k a lo n g w it h th e p r e lim in a r y w e ig h in g th e p e r c e n t d r y w e ig h t o f th e t i s s u e ( u s u a l l y 3 0 $ ) • A 5$ b y w e t w e ig h t s o l u t i o n o f th e t i s s u e was fo u n d to b e th e b e s t . No s e t t l i n g o u t w as n o t e d and th e r a t e o f o x y g e n co n su m p tio n w as r a p id en ou gh t o e li m in a t e e r r o r s t h a t may o c c u r w it h lo n g r u n s . CHAPTER IV RESULTS I . RELATION BETWEEN RESPIRATION RATE AND AM OUNT OP HOM OGENATE I f th e enzym e c o n c e n t r a t io n i s th e l i m i t i n g f a c t o r i n t h e r a t e o f o x y g e n c o n su m p tio n , th e r a t e o f o x y g e n c o n su m p tio n s h o u ld h e p r o p o r t io n a l t o t h e am ount o f hom ogen ate em p loyed w it h in c e r t a i n l i m i t s * The u p p e r l i m i t i s a c o n c e n t r a t i o n a t w h ic h th e r a t e i s to o r a p id f o r v i s u a l r e c o r d in g o f t h e g a lv a n o m e te r d e f l e c t i o n s and th e lo w e r l i m i t a t a c o n c e n t r a t io n w h e r e th e r a t e i s so slo w t h a t o t h e r u n p r e d ic t a b l e e r r o r s r e s u l t . S u ch t h in g s a s s e t i l i n g o u t o f th e h o m o g en a te, d e s t r u c t i o n o f h e a t l a b i l e e n e m a s o r s u b s t r a t e s and te m p e r a tu r e f l u c t u a t i o n s e f f e c t th e lo w e r l i m i t s o f c o n c e n tr a t io n * T a b le I show s a t a b u l a t i o n o f th e s o l u t i o n s u s e d i n a t y p i c a l e x p e r im e n t i n v e s t i g a t i n g t h e in c r e a s e i n th e r a t e C F f r e s p i r a t i o n o f th e cy to eh ro m e o x id a s e sy ste m w it h enzym e c o n c e n tr a t io n * I n a l l c a s e s a f r e s h enzym e s o l u t i o n w as made up f o r e a c h d e te r m in a t io n u s in g a l i q u o t s o f th e o r i g i n a l t i s s u e hom ogenate* T a b le I I show s th e g a lv a n o m e te r r e a d in g s a s a f u n c t io n o f tim e f o r e a c h o f th e t h r e e ru n s o f T a b le I* R e a d in g s w ere made on t h e 1 /2 o r l / 4 m in u te a s deem ed n e c e s s a r y . The a i r v a lu e i n t h e s e e x p e r im e n ts w as 1 00 and th e n it r o g e n v a lu e w as 4 . F ig u r e I I i s a g ra p h o f th e g a lv a n o m e te r r e a d in g s a g a i n s t tim e a s r e c o r d e d i n T a b le I I . The r e a d in g s d u r in g th e f i r s t i n i t i a l m in u te a r e i n v a l i d due t o tu r b u le n c e o f t h e s u s p e n s io n . A f t e r t h i s p e r io d th e s lo p e o f th e c u r v e s i n c r e a s e i n th e i n c r e a s i n g enzym e c o n c e n t r a t io n . I t w i l l a l s o b e n o te d t h a t t h e r a t e s lo w s down rem ark a b ly a t a b o u t a r e a d in g o f 2 3 on th e g a lv a n o m e te r . The r e a s o n f o r t h i s i s unknown b u t i t i s s u s p e c t e d t o h in g e upon t h e o x y g e n d i s s o c i a t i o n c o n s t a n t o f th e enzym e s y s te m . F u r th e r work w i l l h a v e t o b e d on e a lo n g t h e s e l i n e s . T h ese e x p e r im e n ts d e m o n str a te th e n a l i n e a r r e l a t i o n s h ip b e tw e e n enzym e c o n c e n t r a t io n and r a t e o f o x y g en consum p t i o n . TABLE I TABULATION OP SOLUTIONS USED IN CYTOCHROM E OXI DASE ENZYME SYSTEM S o lu t io n 1 2 3 . . . * . ^ o o c c c c 1 H om ogenate* 0 .5 0 1 .0 0 2 .0 0 5 m g /cc C ytochrom e C 0 .2 5 0 .2 5 0 .2 5 0 .1 1 4 M pH 7 .4 Na A s c r o b a te 0 .3 0 0 .3 0 0 .3 0 A1C13 4 x 1 0 ” 3 M 0 .3 0 0 .3 0 o . o pH = 7 .4 5 P h o sp h a te b u f f e r 1 .0 0 1 .0 0 1 .0 0 h2 o 0 .6 5 0 .6 5 0 .6 5 T o t a l volum e 3 .0 0 3 .7 5 5 .0 0 * H om ogenate - 0 .0 2 7 1 gms d r y w t . / c c . . ■ . : . : : : : ’ • ....... - - 10 TABLE I I TABULATION OF GALVANOMETER DEFLECTIONS VS, TIME IN MINUTES ________ . . M in u te s S o l n .r * S o ln . S o ln 1 2 3 0 109 1 13 7 6 1 / 4 - 99 56 1 /2 86 76 40 3 / 4 . « ► 67 2 4 1 6 3 55 19 1 1 / 4 - 44 16 1 1 / 2 49 36 « » 2 36 2 4 „ 2 1 / 4 - 21 - 2 1 /2 25 1 1 8 - 2 3 / 4 - 17 - 3 19 15 — 3 1 / 4 - 14 - 3 1 /2 15 12 - R ate**' S .D ./M in . 2 7 43 66 Q08 ‘ 9 4 .4 9 4 .0 9 7 . * H om ogenate - 0 .0 2 7 1 gms d r y w t , / c c , * * R a te I n d ic a t e d I s i n s c a l e d i v i s i o n on th e g a lv a n o m e te r p e r m in u te . galvanometer d e f l e c t i o n s GRAPH-I THE EFFECT OF CONCENTRATION ON THE RATE OF RESPIRATION ♦ 0 .5 CC HOMOGENATE I.OOCC " 2 .0 0 CC - MA&Ot MIN 02 TIME IN MINUTES I I . COMPARISON OF POLAROGRAPHIC AND W ARBURG METHODS 12 I n an e f f o r t t o c h e ck th e v a lu e s o b ta in e d w ith t h i s m ethod i t w as d e c id e d t o com pare th e r e s u l t s w it h a c o n t r o l e x p e r im e n t u s in g th e sta n d a r d W arburg tec h n iq u e-. T h rou gh ou t th e e n t i r e i n v e s t i g a t i o n th e v a lu e s o b t a in e d f o r th e Qog o f t h e enzym e sy s te m had b e e n lo w when com pared to t h o s e r e p o r t e d i n th e l i t e r a t u r e . The c o m p a r iso n w it h t h e W arburg te c h n iq u e r e s u l t s w as u s e d In an e f f o r t t o d is c o v e r th e r e a s o n f o r t h e lo w v a l u e s . The same s o l u t i o n s I n th e same r e l a t i v e c o n c e n t r a t i o n s w ere u s e d i n b o th W arburg and p o la r o g r a p h ic s t u d i e s . T a b le ..I I I g i v e s a t a b u l a t i o n o f th e s o l u t i o n s u s e d i n th e W arburg e x p e r im e n t. The Q .O g v a lu e s f o r th e cy to ch ro m e o x id a s e sy s te m a s o b t a in e d i n t h i s e x p e r im e n t w ere 1 7 2 .5 ram^Og/hr/mg d r y w e ig h t and 1 8 2 .9 m m 30 g /h r /m g d r y w e ig h t f o r f l a s k s num bers 1 and 2 . Im m e d ia te ly a f t e r th e W arburg ru n w as c o m p le te d a p o la r o g r a p h ic d e t e r m in a t io n w as made on a s o l u t i o n made up a s i n f l a s k number 1 o f T a b le I I I . The Qog v a lu e o b t a in e d w as 1 6 4 .5 mm®/hr/mg d r y w e ig h t . The a g r ee m e n t b e tw e en t h e two m eth od s th u s w as n o t p e r f e c t b u t c a n b e c o n s id e r e d s a t i s f a c t o r y f o r p r e lim in a r y e x p e r im e n ts . 1 5 TABLE I I I SOLUTIONS USED IN W ARBURG EXPERIMENT F la s k * ............. ........... i " 3 S o l u t io n s ■ - T h erm o-- - . b a ro m eter HgO t o make 3 c c . . . . . . . . 1 .0 5 ’ ITS ~~ " 0 .9 5 3 .0 0 P04 b u f f e r pH 7 .4 5 • 1 .0 0 1 .0 0 mm C ytochrom e C 5 m g /c c . 0 .2 5 0 .2 5 mm- A1C13 . 4 x 1 0 “% 0 .3 0 0 .3 0 A s c o r b a te 0 .1 1 4 M 0 .3 0 0 .3 0 mm 1% l i v e r hom ogenate 0 .1 0 .2 0 mm * 0* 1 m l. 2N KO H i n c e n t e r cup w it h 1 cm2 f i l t e r p a p er f o ld e d CHAPTER V DISCUSSION AND SUM M ARY T h ese e x p e r im e n ts c a n n o t b e c o n s id e r e d e x t e n s i v e . en ou gh t o e s t a b l i s h t h e v a l i d i t y o f t h e .p o la r o g r a p h ic m ethod f o r d e te r m in a tio n o f th e cy to ch ro m e o x id a s e a c t i v i t y o f th e t i s s u e h o m o g e n a te s, The work w as d is c o n t in u e d b e f o r e su c h d e f i n i t i v e e x p e r im e n ts w ere a tte m p te d . H ow ever, th e r e s u l t s a r e s u f f i c i e n t l y e n c o u r a g in g t o i n d i c a t e t h a t th e p o la r o g r a p h ic m eth od may h a v e p r o m ise f o r th e s tu d y o f t h e m eta b o l is m o f t i s s u e h om ogen ates* PART IX STUDIES ON THE EFFECT O F THYROXINE ON RESPIRATION OF ISOLATED RAT DIAPHRAGM CHAPTER VI INTRODUCTION An o u t s ta n d in g e f f e c t o f h y p e r th y r o id is m i n man and e x p e r im e n ta l a n im a ls i s a m arked i n c r e a s e i n t h e r a t e o f m eta b o lism * T h is i n c r e a s e i s o x y g e n co n su m p tio n i s m a n ife s te d b y a n i n c r e a s e on t h e r e s p i r a t o r y r a t e o f t h e i n d iv i d u a l t i s s u e s and i t c a n r e a d i l y b e d e m o n str a te d t h a t t h e r e s p i r a t i o n o f t i s s u e s e x c i s e d from h y p e r th y r o id a n im a ls consum e o x y g e n m ore r e a d i l y th a n t i s s u e s fro m n orm al a n im a ls* T h ere h a s b e e n no e x p la n a t io n f o r th e i n c r e a s e i n t h e t i s s u e m etab o l i s m o f h y p er t h y r o id a n im a ls* Many e x p e r im e n ts h a v e b e e n c a r r ie d o u t i n e f f o r t s t o d e m o n s tr a te a n Im m ed iate e f f e c t o f t h y r o x in e o r o f t h y r o g lo b u lin on t i s s u e s , th e r e s u l t s o f t h e s e e x p e r im e n ts b e in g i n g e n e r a l c o n f l i c t i n g * I t i s th e p u r p o se o f t h i s t h e s i s t o r e p o r t a n e x p e r i m ent o n t h e e f f e c t o f t h y r o x in e on th e r e s p i r a t i o n o f e x c i s e d r a t d iap h ragm , a t i s s u e w h ic h h a s n o t p r e v io u s l y b e e n s t u d ie d fro m t h i s p o in t o f view * T h is t i s s u e h a s th e a d v a n ta g e o f b e in g t h i n en ou gh t h a t s l i c i n g and uncom m it a n t i n j u r y t o t h e t i s s u e ca n b e a v o id e d * I t seem s p o s s i b l e t h a t s u c h i n j u r y may p r e v e n t th e r e s p o n s e o f a t i s s u e t o added th y r o x in e * CHAPTER V II LITERATURE S in c e t h e y e a r 1 8 9 5 , when Magnus -L e v y show ed t h a t th e t h y r o id g la n d in f lu e n c e d m e ta h o lis m , i n v e s t i g a t o r s h a v e a tte m p te d t o e l u c i d a t e th e m ode o f a c t i o n o f t h e g la n d . The e x a c t m anner i n w h ic h th e th y r o id horm one a f f e c t s m e ta b o lism i s s t i l l unknow n. I n v e s t i g a t o r s h a v e a tte m p te d t o b r e a k th e p ro b lem down i n t o v a r io u s p h a s e s . The s t i m u l a t i o n o f m e ta b o lism by i n v iv o a d m in is t r a t io n o f th y r o x in e and o t h e r th y r o t r o p h ic com pounds h a s b e e n f i r m l y e s t a b l i s h e d . T h y ro x in e when i n j e c t e d i n t o th e b lo o d str e a m o r ta k e n o r a l l y c a u s e s a g r a d u a l I n c r e a s e i n t h e m e ta b o lis m o f t h e t i s s u e s r e a c h in g a maximum i n a b o u t s e v e n ty -tw o h o u rs (H opp in g 1 9 5 1 , M ayer e t a l . 1 9 5 5 , T ip to n e t a l . 1 9 4 6 ) • An i n v i t r o e f f e c t o f t h y r o x in e I s s t i l l n o t s a t i s f a c t o r i l y e s t a b l i s h e d , but th e c o n c e n su s o f p a p e r s w o u ld i n d i c a t e t h a t t h e r e i s no Ln v i t r o t h y r o x in e e f f e c t . T h is may b e p r im a r ily d u e t o t h e " tim e -la g " e f f e c t n o te d i n t h e th y r o x in e i n j e c t i o n e x p e r im e n ts . E x c e l le n t r e v ie w s o f t h e l i t e r a t u r e c o n n e c te d w it h th e p r o b le m o f i n v iv o and i n v i t r o e f f e c t s o f th y r o x in e and o t h e r t h y r o t r o p ic compounds a p p e a r i n p a p e r s o f H opping ( 1 9 5 1 ) , M eyer et^ a l . ( 1 9 5 5 ) , an d C r a ig e t, a l . ( 1 9 4 5 ) . A l l a g r e e t h a t t h e r e s u l t s t o d a t e a r e c o n f u s in g an d som ew hat c o n t r a d ic t o r y , 17 b u t r e p o r t I n t h e i r p a p e r s t h a t t h y r o x in e , a s s u c h , had no e f f e c t u p on t h e m e ta b o lism o f norm al i s o l a t e d t i s s u e when added i n v i t r o . Two f a c t s h a v e s t im u la t e d r e s e a r c h i n t o a n o th e r p h a se o f t h e t h y r o id g la n d * s a c t i o n ; f i r s t , t h e " t im e - la g ” e f f e c t o f i n j e c t e d t h y r o x in e t o in d u c e e le v a t e d m e t a b o lic p r o c e s s e s and s e c o n d , t h e n o n -r e s p o n s e o f t i s s u e t o th y r o x in e a s s u c h i n v i t r o . S in c e t h e a fo r e m e n tio n e d f a c t s h a v e b e e n f a i r l y u n i v e r s a l l y a c c e p t e d , and s i n c e th e p r o t e i n bound i o d in e o f t h e b lo o d o f h y p e r th y r o id a n im a ls i s in c r e a s e d ( B a s s e t e t a l . 1 9 4 1 ) , t h e p o s s i b i l i t y e x i s t s t h a t t h i s h y p e r th y r o id b lo o d m ig h t I n c r e a s e th e m e ta b o lism o f norm al i s o l a t e d t i s s u e , i n v i t r o . The f i r . s t o f w h at may p r o v e t o b e a lo n g l i n e o f p a p e r s on t h i s s u b j e c t was p u b lis h e d b y C r a ig and S a l t e r ( 1 9 4 5 ) . T h ese w o r k e rs r e p o r t a n i n c r e a s e o f tw e n ty p e r c e n t i n t h e m e ta b o lis m o f sarcom a 1 8 0 when t h e t i s s u e w as In c u b a te d i n t h e b lo o d o f h y p e r th y r o id g u in e a p i g s f o r tw o h o u r s a t 2 0 °C . I n t h e sam e p a p er i t w as shown t h a t Qpg o f m ouse l i v e r s u s p en d ed i n h y p e r th y r o id m ouse b lo o d was 13 t o 3 7 $ h ig h e r th a n c o n t r o l t i s s u e su sp e n d e d i n norm al m ouse b lo o d . T h is f i n d i n g , i f s u b s t a n t i a t e d , w o u ld be a m o st im p o r ta n t o b s e r v a t io n . A m ajor d e f e c t i n t h e ex p e r im e n ts o f C r a ig and S a l t e r w as t h a t t h e d i f f e r e n c e b e tw e en t h e tw o g ro u p s w as n o t a s t i m u l a t i o n o f r e s p i r a t i o n but a p a r t i a l p r o t e c t i o n a g a in s t t h e r a p id f a l l i n r a t e w it h tim e n o te d i n t h e c o n t r o l s . T h is f a l l i n 1 8 r e s p i r a t i o n w it h I n c u b a tio n tim e i s c h a r a c t e r i s t i c o f a l l s l i c e d t i s s u e s and i t w as h o p ed i n th e p r e s e n t s t u d i e s t h a t i t c o u ld b e a v o id e d b y u s in g u n s l i c e d r a t diaphragm * CHAPTER V I I I METHODS E ach ex p erim en t w as s e t up s e p a r a t e l y and c a r r ie d o u t t o p r o v e o n e p o in t and s o th e y w i l l b e d is c u s s e d I n d i v i d u a l l y . T here a r e a few te c h n iq u e s common t o a l l and t h e s e w i l l b e d is c u s s e d i n g e n e r a l . The t i s s u e u s e d i n a l l c a s e s was th e diap h ragm o f w h it e r a t s w e ig h in g n o t more th a n 1 0 0 g r a m s. T h is t i s s u e w as s e l e c t e d f o r tw o r e a s o n s . F i r s t , b e c a u s e o t h e r i n v e s t i g a t o r s i n th e d ep a rtm en t w ere c o n d u c tin g r e l a t e d e x p e r im e n ts i n th e t h y r o id f i e l d w it h t h i s t i s s u e , and r e s u l t s c o u ld b e c o r r e l a t e d . S e c o n d , t h e t i s s u e w i l l rem ain a l i v e and r e s p i r e a t a n u n - d lm in is h e d r a t e f o r p e r io d s o f tim e up t o t w e lv e h o u r s . T h is p o in t i s o f param ount im p o r ta n c e t o t h i s w o rk . The a n im a ls w e re s a c r i f i c e d b y d e c a p i t a t i o n , a f t e r w h ic h th e r a t w as s e v e r e d c o m p le te ly th r o u g h a l i n e j u s t b elo w t h e lo w e r r i b . W ith s m a ll c u r v e d s c i s s o r s th e v e n t r a l p o r t io n o f t h e d iap h ragm was s e v e r e d from t h e sep tu m (F ig u r e 3 , I n c i s i o n N o. 1 ) . G ra sp in g t h e t i s s u e h e r e w it h f l a t t ip p e d f o r e e p s , th e m u sc u la r p o r t io n o f on e h a l f o f t h e diap hragm was s e v e r e d from t h e c e n t r a l band o f c o n n e c tiv e t i s s u e ( I n c i s i o n N o. 2 ) . By c a r e f u l l y c u t t in g th e r e m a in in g bond ( I n c i s i o n N o. 3 ) a lo n g t h e r i b s th e e n t i r e h a l f o f t h e m u s c le w as 20 rem oved . T h is h a l f w as t r a n s p o r te d t o t h e a p p r o p r ia te Warburg f l a s k and th e b o tto m ( d o r s a l) q u a r te r w a s rem oved ( I n c i s i o n Ho. 4 ) and p la c e d i n th e r e s p i r a t i o n m e d ia . The r e m a in in g q u a r te r (v e n tfr a l) w as ad d ed to a n o th e r f l a s k . The o t h e r h a l f o f t h e diaphragm was rem oved i n a s i m i l a r m anner and th e d o r s a l and v e n t r a l q u a r t e r s ad d ed t o t h e p r o p e r f l a s k s ( I n c i s i o n s N o. 5 , 6 , and 7 ) . T h is m ethod o f rem ovin g th e diap h ragm h a s many d i s t i n c t a d v a n t a g e s . I t a llo w s f o r th e rem oval w ith a minimum o f h a n d lin g and t h e r e f o r e a minimum o f damage to t h e t i s s u e c e l l s . I t i s * r a p i d and a f t e r a fe w t r i e s t h e e n t i r e m u s c le c a n b e rem oved and p la c e d i n t h e f l a s k s i n l e s s th a n f i v e m in u te s a f t e r d e c a p i t a t i o n . By p a r in g o f f t h e d o r s a l and v e n t r a l q u a r t e r s , th e t i s s u e I t s e l f i s i t s own c o n t r o l . E x a m in a tio n h a s shown t h a t th e d o r s a l q u a r t e r s a r e t h i c k e r th a n th e v e n t r a l q u a r te r s and so sh o u ld h a v e s l i g h t l y lo w e r Qo2 *s* T h is i s show n t o b e t h e c a s e , g e n e r a l l y . Hyper t h y r o id seru m , when r e f e r r e d t o i n t h i s p a p e r , w as o b t a in e d b y d e c a p i t a t i o n o f r a t s f e d a d i e t c o n t a in in g t h r e e p e r c e n t d e s s i c a t e d t h y r o id g la n d f o r s e v e n d a y s . B o th h y p e r th y r o id serum and norm al seru m w ere p r e p a r e d f o r t h e Warburg f l a s k s a f t e r t h e m eth od o f W arren ( 1 9 4 2 ) . The in o r g a n ic in c u b a t io n m ed ia w as p r e p a r e d a s f o l l o w s : 21 INCISION N O . I MUSCLE BA N D OF CONNECTIVE TISSUE I Ventral Quarter Ventral Quarter | | Dorsal Quarter Dorsal Quarter Fig. I Schematic diagram of rat diaphragm showing the method of removal of tissue. 22 P h o sp h a te b u f f e r ( 1 /1 5 M ) 0 .2 8 m l. NaCl (1 8 m g ./ e e .) 1 . 2 m l. G lu c o s e (2 .5 /6 ) 0 . 2 m l. S a l t s o l u t i o n (2 0 p t s KC1 0 .2 m l. 0 .1 5 4 M; 1 5 p t s OaCl 0 .1 1 1 M ; 1 0 p t s MgCl 0 .1 1 1 M ) KOH (1 0 N ) - c e n t e r w e ll _______0 .2 m l. H 0 t o make up t o t o t a l v o l . 3 .0 m l. The t h y r o x in e w as addled so t h a t t h e f i n a l c o n c e n t r a t io n i n t h e I n c u b a tin g m ed ia w ou ld be 4 x 1 0 “® gram s p e r m l l l l l e t e r . T h is i s t h e p lasm a c o n c e n t r a t io n o f th y r o x in e i o d in e i n norm al b lo o d . The s ta n d a r d Warburg s i n g l e v e s s e l m ethod o f d e te r m in in g t i s s u e r e s p i r a t i o n w as u s e d . R e a d in g s f o r th e m ost p a r t w ere made o v e r a n in e h ou r p e r io d . The t i s s u e w as rem oved from th e f l a s k , d r ie d a t 9 8 °C . f o r t w e n t y - f o u r h o u rs and w e ig h e d t o o b t a in t h e d r y w e ig h t f o r c a l c u l a t i n g t h e Q og. CHAPTER IX RESULTS The r e s u l t s o f t h e f i r s t ex p e rim en t c a r r ie d o u t i s g iv e n i n T ab le 4 and F ig u r e 4 . U n p a ired r a t d iap h ragm was su sp en d e d i n r in g e r p h o sp h a te g lu c o s e o r i n norm al r a t serum and t h y r o x in e w as add ed from th e s i d e arm a f t e r 1 h o u r o f in c u b a t io n . I t i s s e e n t b a t t h e r e w as no te n d e n c y f o r th e t h y r o x i n e - t r e a t e d t i s s u e s t o in c r e a s e t h e i r m e ta b o lis m o v e r t h e u n t r e a t e d c o n t r o l s . A s l i g i t I n c r e a s e a t th e s i x t h h ou r s u g g e s t e d t b a t t h e in c u b a t io n tim e sh o u ld b e p r o lo n g e d . The se c o n d e x p e r im e n t was c a r r ie d o u t i n a m anner s i m i l a r t o t h e f i r s t b u t t h e in c u b a t io n tim e w as e x te n d e d t o e i g h t h o u r s . The r e s u l t s shown i n T able 5 and f i g u r e 5 i n d i c a t e t h a t a m arked r i s e i n m e ta b o lism o c c u r s a f t e r f i v e h o u r s b u t t h a t t h e a d d i t i o n o f th y r o x in e h ad n o th in g t o d o w it h t h i s phenom enon. I t seem ed m ost l i k e l y t h a t t h i s i n c r e a s e w as due t o t h e g r o w th o f b a c t e r i a and su b s e q u e n t e x p e r im e n ts w ere c a r r ie d o u t u s in g m ore s t e r i l e m e th o d s. F a ir in g o f t h e d o r s a l and v e n t r a l q u a r te r s o f t h e t i s s u e w as u s e d i n t h i s e x p e r im e n t. The d o r s a l q u a r te r s show ed a lo w e r ^ 0 3 th a n th e v e n t r a l . V ery c l o s e a g reem en t w as o b t a in e d w it h t h e two d o r s a l q u a r te r s and a b s o l u t e l y no d i f f e r e n c e b e tw e e n t h e c o n t r o l and t h y r o x i n e - t r e a t e d t i s s u e was a p p a r e n t. 24 TABLE IV THE IN VITRO EFFECT O F THYROXINE IN RINGER PHOSPHATE GLUCOSE (R .P .G .) AND NORM AL SERUM ( N .S .) Hour Qpg* R in g e r p h o sp h a te g lu c o s e Norm al serum C o n tr o l T h y ro x in e C o n tr o l T h y ro x in e 1 4 .2 5 3 .8 5 4 .1 2 5 .9 3 2 3 .7 6 3 .4 6 3 .3 5 4 .3 6 3 3 .5 7 ... 3 .7 0 2 .7 9 3 .5 5 4 3 .8 4 3 .9 5 3 .0 6 3 .7 3 5 3 .6 8 4*06 2 .6 7 3 .3 2 6 3 .8 1 4 .3 4 2 .8 7 3 .7 3 * Qog 2 vsn p Q 2 /h r/m g d r y w t . t i s s u e . '25 GRAPH- I THE INVITRO EFFECT OF THYROXIN IN RINGER PHOSPHATE GLUCOSE AND NORMAL SERUM if-THYROXIN ADDED AFTER FIRST HOUR * X CL “ O --- _L_______________1 ____________ 2 3 TIME IN HOURS O — — oCONTROLjRPG •------ -•THYROXINE,RPG * o — oCONTROL, SERUM •---- — • THYROXINE, S ERUM * 2 6 TABLE V STUDIES ON THE IN VITRO EFFECT OF TH2ROXINE IN RINGER PHOSPHATE GLUCOSE OVER AN EIGHT-HOUR PERIOD Hour “ " " ^ 0 2 ■ D o r s a l V e n tr a l T h y ro x in e C o n tr o l T h y ro x in e 1 4 .4 4 4 .0 0 6 .0 5 2 3 .9 0 3 .7 5 6 . 1 2 3 3 .8 0 3 .7 9 5 .9 0 4 3 .9 6 4 .0 7 6 .0 5 5 3 .9 0 3 .9 2 5 .5 1 6 4 .2 8 4 .2 8 6 . 1 2 7 5 .0 8 5 .1 0 6 .8 0 8 6 .9 5 6 .9 8 9 .0 0 27 GRAPH- 2 THE IN VITRO EFFECT OF THYROXIN IN RINGER PHOSPHATE GLUCOSE * THYROXINE RP© A AU VHAl A n OORSAL/ ■ O CONTROL, RP<g / -o THYROXINE RP6}VENTj^AL 8 - 1 6 - JIM E IN HOURS 2 8 The t h i r d e x p e r im e n t w as c a r r ie d o u t t o s e e i f th e p r e s e n c e o f serum w o u ld p e r m it a th y r o x in e e f f e c t o n r a t d iap hragm o v e r lo n g e r in c u b a t io n t im e s u s in g s t e r i l e m eth ods* I t w as f e l t t h e u s e o f serum w ou ld p la c e th e t i s s u e and t h y r o x in e i n a more n a t u r a l h a b i t a t and i f a n I n c r e a s e i n Qog r e s u l t e d , t h e n on e w o u ld p o s t u l a t e t h a t some b lo o d serum c o n s t i t u e n t i s n e c e s s a r y t o p rom ote t h e a c t i o n o f t h e th y r o x in e * The s t e r i l e t e c h n iq u e d e v e lo p e d c o n s i s t e d o f two p a r ts * F i r s t t h e f l a s k s w ere a u t o d a v e d f o r f i f t e e n m in u te s a t 15 l b s and s e c o n d , a p p r o x im a te ly 0 * 1 gram o f c r y s t a l l i n e p e n i c i l l i n ad d ed t o e a ch a f t e r t h e serum w as added* One c o n t r o l f l a s k was u s e d w ith o u t th e p e n c l l l i n * The r e s u l t s a r e show n i n T a b le 6 and F ig u r e 6 * From a n e x a m in a tio n o f t h e r e s u l t s , i t i s s e e n t h a t th y r o x in e d id n o t i n c r e a s e th e r e s p i r a t i o n o f r a t d iap h ragm i n v i t r o e v e n th o u g h su sp e n d e d i n seru m . T h ree p o i n t s a r e b r o u g h t o u t from th e g ra p h s* The c h a r a c t e r i s t i c r i s e i n % ©g a f t e r f i v e h o u rs i s l a c k in g e v e n i n t h e f l a s k c o n t a in in g no p e n i c i l l i n * The a u t o c la v in g t h e r e f o r e r e d u c e d t h e i n i t i a l number o f b a c t e r i a and i n t h e p e r io d c o v e r e d b y t h e e x p e r im e n t th e b a c t e r i a l g r o w th d id n o t r e a c h a s u f f i c i e n t l y h ig h r a t e t o e f f e c t t h e r e s u l t s * 2 9 TABLE VI THE IN VITRO EFFECT OF THYROXINE IN NORM AL TEflUM OVER A NINE-HOUR PERIOD Qo2 H ours V ent r a l D o r s a l C o n tr o l C o n tr o l No Pen* And Pen* K iy r o x tn e And Pen* 1 9*04 1 0 .0 4 .3 6 2 5*22 6 .0 3 2 .4 8 3 4 * 6 6 5 .9 5 2 .0 8 4 3 * 9 5 4 .7 3 1 .4 9 5 4*09 4 .4 5 1 .3 9 6 - 4 .2 4 4 .1 9 1 *59- 7 4 .0 9 3 .5 1 1 .3 9 8 3 .5 3 3 .3 8 0 .9 9 9 3 .9 6 3 .3 8 1 .3 9 30 MM1 q /H f^ M G D R Y W T G R A P H - 4 11- THE INVITRO EFFECT OF HYPERTHYROID SERUM 1 0- 9- 8 - 7- 6 - 8 - 4- 2h 2- I- — -oCONTROL NORMAL SERUM*VENTRAL — •HYPERTHYROID SERUM OHYPERTHYROIO SERUM DORSAL VENTRAL 6 8 e TIME IN HOURS 31 The s e c o n d p o in t I s t h e d i f f e r e n c e I n Qog b e tw e e n th e d o r s a l and v e n t r a l p a r t s o f t h e d iap h ragm . A g a in t h e v e n t r a l q u a r te r s sh ew ed a g r e a t e r Qpg th a n d id t h e d o r s a l . The t h i r d p o i n t , t h a t w as m en tio n e d in t h e d i s c u s s i o n o f E x p erim en t n o . 2 , i s t h e n o n - u n ifo ir o it y o f r e s p i r a t i o n e s p e c i a l l y d u r in g th e f i r s t t h r e e h o u r s . I t w i l l b e n o te d t h a t t h e r e i s a d i s t i n c t d ro p i n Qo2 d u r in g th e f i r s t th r e e h o u r s . The r e s p i r a t i o n t h e n r e a c h e s a c o n s t a n t v a lu e and m a in ta in s t h a t v a lu e fo r th e b a la n c e o f t h e e x p e r im e n t. The seru m seem s t o h a v e a s t i m u l a t i n g e f f e c t o n t h e r e s p i r a t i o n d u r in g th e f i r s t h o u r , f o llo w e d b y a g r a d u a l d e c r e a s e u n t i l a c o n s ta n t r e s p i r a t i o n i s r e a c h e d f o r th e b a la n c e o f t h e r u n . C om parison o f th e Qo2 a f t e r th e f i r s t h o u r b e tw e e n t h e s y n t h e t i c and serum m ed ia show s t h a t th e serum m edia y i e l d e d an i n i t i a l Qog v a lu e much h ig h e r th a n th e s y n t h e t i c . The e x a c t r e a s o n f o r t h i s i s unknown. The f o r e g o in g e x p e r im e n ts h a v e add ed f u r t h e r e v id e n c e t o th e f a c t t h a t th y r o x in e a s s u c h , d id n o t , u n d e r t h e s e c o n d i t i o n s , i n c r e a s e th e r e s p i r a t i o n o f r a t t i s s u e i n v i t r o . The b a la n c e o f t h e w ork w i l l b e o n t h e e f f e c t o f h y p e r th y r o id serum i n v i t r o . I n th e f o u r t h e x p e r im e n t, two o f t h e f l a s k s c o n ta in e d h y p e r th y r o id serum c o l l e c t e d and p r e p a r e d a s s e t f o r t h u n d er M eth od s. The t h i r d f l a s k c o n ta in e d n orm al seru m . The r e s u l t s 32 a r e show n i n F ig u r e 7 and T a b le 7* A lth o u g h t h e tw o f l a s k s c o n t a in in g h y p e r th y r o id serum show ed an i n c r e a s e o f a b o u t f i f t y p e r c e n t r e s p i r a t i o n o v e r t h e c o n t r o l , t h i s i n c r e a s e i s q u e s t io n a b le b e c a u se o f t h e ex trem e v a r i a t i o n i n t h e $Og o b t a in e d w it h t h i s and o t h e r e x p e r im e n ts u s i n g serum ( 1 ,4 t o 4 i n E xp erim en t n o . 3 j 2 .8 5 i n E xp erim en t n o . 1 ; and 3 .2 5 t o 4 .2 i n e x p e r im e n t n o . 5 ) . The h y p e r th y r o id serum f l a s k s show ed a QOg o f 3 . 2 5 . D e f i n i t e c o n c lu s io n s c a n n o t b e n a d e fro m o n e r u n o f t h i s n a t u r e . The c h a r a c t e r i s t i c d ro p i n Qog o v e r t h e f i r s t t h r e e h o u rs i s shown a g a in i n t h e s e r u n s . The p a i r in g o f t h e diap hragm q u a r t e r s show ed no s i g n i f i c a n t d i f f e r e n c e i n t h e r e s p i r a t i o n o f v e n t r a l o v e r d o r s a l . S in c e i n p roceeding e x p e r im e n ts no s i g n i f i c a n t I n c r e a s e i n Qo0 o f h y p e r th y r o id seru m o v e r n orm al c o u ld be sh ow n , i t was d e c id e d t o t r y a n o th e r a p p r o a c h . T h is w as t o s t a r t a l l f o u r f l a s k s o u t u s in g th e s y n t h e t i c medium. A f t e r e s t a b l i s h i n g t h e norm al QOg d u r in g t h e f i r s t h o u r , e q u a l v o lu m es o f h y p e r t h y r o id serum w ere added to th e o th e r tw o . In t h i s w ay, an y i n c r e a s e i n QOg o f th e h y p e r th y r o id seru m f l a s k s o v e r t h e n orm al c o u ld be a t t r i b u t e d t o t h e e f f e c t o f t h i s seru m . The r e s u l t s o b t a in e d a s shown i n T a b le 8 and F ig u r e 8 a r e d i f f i c u l t t o i n t e r p r e t . The v e n t r a l c o n t r o l show ed a h ig h e r S & O g th a n t h e d o r s a l a s i n t h e o t h e r e x p e r im e n ts . The 33 d o r s a l h y p e r th y r o id f l a s k d rop p ed o f f a t a c o n s t a n t r a t e o v e r t h e n in e h ou r p e r io d and t h e v e n t r a l h y p e r th y r o id f l a s k show ed a l a r g e i n i t i a l I n c r e a s e a f t e r t h e a d d i t i o n o f th e h y p e r th y r o id serum f o llo w e d by a g r a d u a l d e c r e a s e o v e r t h e b a la n c e o f t h e ex p erim en t* The e x a c t c a u se o f t h i s trem en dou s i n c r e a s e i s unknown b u t i t i s th o u g h t t o b e due t o a s t a t e o f c o n t r a c t io n o r r i g o r i n t h e m u scle* The r e s u l t s a s p r e s e n te d can n o t be i n t e r p r e t e d a s e v id e n c e t h a t h y p e r t h y r o id serum s t im u la t e s t h e o x y g e n co n su m p tio n o f m u s d e t i s s u e * 34 TABLE V II THE IN VITRO EFFECT OF HYPERTHYROID SERUM Hour Stag V e n tr a l* D o r s a l* V e n tr a l* Normal seru m H y p e r th y r o id H yp erth iyroid 1 4 .9 5 7 .3 6 8 .9 0 2 4 .1 4 5 .4 1 7 .0 6 3 2 .9 3 4 .0 3 4 .1 8 4 3 .4 4 4 .8 6 5 .2 2 5 3 .3 4 4 .8 6 5 .5 5 6 3 .7 4 5 .2 8 6 .0 0 7 2 .9 3 4 .5 8 4 .4 5 8 3 .3 4 5 .1 4 5 .2 4 9 3 .0 3 4 .7 2 4 .4 5 P e n i c i l l i n u s e d i n a l l f l a s k s 35 TABLE V I I I THE IN VITRO EFFECT OF ADDING HYPER THYROID SERUM TO R .P .G * Hour V e n tr a l D o r sa l T h y r o x in e ’”* ' Control'***®’ C o n tr o l1 ” ” * T h y ro x in e - * ”* 1 7 .0 6 5 .5 0 5 .2 5 6 .4 5 2 1 1 .3 2 6 . 1 0 4 .9 5 5 .4 5 3 8 .9 1 4 .4 7 3 .4 5 4 .1 8 4 7 .7 0 4 .2 6 3 .4 5 4 .0 5 5 7 .9 5 4 .2 6 3 .3 4 3 .9 3 6 7 .0 0 4 .2 6 3 .1 1 3 .5 5 7 6 .2 6 4 .2 6 3 .2 2 3 .5 5 8 5 .7 7 3 .8 6 3 .1 1 3 .3 0 9 6 . 0 2 3 .8 6 3 .1 1 3 .0 5 * 1 . 5 c c . R .P .G . u s e d f i r s t h o u r . * * 1 . 5 c c . H .T .S . added t o f l a s k s a f t e r f i r s t h o u r . * * * * * 1 . 5 c c . N .S . ad d ed t o f l a s k s a fter* f i r s t h o u r . 1 0 9 8 7 6 5 4 3 2 I 0 GRAPH - 3 t h e i n v i t r o e f f e c t o f THYRO XIN IN NORMAL SERUM o -o CONTROL SERUM —• CONTROL SERUM THYROXINE SERUM -o- o o o 8 T IM E IN HOURS mm3 ojnn/m dry w t GRAPH - 5 THE INVITRO EFFECT OF HYDER THYROID SERUM AND NORMAL SERUM 12- 11- 1 0- 9- 0 - 7- 6 - 5- 4- * - 2 “ I- 0 A o—: o RPG + NS DORSAL • -------- • RPG +HTS VENTRAL o— ---o RPG + NS VENTRAL * RPG +HTS DORSAL JL 4 TIME 8 9 IN HOURS 36 37 Sj3P fj?**S S CHAPTER X DISCUSSION Under t h e c o n d it io n s o f t h e s e e x p e r im e n ts , t h y r o x in e , a s s u c h , o r a s h y p e r th y r o id serum show ed no d e f i n i t e s tim u l a t i n g a c t io n , on o x y g en c o n su m p tio n b y e x c i s e d r a t d iap h ragm i n e i t h e r s y n t h e t i c o r seru m m e d ia . I n v e s t i g a t o r s w o rk in g 5 * w it h th e " t im e - la g ” e f f e c t o f th y r o x in e I n j e c t e d i n t o a n im a ls r e p o r t p e r io d s up t o t h r e e d a y s b e f o r e a n y * I n c r e a s e i n r e s p i r a t i o n o f t h e t i s s u e s ta k e s p l a c e . I t w o u ld a p p ea r t h a t th y r o x in e m u st go th r o u g h som e m e t a b o lic p r o c e s s b e f o r e i t c a n s t i m u l a t e th e t i s s u e r e s p i r a t i o n . I n th e m e ta b o lism o f t h e th y r o id g la n d th e th y r o x in e may s t im u la t e o t h e r enzym es o r enzym es w h ich m a n if e s t them s e l v e s s e c o n d a r ily i n an I n c r e a s e i n t h e r e s p i r a t i o n o f th e t i s s u e s c o n t a in in g th em . BIBLIOGRAPHY A n se lm in o , K. J . , 0* E i c h l e r , and H. S e h lo ssm a n , B lo ch em ieh e Z e l t s c h r l f f t . 2 0 5 s 4 8 1 , 1 9 2 9 . B a s s e t t , A. M ., A. H. C o o n s, and W. T. S a l t e r , A m erican J o u r n a l o f M ed ic a l S c i e n c e . 2 0 2 . 5 1 6 , 1 9 4 1 . B aum berger, J . B ., C old S p r in g H arbor Sym posium . 7 s 1 9 5 , 1 9 3 9 . C r a ig , P . N. and W . T . S a l t e r , E n d o c r ln o lo g y . 3 7 s 1 1 9 , 1 9 4 5 . D a v is , J . E ., E . D a C o sta , and A. B . H a s t in g s , A m erican J o u r n a l o f P h y s io lo g y . 1 1 0 s 1 8 7 , 1 9 3 7 . Du B o ls , K. P . , and V. R . P o t t e r , J o u r n a l o f B i o l o g i c a l C h em istr y 1 5 0 s 1 8 5 , 1 9 4 3 . D ye, J . A ., A m erican J o u r n a l o f P h y s io lo g y . 105$ 5 1 8 , 1 9 3 3 . P o s t e r , G. L ., P r o c e e d in g s o f S o c i e t y f o r E x p e r im e n ta l B io lo g y and M e d ic in e . 2 4 s 3 5 4 . 1 ^ 2 7 . G erard , R . ff. and M. M c In ty r e , A m erican Jou rn a l o f P h y s io lo g y . 103S 2 2 5 , 1 9 3 3 . H ey ro v sk y , J . and M. S h lk a t a , R e c . T r a y . C hem .. 4 4 s 4 9 6 , 1 9 2 5 . H op p in g, A ., P r o c e e d in g s o f t h e S o c i e t y f o r E x p e r im e n ta l B io lo g y and M e d ic in e . 2 8 s 726*1 l& S l. Magnus - L ev y , B e r l in e r K le in e r W o c h sc h a ft, 3 2 s 6 5 0 , 1 8 9 5 . M eyer, 0 . 0 . , C. M cT iernan , and J . C. Aub, J o u r n a l o f C l i n i c a l I n v e s t i g a t i o n . 1 2 s 7 2 3 , 1 9 3 3 . M i l l e r , D. H ., R e p o r t a t W in ter M e e tin g s o f t h e W estern S o c i e t y o f N a t u r a l i s t s . S ta n fo r d t J n lv e r s it y . 1 5 3 5 . M i l l e r , D . H ., C old S p r in g H arbor Sym posium . 7 s 5 9 , 1 9 3 9 . P e t e r i n g , H. B ., and P . D a n ie l s , J o u r n a l o f t h e A m erican C h em ical S o c i e t y , 6 0 s 2 7 9 6 , 1 9 3 8 . P o t t e r , V . R ., J o u r n a l o f B i o l o g i c a l C h e m istr y . 114$ 4 9 5 , 1 9 3 6 . 40 -___________, I b i d , 1 3 4 s 4 1 7 , 1 9 4 0 . ______________I b i d , 1 4 2 : 5 4 3 , 1 9 4 2 . S c h n e id e r , W. C. and V . R . P o t t e r , J o u r n a l o f B i o l o g i c a l C h e m istr y . 1 4 9 : 2 1 7 , 1 9 4 3 . S c o t t , A. H ., -American J o u r n a l o f P h y s io lo g y . I l l : 1 0 7 , 1 9 3 5 . T a v ro g , A ., I . L . C h a ik o ff , and. C. Entenm an, E n d o c r in o lo g y . 4 0 : 8 6 , 1 9 4 7 . T ip to n , S . R ., and W. L . M ixon, E n d o c r in o lo g y . 3 9 s 3 0 0 , 1 9 4 6 . U m b r e lt, W. W ., R. H. B u r r is , an d P . J . S t a u f f e r , M anom etrie T e ch n iq u es and R e la te d M ethods f o r t h e S tu d y o f T is s u e M e ta b o lism . 1 9 4 5 , 1 9 4 .p p . W arren, C. 0 . , J o u r n a l o f C e l lu la r an d C om p arative P h y s io lo g y . 1 9 : 2 , 1 9 4 2 . W in z le r , R . J . , J o u r n a l o f C e l lu la r and C om p arative P h y s io lo g y . 1 7 : 2 6 3 , 1943T W in z le r , R. J . , I b i d . , 2 1 : 2 2 9 , 1 9 4 3 . «*>vorefty of SQMtftorra Colfferota OEscfn?

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

The effect of incubation on the cholesterol content of rat liver homogenates

PDF

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

PDF

The influence of thyroxine and desiccated thyroid preparation on the metabolic and heart rates of the rat

PDF

The effect of choline on phospholipid metabolism: A literature review and preliminary in vitro experiment

PDF

A study of the alterations in the amount of cholesterol present in rat liver homogenates after incubation

PDF

Glycoproteins in saline extracts of tissues of normal adult rats

PDF

Studies of fatty acids: Effects on nutritional status and tissue cholesterol levels of rats

PDF

The effect of thyroxine and thyroxine analogs on resting cells of yeast.

PDF

The electrolyte composition of the soluble phase of cell homogenates

PDF

Some aspects of the metabolism of nervous tissue

PDF

Study of cytochrome oxidase by the use of resonance Raman spectroscopy

PDF

A study on the comparison of the cholesterol content of rat liver homogenates before and after incubation

$The effect of L-thyroxine on oxidation and phosphorylation in a subcellular particulate fraction of baking yeast$

PDF

The effect of L-thyroxine on oxidation and phosphorylation in a subcellular particulate fraction of baking yeast

PDF

The metabolism of the hexitols

PDF

The synthesis and biological activity of compounds structurally related to thyroxine

PDF

Studies on hexose mono-phosphate oxidation

PDF

Studies of iron binding by the human serum protein, transferrin

PDF

A study of the ketone body metabolism of excised rat tissues with respect to labile phosphate content

PDF

The effect of lutein on carotene utilization as measured by growth of the rat

PDF

Studies of aminodeoxyuridine, methylaminodeoxyuridine and dimethylaminodeoxyuridine

Asset Metadata

Creator Fasolas, James Eugene (author)

Core Title Studies on metabolism of tissues. I. The polarographic measurement of cytochrome oxidase in liver homogenates. II. Studies on the effect of thyroxine on respiration of isolated rat diaphragm

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

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-776654

Unique identifier UC11348323

Identifier EP41303.pdf (filename),usctheses-c17-776654 (legacy record id)

Legacy Identifier EP41303.pdf

Dmrecord 776654

Document Type Thesis

Rights Fasolas, James Eugene

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