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

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

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Content A . STUDY OF THE KETOHE BODY METABOLISM
OF EXCISED RAF TISSUES; WITH RESPECT.
TO LABILE PHOSPHATE COETEUT
A. T h esis:
P r e s e n t e d t o
th e F a c u lt y o f t h e G rad u ate S c h o o l
The U n i v e r s i t y o f S o u th e r n C a l i f o r n ia
In P a r t i a l F u l f il l m e n t
o f th e R e q u ir e m e n ts f o r th e D e g r e e
M a ster o f S c ie n c e
by
J a s p e r B u lb e r g
A u g u st 1948
Bi's [ Y f P f i f
This thesis, written by
JASPER DULBERG-
under the guidance of Faculty Committee,
and approved by all its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fulfill
ment of the requirements fo r the degree of
MASTER 0F SCIENCE
E. S. BOGARDUS
Dean
Date AtXgUS.fc...l.9k&....................
Faculty Committee
TABLE OF CONTENTS
CHAPTER PAGE
I . INTRODUCTION........................................................................ 1
I I . HISTORY A M D REVIEW OF THE LITERATURE.......... 4
I I I . STATEMENT OF PROBLEM............................................................ 12
IV . EXPERIMENTAL PROCEDURE AM D METHODS.............................. 17
I n t a c t A n im al M ethod . . . . . . . . . . . . 1 7
D iaphragm T is a n e S l i c e S t u d ie s . . . . . . . 18
G e n e r a l p ro ced u re .. .. »»»••••» 19
P r e p a r a t io n s . » •« •• » .. .» • » . 20
I n c u b a t io n m edia . . • • • • • • » • • • • 21
A n a ly s i s o f ch an ge in k e to n e body s u b s t r a t e 22
C h em ica l m ethods f o r g ly c o g e n and l a b i l e
p h o sp h a te a s s a y s . . . » . • • • . » • 25
L iv e r S l i c e S t u d ie s •• •• • ••• •• • 27
Y . RESULTS .................................................................. 28
TABLE I E f f e c t o f ATP on o x y g en co n su m p tio n
o f r a t diaphragm .. . . « • • • » 28
TABLE I I U t i l i z a t i o n o f L -b e ta h y d r o x y b u ty r a te
and o xygen by r a t diap h ragm . . . . 29
TABLE I I I U t i l i z a t i o n o f a o e t o a o e t a t e and
o x y g en by r a t diap hragm . . . . . . 30
TABLE IY G ly c o g en c o n te n t . • • . • • • • • • 31
TABLE V L a b ile p h o sp h a te c o n te n t • • • . • • 32
TABLE VI L a b ile p h o sp h a te e f f e c t on th e u p ta k e
o f k e to n e b o d ie s by l i v e r s l i c e s • 33
V I. DISCUSSION OF RESULTS 34
V I I . SUM M ARY AND CONCLUSIONS.................................................. . 48
BIBLI0 GRAPHY .................................................................................................. 50
CHAPTER I
INTRODUCT I O H
The f a s t t h a t c a r b o h y d r a te s h ay a a n e f f e c t on t h e o x id a
t i o n o f f a t s by I n t a c t a h im a ls i s w a l l e s t a b l i s h e d . I n humans
th e a b s e n c e o f d i e t a r y c a r b o h y d r a te and s e v e r a l c o n d i t i o n s i n
v o lv in g f a u l t y c a r b o h y d r a te m e ta b o lism r e s u l t i n k e t o s i s , a
c o n d it io n i n w h ich abnorm al am oun ts o f b e ta h y d r o x y b u ty r a te ,
a c e t o a e e t a t e and a c e to n e a c cu m u la te i n t h e body f l u i d s and may
be e x c r e t e d i n t h e u r i n e . T h e se com pounds a r e known a s k e to n e
b o d ie s and a r e form ed p r i n c i p a l l y from f a t t y a c i d s , a lth o u g h
c e r t a i n am ino a c i d s a r e p o s s i b l e p r e c u r s o r s t o a l e s s e r e x t e n t .
The l i v e r a p p e a r s t o b e t h e p r i n c ip a l s i t e o f fo r m a tio n o f ,
t h e k e to n e b o d ie s i n a n im a ls , and t h e p e r ip h e r a l t i s s u e , th e
p r i n c i p a l s i t e o f o x id a t io n o f th e com pounds. K eto n e b o d ie s
a r e b e l ie v e d t o be th e norm al in t e r m e d ia t e s o f f a t t y a c id
o x id a t io n in th e l i v e r . The e x c e s s i v e a c c u m u la tio n o f t h e
k e to n e b o d ie s t h a t d e v e lo p s In k e t o s i a , h o w ev er, i s ab n o rm a l.
The d ev elo p m en t o f k e t o s i a in humans a p p e a r s t o be
som ewhat d i f f e r e n t from t h a t o f o th e r s p e c i e s su ch a s th e r a t ,
d ag o r r a b b i t . W hereas man and m onkeys r e a d i l y d e v e lo p a
k e t o s i a d u r in g f a s t i n g , r a t s a r e v e r y r e s i s t a n t t o t h e d e v e l
opm ent o f a k e t o s i s . I t i s f e l t b y many c l i n i c i a n s t h a t th e
i n c l u s i o n o f t w ic e a s much c a r b o h y d r a te a s o f f a t i n t h e d i e t
i s n e c e s s a r y t o a v o id a k e t o s i s i n hum ans. A . k e t o s i s d e v e lo p s
£
l a I n s u l i n I n s u f f i c i e n c y i n b o th r a t s and humans p r o b a b ly due
t o f a u l t y c a r b o h y d r a te m etab olism , w h ich i s b e lie v e d t o e f f e c t
th e m e ta b o lism o f f a t s . A lth o u g h n orm al r a t s do n o t d e v e lo p
o r d in a r y f a s t i n g k e t o s i s , i t h a s b een fou n d t h a t r a t s f e d
b u t y r a t e o r t h o s e w it h d ie t a r y f a t t y l i v e r s may d e v e lo p a
f a s t i n g k e t o s i s . I t i s b e l ie v e d t h a t t h e d i f f e r e n c e b e tw e en
human and r a t k e t o s i s i s due t o q u a n t i t a t i v e r a th e r th a n
q u a l i t a t i v e b io c h e m ic a l c a u s e s , b u t t h e o v e r a l l e f f e c t m akes
. f o r a q u a l i t a t i v e d i f f e r e n c e In th e f a s t i n g c o n d i t i o n .
I n s u l i n i s known t o c o r r e c t th e k e t o s i s r e u l t i n g from
d i a b e t e s m e l l i t u s , s u g g e s t in g a r e l a t i o n b etw een c a r b o h y d r a te
m e ta b o lism and f a t t y a c id o x i d a t i o n . A c o n n e c tio n b etw een
c a r b o h y d r a te m e ta b o lism and t h e a c t i o n o f i n s u l i n h a s r e c e n t l y
b e e n e s t a b l i s h e d b y th e G o r is , w ith a d e m o n s tr a tio n o f th e
a c t i v a t i o n o f t h e c a r b o h y d r a te enzym e, h e x o k in a s e , b y I n s u l i n .
I t may be th r o u g h t h i s m echanism t h a t c a r b o h y d r a te a f f e c t s
k e to n e bod y m e ta b o lis m . Horm al c a r b o h y d r a te m e ta b o lism p o s s e s s
e s t h e c h a r a c t e r i s t i c a b i l i t y o f t h e a n a e r o b ic g e n e r a t io n o f
e n e r g y r i c h or l a b i l e p h o sp h a te com pounds i n a d d i t io n t o i t s
a b i l i t y common t o o t h e r m e t a b o l it e s o f f u r n is h in g l a b i l e p h o s
p h a te b y a e r o b ic o x i d a t i o n . I t a p p e a r s p o s s i b l e t h a t o x id a
t i o n o f k e to n e b o d ie s may in v o lv e a p h o s p h o r y la tio n b y su ch
h igh , e n e r g y p h o sp h a te com pounds, from l e h n i n g e r 1 s r e c e n t s t u d i e s
on th e o x id a t io n o f f a t t y a c i d s . I t i s o u r b e l i e f t h a t t h e
p r e c i s e mode o f i n t e r a c t i o n o f c a r b o h y d r a te and f a t , w h ic h i s
in d ou b t a t p r e s e n t , may p o s s i b l y be. r e l a t e d t o th e l a b i l e
p h o sp h a te com pounds o r i g i n a t i n g from t h e g l y c o l y s i s o f c a r
b o h y d r a te . I f t h i s i s t h e c a s e , a d d i t io n o f su c h h ig h e n e r g y
p h o sp h a te compounds su ch a s a d e n o s in e t r ip h o s p h a t e may f a c i l
i t a t e t h e o x id a t io n o f k e to n e b o d ie s *
® h is p a p e r r e p o r t s a s tu d y o f some e f f e c t s o f l a b i l e
p h o sp h a te com pounds on t h e k e to n e bod y m e ta b o lism o f e x c i s e d
r a t t i s s u e s , f h e o r i g i n a l i n t e n t i o n w as t o make t h i s w ork a
p r e lim in a r y t o t h e s tu d y o f t h e r e l a t i o n o f l a b i l e p h o sp h a te
o r i g i n a t i n g from c a r b o h y d r a te t o th e m e ta b o lism o f f a t s b u t
t h e u lt im a t e o b j e c t i've h a s n o t y e t b een a tte m p te d .
CHAPTER II
HISTORY M D RETIEO OF THE LITERATURE
H i s t o r i c a l l y th e s tu d y o f f a t m etab olism , b egan w ith t h e
u s e o f I n t a c t a n im a ls a s t e s t o r g a n ism s; p r o c e e d e d t o t h e i n
•v itr o u s e o f s u r v iv in g i n t a c t o r g a n s and e v o lv e d t o th e more
s e l e c t i v e u se o f s u r v iv in g t i s s u e s l i c e s , h o m o g en a tes and
t i s s u e e x t r a c t s i n an a tte m p t t o i s o l a t e s p e c i f i c r e a c t i o n s
and enzyme sy ste m s*
Some o f th e m ost v a l i d and l a s t i n g b io c h e m ic a l r e s e a r c h
h a s b een done w ith I n t a c t a n im a ls . Knoop ( 1 9 0 4 ) , i n h i s c l a s s
i c a l e x p e r im e n ts , f e d d o g s h om ologu es o f b e n z o ic a c id and
fou n d t h a t t h e u r in e c o n ta in e d h ip p u r lo or p h e n y la c e t u r ie
a c i d , d e p e n d in g on w h eth er t h e f a t t y a c id s i d e c h a in o f t h e
a d m in is te r e d compound w as a n odd o r an e v en num bered carb on
c h a in * Khoop in t e r p r e t e d t h e s e r e s u l t s a s an i n d i c a t i o n t h a t
f a t s a r e b rok en down by u n i t s o f two ca rb o n s f o llo w in g o x id a
t i o n o f th e b e t a carb on * I t i s now g e n e r a l ly a c c e p te d t h a t
f a t s a r e d egrad ed and b u i l t up p r e d o m in a te ly b y u n i t s o f two
c a r b o n s ; h o w ev e r, t h e r e i s some e v id e n c e t h a t la r g e r u n i t s
may be in v o lv e d a s w e l l . M orehouse and D e u e l (1 9 4 1 ) fe d
o a p r o ic a c id la b e lL e d w ith d e u te riu m to r a t s and i s o l a t e d th e
k e to n e b o d ie s from t h e u r in e * The p o s i t i o n o f th e d e u te r iu m
i n t h e k e to n e b o d ie s w as su ch a s t o i n d i c a t e t h a t f a t t y a c id s
a r e b ro k en down b y u n i t s o f fo u r ca rb o n s a s w e l l a s two c a r
bon u n i t s * S ch oen h eim er and R it te n b e r g (1 9 3 7 , 194Q) h ave
d e m o n stra te d d e g r a d a tio n and le n g t h e n in g o f f a t t y a c i d s b y
f e e d in g d e u t e r iu m - la b e lle d s t e a r i c a c id t o l i v e m ice and i s o
l a t i n g l a b e l l e d p a l m i t ic a c id from th e c a r c a s s f a t * R i t t e n -
h e r g and B lo c k (1 9 4 4 ) f e d a c e t i c a c id l a b e l l e d w ith d e u te r iu m
and h e a v y ca rb o n t o m ice and o b ta in e d e v id e n c e t h a t th e a c e t i c
a e id had b een con d en sed t o form f a t t y a c i d s . B o th D e u e l (1 9 3 8 )
and i-SvhapIro (1 9 3 b ) h a v e shown t h a t a d m in is t r a t io n o f g lu c o s e
and g lu c o s e fo r m in g compounds w i l l r e s u l t In d e p o s i t i o n o f
l i v e r g ly c o g e n and a r e d u c t io n o f k e t o n u r ia i n b u t y r ic a c id
f e d r a t s . I h e s e l a s t two c i t a t i o n s a r e o n ly a sam ple o f th e
la r g e bod y o f l i t e r a t u r e on t h e t e s t i n g o f th e r e l a t i o n s h i p s
o f c a r b o h y d r a te and f a t m e ta b o lism by t h e u se o f t h e i n v iv o
m eth od . D eu el* s work (1 9 3 7 ) d e m o n stra te d t h a t r a t a w h ich do
n o t e x h i b i t an o r d in a r y f a s t i n g k e t o s i s w i l l d e v e lo p k e t o s i s
i f a f a t t y l i v e r i s In d u ced p r io r t o th e f a s t . S in c e th e
p r i n c i p a l d i f f e r e n c e b e tw e en o r d in a r y f a s t e d r a t s and D e u e l’ s
f a s t e d r a t s Is: th e f a t t y l i v e r , th e abnorm al f a t m ust be im
p l i c a t e d a s th e c h i e f p r e c u r s o r o f th e k e to n e b o d ie s in th e
p a r t i c u la r k e t o s i s . Boeb* s (1912.) w ork d e m o n stra te d t h a t
a c e t a t e c o n d e n s e s t o a c e t o a c e t a t e by p e r f u s io n o f a c e t a t e
th r o u g h an i n t a c t l i v e r . A m odern in v iv o c o n fir m a tio n o f
o f a c e t a t e c o n d e n s a tio n t o a c e t o a c e t a t e h a s b een made by
S w e n se id e t a l (1 9 4 2 ) b y f e e d in g a c e t i c a c id ■ la b e l l e d w ith
h e a v y carb on t o r a t s and d e m o n s tr a tin g th e p r e s e n c e o f h e a v y
carb on i n t h e i s o l a t e d a c e t o a c e t i c a c i d . S o s k in (1 9 4 6 ) h a s
d e m o n str a te d t h a t th e p e r ip h e r a l t i s s u e s o f b o th n orm al and
d ia b e t ic - mammals; o x i d i z e k e to n e b o d ie s r e a d i l y w it h no ap p ar
e n t l o s s o f o x id a t iv e a b i l i t y in d i a b e t e s , a t l e a s t n o t in
t h e c a s e o f th e dog:. One may sum m arize b y th e o b s e r v a t io n
t h a t when th e e f f e c t s n o te d by th e use; o f th e i n t a c t org an
o r t h e i n v iv o m ethod a r e r e c o r d e d f-a s s u c h , t h e y make v a l i d
and perm anent r e c o r d s .
!Ehe a d v en t o f t h e _in v i t r o t i s s u e s l i e e and W arburg
m ethod l e d t o more p r e c i s e e v id e n c e t h a t l i v e r p r o d u c e s k e to n e
b o d i e s a s th e end p r o d u c ts o f f a t t y a c id m e ta b o lis m . Q u a s te l
(1 9 3 3 ) u se d l i v e r s l i c e s and th e Warburg m ethod t o d e m o n str a te
th e p r o d u c tio n o f k e to n e b o d ie s by th e l i v e r . E d son (1 9 3 5 )
r e p e a te d t h i s work w ith l i v e r s l i c e s w ith some v a r i a t i o n s in
th e c o n d it io n s and e x te n d e d th e o b s e r v a t io n s o f Q u a s t e l.
B o b b itt and D e u e l (194E ) u s in g a n o th e r var i a t i o n w ith l i v e r
s l i c e s c o n fir m e d th e p r o d u c tio n o f k e to n e b o d ie s b y l i v e r and
made t h e a d d i t i o n a l d is c o v e r y t h a t g ly c o g e n c a u s e s an i n c r e a s
ed u p ta k e o f k e to n e b o d ie s b y l i v e r * W ein h o u se, M edes and
E lo y d (1 9 4 4 , 1945) in c u b a te d l i v e r s l i c e s w ith o c t a n o ic a c id
l a b e l l e d w it h i s o t o p l c ca rb o n i n th e c a r b o x y l group and o b t a in
ed c le a r c u t e v id e n c e t h a t f a t t y a c id s d is m u ta te b y fr a g m e n ts
o f two ca rb o n s w h ich condense, t o form a c e t o a c e t a t e . I.h ese
same w o rk ers a l s o in c u b a te d c a r b o x y l- l a b e ll e d a c e t a t e w it h
liv e r : s l i c e s and o b ta in e d a c e t o a c e t a t e a s th e c o n d e n s a tio n
p r o d u c t; t h i s c o n d e n s a tio n d id n o t o c cu r a s r e a d i l y i n k id n e y
s l i c e s . F u r th e r e x a m in a tio n o f th e p r o d u c ts o f i s o t o p i c a c e
t a t e in k id n e y t i s s u e d is p la y e d e v id e n c e t h a t in t i s s u e s o th e r
th a n l i v e r , a c e t a t e g o e s th r o u g h th e K rebs c y c le on o x id a t io n .
The w o r k -o f W ein h ou se, M edes and F lo y d i s im p o r ta n t n o t o n ly
i n i t s i s o t o p i c e v id e n c e o f th e two ca rb o n d is m u ta tio n o f f a t s
f o llo w e d by c o n d e n s a tio n t o a c e t o a c e t a t e in l i v e r , b u t i s more
s i g n i f i c a n t in i t s e v id e n c e th a t in o th e r t i s s u e s th e i s o t o p i c
a c e t a t e may be o x id iz e d by th e same r o u te ta k e n by c a r b o h y d r a te .
A d d it io n a l e v id e n c e o f t h e o x id a t io n o f f a t s th ro u g h th e K rebs
c y c le by th e u se o f i s o t o p e s i s g iv e n b elo w in B uchananr s
hom ogenate s t u d i e s , K rebs and E g g le s t o n (1 9 4 5 ) u s in g a n a ero b
i c c o n d i t i o n s ( a v a r i a t i o n from t h e s e c i t a t i o n s , ) o b ta in e d a
q u a n t it a t iv e r e d u c t io n o f a c e t o a c e t a t e to* b e ta h y d r o x y b u ty r a te
i n a s u s p e n s io n o f m inced m u s c le - T h e ir a n a ly s e s o f th e p r o
d u c t s d i s c l o s e d t h a t t h e a c e t o a c e t a t e d id n o t e n t e r t h e K reb s
c y c le a n a e r o b i c a l l y , and t h e y c o n c lu d e d t h a t a c e t o a c e t a t e
d o e s n o t e n t e r th e K reb s c y c l e a t a l l * I t t h u s a p p e a r s t h a t
th e m ethod o f s tu d y o f t i s s u e s w ith i n t a c t c e l l s i n th e War
b u rg a p p a r a tu s i s one o f t h e m ost e x c e l l e n t m eth od s f o r t h e
s e l e c t i v e s t u d y o f c e l l u l a r m e ta b o lic p r o c e s s e s and h a s l e d
t o a w e a lth o f l i t e r a t u r e on f a t t y a c id m e ta b o lis m .
The u s e o f t h e n o n - o e l l u l a r h o m o g en a tes and t i s s u e e x t r a c t s
a s s tu d y m a t e r i a ls w as i n i t i a t e d in an e f f o r t t o g a in b e t t e r
d i f f u s i o n and b e t t e r i s o l a t i o n o f s p e c i f i c r e a c t i o n s and e n
zyme s y s t e m s . I n a d d i t io n , t h e n o n - c e l l u l a r m ethod p e r m its
v a r i a t i o n In c o n d i t i o n s n o t p o s s i b l e i n c e l l u l a r m e ta b o lism ,
and le a d s t o I s o l a t i o n o f I n t e r m e d ia t e s w h ich a r e d i f f i c u l t
t o o b t a in b y th e e n t i r e c e l l u l a r p r e c e d e s , I h a t th e c o n v e r -
s l a n o f a c e t o a c e t a t e to a c e t a t e I s r e v e r s i b l e i s s u g g e s te d
b y t h e w o r t o f B e h n ln g er (1 9 4 2 } who d e m o n str a te d tb e s p l i t t
i n g o f a c e t o a c e t a t e t o a c e t a t e b y a f e e b l e enzym e e x t r a c t
o b ta in e d from r a b b i t k id n e y and m u s c le , b o th o f t h e s e t i s s u e s
b e in g a b le t o o x i d i z e a c e t o a c e t a t e when i n t a c t . I h i s may be
ta k e n a s a d d i t i o n a l e v id e n c e o f a c e t a t e c o n d e n s a tio n t o a c e t o -
a c e t a t e a s a r e v e r s i b l e p r o c e s s u n d er c e r t a i n c o n d it io n s j t h e
c o n d i t i o n s i n L e h n in g e r ’ s e x p e r im e n ts e v i d e n t l y fa v o r e d t h e
s p l i t t i n g o f a c e t o a c e t a t e t o a c e t a t e a s t h e ma^or r e a c t i o n .
B uchanan e t a l (1 946} a l t e r n a t e l y in c u b a te d i s o t o p i c a c e t a t e
and i s o t o p i c a c e t o a c e t a t e i n k id n e y h o m o g en a te. B e s id e s ob
t a i n i n g e v id e n c e t h a t t h e K reb s c y c le i s in v o lv e d i n t h e o x i
d a t io n o f a c e t a t e and a c e t o a c e t a t e by t h e i r a n a l y s e s o f th e
in t e r m e d ia t e s , B uchanan e t a l w ere a b le t o d e m o n str a te t h a t
t h e a c e t o a c e t a t e w h ic h rem a in ed u n o x id lz e d u n d er t h e i r c h o se n
c o n d i t i o n s had a c o m p o s itio n w h ich c o u ld o n ly be e x p la in e d on
t h e b a s i s o f t h e s p l i t t i n g o f t h e i n i t i a l a c e t o a c e t a t e and
t h e r e c a n d e n s a t io n o f t h e tw o ca rb o n fc a g m e n ts t o a c e t o a c e t a t e
a g a in . W h ile B uchanan d o e s n o t s t a t e t h a t t h i s I s e v id e n c e o f
a n e q u ilib r iu m b e tw e en a c e t a t e and a c e t o a c e t a t e a t h i s c o n d i
t i o n s , we b e l i e v e i t may b e i n t e r p r e t e d a s e x h i b i t i n g an e q u i
lib r iu m . f h e i n t a c t k id n e y o x i d i z e s a c e t o a c e t a t e c o m p le t e ly .
Lipmann (1 9 4 4 , 1 9 4 5 ) h a s d e m o n stra te d t h e fo r m a tio n o f a c e t y l
p h o sp h a te i n r a t and p ig e o n l i v e r h om ogen ates b y a r e a c t i o n
b e tw e en a c e t a t e and a d e n o s in e t r ip h o s p h a t e , h a s b e e n a b le t o
o b t a in t h e e q u ilib r iu m c o n s t a n t o f t h e r e a c t i o n , h a s su c c e e d e d
i n tr a p p in g th e a c e t y l p h o sp h a te f o r a s s a y , and h a s d em o n stra
t e d t h a t t h e enzym e, a c e t y l p h o s p h o ta s e , i s p r e s e n t i n v a r io u s
e x t r a c t s o f m u s c le , l i v e r and o t h e r mammalian t i s s u e s . L ip -
m ann 's s tu d y o f t h e p o s s i b l e e n e r g y r e q u ir e m e n ts o f a r e a c t i v e
two ca rb o n l i n k b etw een a c e t a t e and a c e t o a c e t a t e c l e a r l y i n d i
c a t e s t h a t a c e t y l p h o sp h a te f u l f i l l s t h e s e r e q u ir e m e n ts .
H ow ever, he h a s n o t y e t b e e n a b le t o d e m o n str a te a c o n d e n sa
t i o n - s y n t h e s i s o f a c e t y l p h o sp h a te t o a c e t o a c e t a t e i n a t i s s u e
h o m o g en a te. Buchanan and H a s tin g s (1 9 4 6 ) i n g i v in g t h e i r
o p in io n o f t h e p r e s e n t la c k o f s u c c e s s o f a tte m p ts t o u s e a c e t y l
p h o sp h a te i n hom ogen ate e x p e r im e n ts s t a t e , "K now ledge, h o w ev e r,
o f t h e r e a c t i o n s o f a c e t y l p h o sp h a te i n mammalian t i s s u e s i s
s o l i m i t e d t h a t i t i s im p o s s ib le t o a s s i g n i t a r o l e i n t h e
o x i d a t i v e r e a c t i o n s a t t h i s tim e " . Wood (1 9 4 6 ) com m enting on
e x p e r im e n ts w ith a c e t y l p h o sp h a te s a y s , " . , s i n c e a c e t y l p h o s
p h a te i s r a p id ly b rok en down by a n im a l t i s s u e i t i s q u e s t io n
a b le w h eth er t h e t e s t w i l l h a v e an y d e f i n i t e s i g n i f i c a n c e ,
u n t i l i t i s d e m o n str a te d t h a t t h e e s t e r w as s u f f i c i e n t l y s t a b l e
u n d er th e c o n d it io n s o f t h e t e s t and a v a i l a b l e f o r enzym e
a c t i o n " . Wood m e n tio n s t h e f a c t t h a t a c e t y l p h o sp h a te d o e s
n o t d i f f u s e e a s i l y i n t o i n t a c t c e l l s and t h a t t h i s a d d s t o
10
t h e d i f f i c u l t i e s In e x p e r im e n ts w ith th e e s t e r * l e h n i n g e r (1 9 4 5 )
u s e d a w ashed l i v e r hom ogen ate t o d e m o n str a te t h a t f a t t y a c i d s
r e q u ir e p h o s p h o r y la tio n t o a c y l p h o s p h a te s p r i o r t o t h e i r o x i
d a tio n * The s p e c i f i c p h osp h or y l a t i n g compound fou n d t o he
e s s e n t i a l i n h i s e x p e r im e n ts w as a d e n o s in e t r ip h o s p h a t e (ATP)
e i t h e r in tr o d u c e d a s su c h o r g e n e r a te d w it h in th e hom ogen ate
b y t h e c o o x id a t lo n o f a n o th e r m e t a b o lit e . The end p r o d u c t o f
f a t t y a c id o x i d a t i o n i n t h e l i v e r h om ogenate w a s an accu m u la
t i o n o f h e t o n e b o d ie s * le h n l n g e r fou n d t h a t t h e minimum o f
ATP r e q u ir e d f o r d is m u t a tio n o f f a t t y a c i d s w as 10~^M ., and
t h a t i t w a s d i f f i c u l t t o m a in ta in t h e c o n c e n t r a t io n o f ATP a t
t h a t l e v e l I n h i s h o m o g e n a te . L e h n in g e r b e l i e v e s t h a t a c e t y l
p h o sp h a te i s a p o s s i b l e in te r m e d ia t e b u t p r e f e r s t o s t a t e t h a t
"some r e a c t i v e tw o ca rb o n fra g m en t" i s i n d i c a t e d .
The u s e o f p u re I s o l a t e d en zy m es, w h ic h p e r m it t h e s tu d y
o f t h e i r e n t i r e l y i s o l a t e d r e a c t i o n s and t h e f a c t o r s a f f e c t i n g
th em , h a s b e e n u t i l i z e d b y t h e Gar is, (1 9 4 5 , 1 9 4 6 ) t o d e m o n str a te
th e e f f e c t o f t h e horm one, I n s u l i n , a s an a c t i v a t o r o f h e x o -
k i n a s e , th e enzym e w h ich p h o s p h o r y la te s g lu c o s e and h e n c e a i d s
i t s a n a e r o b ic p h a se o f u t i l i z a t i o n * The o v e r a l l e f f e c t o f
i n s u l i n a d m in is t r a t io n d u r in g d i a b e t e s m e l l i t u s i s t h e a b o l i
t i o n o f k e t o s i s . I n s u l i n may be s a id t o h a v e d i r e c t e f f e c t on
c a r b o h y d r a te m e ta b o lism and p o s s i b l y i n d i r e c t e f f e c t on f a t s *
The c a r b o h y d r a te s t u d i e s o f t h e G o r is and o t h e r s b y v a r i
o u s m eth o d s ( r e v ie w e d b y S o s k in 1946) h a v e e s t a b l i s h e d t h a t
11
a m ole o f g ly c o g e n c a n , a n a e r o b i c a l l y , g e n e r a t e one m ole o f A 2P .
£ h e a n a e r o b ic g e n e r a t io n o f M*P b y g l y c o l y s i s o f c a r b o h y d r a te s
i s u n iq u e , b u t c a r b o h y d r a te s a l s o p o s s e s s t h e p r o p e r ty common
t o o t h e r m e t a b o l it e s o f g e n e r a t io n o f AIEP b y a e r o b ic o x i d a t i o n .
CHS1TEK I I I
ST&TEMEM: OF PROBLEM
The l i t e r a t u r e on f a t m e ta b o lism i s n o t a s s t r a i g h t f o r
w ard a s ou r b r i e f r e v ie w o f i t b y m eans o f s e l e c t e d r e f e r e n c e s
may im p ly . O fte n e m in en t b io c h e m is t s r e a c h c o n c lu s io n s w h ich
a r e a p p a r e n t ly c o n t r a d i c t o r y . More c a r e f u l e x a m in a tio n o f
t h e a p p a r e n t c o n t r a d i c t i o n s may r e v e a l t h a t t h e y a r e b a sed on
♦
e x p e r im e n t a tio n s w h ic h v a r y i n t h e i r c o n d it io n s o r i n t h e i r
m e th o d . The v a r io u s c o n c lu s io n s may f r e q u e n t ly be s a id t o be
th e r e s u l t s o f d i f f e r e n t e x p e r im e n ts r a th e r th a n t r u l y c o n tr a
d i c t o r y c o n c lu s i o n s .
The l i m i t a t i o n s o f one i n d i v i d u a l m ethod o f e x p e r im e n ta
t i o n may a l s o c a u se th e d i f f i c u l t y . The i n t a c t a n im a l g i v e s
a d m ir a b le r e s u l t s b u t l e n d s i t s e l f t o b io c h e m ic a l i n t e r p r e t a
t i o n s w h ic h may b e s a id t o be p r i n c i p a l l y o v e r a l l e f f e c t s
s i n c e th e m ethod l i m i t s t h e s tu d y o f th e in te r m e d ia r y r e a c t i o n s
t o th e n e c e s s i t y o f r e a d in g m echanism s i n f o t h e o v e r a l l p r o
d u c t s o b t a in e d . The more d i r e c t m ethod of. i s o l a t i n g th e i n
te r m e d ia r y s t e p b y i n v i t r o m eans h a s e lu c id a t e d many p ro b lem s
I n in te r m e d ia r y m e ta b o lis m . The m ethod o f u s in g t i s s u e hom o-
g e n a t e s a f f o r d s b e t t e r i s o l a t i o n o f th e b io c h e m ic a l p r o p e r t i e s
o f an in d iv i d u a l t i s s u e and r e s u l t s i n b e t t e r d i f f u s i o n b u t
t h e hom ogenate m ethod d o e s n o t r e p r o d u c e th e c o n d it io n s e x i s t
i n g In c e l l u l a r m e ta b o lis m , a s i s o b ta in e d by -the u s e o f t h e
13
t i s s u e s l i c e m eth o d . A n o th e r d i f f i c u l t y w ith t h e h om ogenate
m ethod i s t h a t v a r i a t i o n i n i t s p r e p a r a tio n may in c lu d e o r
e x c lu d e enough o f a p o r t io n o f t h e t i s s u e so a s t o make t h e
c o m p o s itio n o f one hom ogen ate d i f f e r e n t from t h a t o f a n o th e r
e x p e r im e n te r and h en c e t h e d i f f e r e n t r e s u l t s . The i n t a c t a n i
m al m ethod a p p e a r s t o he b e t t e r s u i t e d f o r b a la n c e s t u d i e s
th a n t h e i n v i t r o m ethod s i n c e b a la n c e s t u d i e s r e q u ir e t h e
o v e r a l l i n t e r a c t i o n o f b io c h e m ic a l s y s te m s t o i n d i c a t e t h e
m a jo r r o u te o f a m e t a b o lit e } t h e u s e o f i s o t o p i c t r a c e r s h a s
p r o v e n c o n c lu s iv e by t h i s m eth o d . The o p in io n h a s b e e n e x
p r e s s e d by a u t h o r i t i e s o f t h e n e c e s s i t y f o r s tu d y in g a b i o
c h e m ic a l f a c t b y s e v e r a l d i f f e r e n t m eth od s t o Judge i t s w o r th .
The n e e d f o r p h o s p h o r y la tio n o f f a t t y a c id s p r io r t o t h e i r
o x id a t io n a s shown by L e h n in g e r i s a u s e f u l id e a f o r t h e r e
c o n c i l i a t i o n o f tw o a p p a r e n tly c o n t r a d ic t o r y c o n c lu s io n s fou n d
i n t h e r e c e n t l i t e r a t u r e . The c o n c lu s io n by K rebs and E g g le s to n
(1 9 4 5 ) t h a t a c e t o a c e t a t e d o e s n o t e n t e r th e K rebs c y c le a p p e a r s
t o c o n t r a d ic t t h e c o n c lu s io n r e a c h e d by b o th Buchanan e t a l
(1 9 4 6 ) and L e h n in g e r (1 9 4 5 ) t h a t a c e t o a c e t a t e d o e s e n t e r t h e
K rebs c y c l e a s i t s o x i d a t i v e r o u t e . K rebs and E g g le s t o n (1 9 4 5 )
in c u b a te d m in ced t i s s u e u n d er a n a e r o b ic c o n d it io n s w h ich e x
c lu d e d t h e a e r o b ic o x i d a t i v e g e n e r a t io n o f l a b i l e p h o sp h a te ; w h ile
Buchanan and a s s o c i a t e s and L e h n in g e r u s e d a e r o b ic c o n d it io n s
and had w hat a p p e a r s t o h ave b een a m ore a d e q u a te su p p ly
14
o f l a b i l e ph.as.ph.ate a s i s a p p a r e n tly r e q u ir e d f o r t h e a c t i v a
t i o n o f a c e t o a c e t a t e . I n ou r o p in io n , both, o f t h e s e a p p a r e n t
l y c o n t r a d ic t o r y c o n c lu s io n s a r e e n t i r e l y v a l i d , w it h p e r h a p s
th e e s s e n t i a l d i s t i n c t i o n t h a t K rebs* r e s u l t be q u a l i f i e d t o
h i s p a r t i c u l a r a n a e r o b ic c o n d i t i o n . K rebs* e x p e r im e n t may be
f r e e l y in t e r p r e t e d , to . mean t h a t th e g iv e n c o n d it io n s e x c lu d e d
s u f f i c i e n t l a b i l e p h o sp h a te ^ to a c t i v a t e a c e t o a c e t a t e , h e n c e
th e a c e t o a c e t a t e w as m e r e ly r e d u c e d , I b i s i s m e r e ly o u r i n
t e r p r e t a t i v e r e c o n c i l i a t i o n o f two e m in e n t, b u t a p p a r e n tly
c o n t r a d ic t o r y ,' a u t h o r i t i e s b a se d on ou r a c c e p ta n c e , o f t h e n eed
f o r f a t t y a c id a c t i v a t i o n by s u f f i c i e n t l a b i l e p h o sp h a te* Lv
Hhe r e l a t i o n o f l a b i l e p h o sp h a te t o f a t t y a c id u t i l i z a t i o n
seem s im p o r ta n t enough t o w a r r e n t f u r t h e r s t u d y b y m eth od s
a d d i t i o n a l t o t h e h om ogen ate on e u se d b y L e h n in g e r . 1 s tu d y
o f t h e e f f e c t o f l a b i l e p h o sp h a te on t h e m e ta b o lism o f i n t a c t
t i s s u e s h a s n o t y e t b e e n r e p o r te d *
®he j n v iv o b a la n c e s t u d i e s in th e l i t e r a t u r e i n d i c a t e
th e im p o r ta n c e o f c a r b o h y d r a te u t i l i z a t i o n t o n orm al f a t m eta
b o lis m . C a r b o h y d r a te s n o t o n ly a f f e c t f a t s , b u t t h e y a r e u n iq u e
i n t h e i r a n a e r o b ic g e n e r a t io n o f l a b i l e p h o s p h a te , i n a d d i t io n
to. t h e i r o x i d a t i v e g e n e r a t io n o f l a b i l e p h o sp h a te l i k e many
m e t a b o l i t e s , f h e a n a e r o b ic g e n e r a t io n o f l a b i l e p h o sp h a te
from c a r b o h y d r a te i s one p o s s i b l e h y p o t h e t ic a l m eans b y w h ic h
c a r b o h y d r a te u t i l i z a t i o n may h a v e a s p e c i a l e f f e c t on n orm al
f a t m e ta b o lis m i n c e r t a i n s p e c i e s , b u t t h i s I s our own u n iq u e
15
id e a and i s e n t i r e l y u n t e s t e d . A n o th e r f a c t s t r e s s i n g t h e
o v e r a l l e f f e c t o f c a r b o h y d r a te u t i l i z a t i o n on n orm al f a t m eta
b o lis m b u t w ith s p e c i a l em p h a sis on t h e a n a e r o b ic p h a se o f
c a r b o h y d r a te u t i l i z a t i o n i s th e r e c e n t d is c o v e r y b y C o r i (1 9 4 5 ,
1 9 4 6 ) t h a t i n s u l i n p ro m o tes t h e h e x o k in a s e r e a c t i o n , p a r t o f th e
a n a e r o b ic p h a se o f c a r b o h y d r a te m e ta b o lis m . The o v e r a l l e f f e c t
o f i n s u l i n on c a r b o h y d r a te u t i l i z a t i o n i s e s t a b l i s h e d and i t s
e f f e c t , i n d i r e c t o r o t h e r w is e , on f a t t y a c id m e ta b o lism i s
a p p a r e n t b y i t s a b o l i t i o n o f k e t o s i s i n t h e d i a b e t i c . B ut i s
t h e i n s u l i n e f f e c t on a k e t o s i s due t o i t s s t im u la t io n o f
p r o d u c tio n o f l a b i l e p h o sp h a te o b ta in e d b y b e t t e r c a r b o h y d r a te
u t i l i z a t i o n ? I n o t h e r w o r d s, d o e s n orm al c a r b o h y d r a te u t i l i z a
t i o n a f f o r d t h e e s s e n t i a l l a b i l e p h o sp h a te f o r norm al f a t t y
a c id a c t i v a t i o n ? T h ese a r e th e q u e s t io n s w h ich i n t e r e s t u s .
The s tu d y o f t h e e f f e c t o f an in t e r m e d ia t e o f c a r b o h y d r a te
m e ta b o lism on f a t s i s a ty p e o f b a la n c e s tu d y w h ich w ould b e s t
be f a c i l i t a t e d b y t h e t r i e d m ethod o f t h e i n t a c t a n im a l as' w as
done i n t h e s t u d i e s o f t h e k e t o s i s - a p a r i n g p r o p e r t ie s o f c a r
b o h y d r a te in t e r m e d ia t e s b y D e u e l (1 9 3 8 ) and S h a p ir o ( 1 9 3 5 ) .
A s i m i l a r m ethod o f d i r e c t a d m in is t r a t io n o f l a b i l e p h o sp h a te
i n t o an i n t a c t a n im a l w as g iv e n some p r e lim in a r y s tu d y b u t was
n o t u se d s in c e t h e p ro p er a n im a ls and m a t e r i a ls w ere n o t immed
i a t e l y a v a i l a b l e . An a c c o u n t o f t h e p r e lim in a r y s tu d y o f t h e
p r o p o sed i n t a c t a n im a l m ethod i s g iv e n i n t h e p r o c e d u r e s e c t i o n
o f t h i s p a p e r .
16
!Ehe bod y o f t h i s s tu d y i s ta k e n up w it h t h e e f f e c t o f
p h o s p h o r y la tio n o f k e to n e b o d ie s i n v a r io u s i n t a c t t i s s u e s
o f th e r a t , i n an e f f o r t t o e x te n d th e h om ogenate s t u d i e s
o f h e h n in g e r to t h e e f f e c t o f l a b i l e p h o sp h a te com pounds
on t h e k e to n e bod y m e ta b o lism o f i n t a c t t i s s u e s . Some s tu d y
o f t h e r e l a t i o n o f c a r b o h y d r a te c o n t e n t t o t h e l a b i l e p h o sp h a te
c o n t e n t i n in d iv i d u a l t i s s u e s i s a n o th e r o b j e c t i v e .
ESPERBOMEAL PEOGEDOEE AHD M E1EEDDS
I n t a c t A n im al M ethod
Ehe i n t a c t a n im a l m eth od , h i s t o r i c a l l y t h e f i r s t m ethod
o f b a la n c e s t u d i e s , w a s c o n s id e r e d f i r s t b a t h a s n o t y e t b e e n
t r i e d a s a m eans o f t e s t i n g w h eth er o r n o t c a r b o h y d r a te s e f f e c t
t h e m e ta b o lism o f f a t s b y t h e i r u n iq u e and a d d i t io n a l g e n e r a
t i o n o f l a b i l e p h o sp h a te com pounds from g l y c o l y s i s . Ehe i n
v iv o m ethod o f D e u e l (1 9 3 8 ) and S h a p ir o (1 9 5 5 ) a p p e a r s t o be
one s u i t a b l e m ethod o f t e s t i n g th e e f f e c t o f a d m in is t r a t io n
o f c a r b o h y d r a te s and c a r b o h y d r a te in t e r m e d ia t e s on t h e m eta
b o lis m o f Ice t o n e b o d ie s b y o b s e r v in g t h e a b o l i t i o n o f k e t o n u r ia
i n r a ts , f e d b u t y r ic a c i d . S i m i l a r l y , I n j e c t i o n o f A 3E P o r
o t h e r l a b i l e p h o sp h a te compounds, i n t o r a t s w ith a s i m i la r e x
o g e n o u s k e t o n u r ia m ig h t b e e x p e c te d t o c a u se a s im ila r r e d u c
t i o n I n th e k e to n u r ia a s w as o b ta in e d by D euel* s a d m in is t r a t io n
o f c a r b o h y d r a te s , i f t h e e f f e c t o f c a r b o h y d r a te is; due t o t h e
l a b i l e p h o sp h a te c o n t e n t . I h e m eth od , h o w ev er, i s n o t a n en
t i r e l y c e r t a i n on e and due t o our l i m i t e d s u p p ly o f AEP w as
n o t u s e d . A d i a b e t i c r a t w it h an en d o g en o u s k e t o n u r ia ,5 w h ic h
may car may n o t be c o m p lic a te d b y horm onal d i f f e r e n c e s , w as n o t
u s e d f o r s i m i l a r r e a s o n s . t h e a p p a r e n tly more v a l i d m ethod
o f t e s t in g ; t h e k e t o s i s — s p a r in g p o s s i b i l i t i e s o f l a b i l e p h o sp h a te
com pounds I n I n t a c t a n im a ls Is: b y th e u se o f a s u i t a b l e a n im a l
18
w ith a .fa s tin g ; k e t o s i a w h ich i s u n c o m p lic a te d and i s e n d o g e n o u s.
A s e a r c h o f th e l i t e r a t u r e d i s c l o s e d few a n im a ls w it h a f a s t
in g k e t o s is ; "besides man and m onkeys; but non e o f th em -proved
s u i t a b l e o th e r th a n m on k eys. , A r e p o r t by C e r v e llo and G firg en ti
(1 9 1 4 ) s t a t i n g t h a t r a b b i t s h ave a k e t o s i s was t e s t e d by u s
but th e e x t e n t o f the: k e t o n u r ia was fou n d t o be t o o s l i g h t t o
be of' any v a l u e . A . r e p o r t by M ir sk y (1 9 4 1 ) s t a t i n g t h a t d u ck s
h a v e a f a s t i n g k e t o s i s also: m e n tio n s p e c u l i a r i t i e s d i f f e r i n g
fro m humans and h en ce m akin g th e stu d y o f du ck s o f l i t t l e
v a lu e t o u s . 3?he m onkeys w ere n o t a v a i l a b l e and t h e t e s t i n g
o f m onkeys b y t h i s i n t a c t a n im a l m ethod h a s n o t y e t b e e n a t
te m p te d . H ow ever, in , v ie w o f cu r su b s e q u e n t e v id e n c e o f th e
s t im u la t o r y e f f e c t o f AEP upon th e u p ta k e o f k e to n e bardies by
r a t l i v e r s l i c e s , t h e f u t u r e p o s s i b i l i t i e s o f th e o r i g i n a l
i n t a c t a n im a l me-fhad o f t e s t i n g a r e e n h a n c e d .
D iaphragm i i s s u e S l i c e S t u d ie s •
I n v iew o f the: ab o v e l i m i t a t i o n s and u n c e r t a i n t i e s w ith
the: i n t a c t a n im a l m eth od , i t w as d e c id e d t o s tu d y th e e f f e c t
o f AEB upon the: k e to n e bod y m e ta b o lism o f i n t a c t r a t t i s s u e
s l i c e s by th e W arburg m eth o d . So f a r , t h e e f f e c t o f l a b i l e
p h o sp h a te compounds upon k e to n e b e d ie s in t i s s u e s h a v e n o t b een
r e c o r d e d in th e l i t e r a t u r e ; o n ly h om ogen ate s t u d i e s h a v e b een
r e p o r t e d . R e l a t i v e l y few t i s s u e s l i c e s t u d i e s o f an y k in d
h ave b e e n r e p o r te d on m u scle t i s s u e , due t o th e d i f f i c u l t y i n
19
s l i c i n g t h e t i s s u e . M u sc le i s , n e v e r t h e l e s s , t h e l o g i c a l f i r s t
t i s s u e to- s tu d y due to' i t s im p o r ta n c e i n t o t a l m e ta h o lism and
th e fa c t, t h a t m u scle i s t h e s i t e o f o x i d a t i o n o f k e to n e b o d ie s .
When, h o w e v e r , th e m u scle s t u d i e s p r o v e d t o be i n c o n c lu s iv e
a s t o t h e l a b i l e p h o sp h a te e f f e c t , t h e s tu d y o f l i v e r s l i c e s
was u n d e r ta k e n w ith some s u c c e s s i n d e te r m in in g th e r o l e o f
l a b i l e p h o sp h a te com pounds i n k e to n e bod y m e ta b o lism w it h in
i n t a c t t i s s u e s .
G e n e r a l p r o c e d u r e — f h e r a t diap hragm i s a m u scle t i s s u e ,
u n iq u e i n i t s a d a p t a b i l i t y t o W arburg s t u d i e s due t o i t s s i z e .
I t i s a p p r o x im a te ly Q .4 mm. t h i c k and i s p erm eab le to an a t
m osphere o f oxy g en w ith o u t, s l i c i n g . I h e d iap hragm may b e h a lv
ed a f t e r i t h a s b een f r e e d b y s c i s s o r s from th e a n im a l, k m edi
um s i z e d r a t t h u s p r o v id e s a t e s t hem idiaph ragm and a c o n t r o l
hem idiaphragm o f a p p r o x im a te ly 3 5 mg. e a c h by d ry w e ig h t . I n
a p p r o p r ia te in c u b a t io n m ed ia , th e diaphragm w i l l s u r v iv e f o r
o v e r t e n h o u r s . S tu d ie s; h ave shown t h a t , w ith a v e r a g e c le a n
l i n e s s , t h e m u scle i s s t a b le f o r o v e r fo u r h o u r s , a f t e r w h ic h
tim e th e lo g a r it h m ic gro w th p h a se o f b a c t e r i a may be e n c o u n te r e d .
I h e t e s t s - w ere s a f e l y l i m i t e d to a b o u t fo u r h o u r s .
£he p r o c e d u r e u s e d in t h e diaphragm s t u d i e s c o n s i s t e d o f
sim u lta n eo u s; c h e m ic a l a n a l y s e s and th e us® o f a W arburg appa
r a t u s . I h e W arburg s t u d ie s , r e s u l t e d i n th e s im u lta n e o u s e v a lu
a t i o n o f t i s s u e c o n s t a n t s f o r th e r a t e o f r e s p i r a t i o n and f o r
th e r a t e o f u p ta k e o f k e to n e b o d ie s from t h e m ed ia , th e
&0
c o n v e n t io n a l Q q2 and o u r Q^.e^.* The c h e m ic a l a n a l y s e s , done
s im u lta n e o u s ly ' w ith or under c o n d it io n s e q u iv a le n t to th e
Warburg t e s t i n g , w ere done to; a s s a y th e g ly c o g e n and th e l a b i l e
p h o sp h a te c o n t e n t s o f th e t e s t e d t i s s u e s * The e f f e c t o f ATP
w as o b ta in e d and n o te d b y i t s a d d i t i o n t o th e in c u b a t io n m edia
o f n orm al and f a s t e d diap hragm , u s in g c o n t r o l s w it h no added
ATP i n e a c h c a s e . The g ly c o g e n r e s e r v e o f th e t i s s u e was d e
p l e t e d by f a s t i n g th e r a t s f o r o v e r a w eek . Norm al and f a s t e d
d iap h ragm s w ere a n a ly z e d t o s tu d y th e e f f e c t o f d e p l e t io n o f
g ly c o g e n on th e l a b i l e p h o sp h a te c o n te n t and a l s o t o f i n d th e
a c t u a l t i s s u e c o n te n t o f l a b i l e p h o sp h a te e x i s t e n t a t th e tim e
o f th e W arburg t e s t in g - .
The W arburg t e s t s w ere m a n ip u la te d in a c o n v e n t io n a l app a
r a t u s in t h e manner o u t lin e d by TJinbreit e t a l (1 9 4 6 ) u s in g 1 5
m l. c o n i c a l v e s s e l s , 3 7 °C . te m p e r a tu r e and an atm osp h ere o f
o x y g e n . The k e to n e body s u b s t r a t e s c o n s is t e d o f th r e e m i l l i
gram s e a ch p er v e s s e l , c a lc u l a t e d on th e f r e e a c id b a s i s .
When addled, t h e ATP w as u sed a t a v e s s e l c o n c e n t r a t io n o f
a . 00-6 J£. A e e t p a c e t a t e and h -b e ta h y d r o x y b u ty r a te w ere th e
a l t e r n a t e k e to n e body s u b s t r a t e s .
P r e p a r a t io n s — S e v e r a l p r e p a r a tio n s w ere made and t e s t e d
a s p r e l i m i n a r i e s to th e W arburg s t u d i e s . The ATP was i s o l a t e d
from r a b b it m u scle a s th e b ariu m s a l t by K o e n ig * s (1 9 4 5 ) p r o
cedure;; i t w as f r e e d o f barium by d i s s o l v i n g a w e ig h e d am ount
o f th e s a l t in d i l u t e h y d r o c h lo r ic a c id f o llo w e d b y a d d it io n
E l
a f th e c o r r e c t amo,ant. o f sod iu m s u l f a t e and by c e n t r if u g in g
o f f th e p r e c i p i t a t e d b ariu m s u l f a t e * Ih e L .-b e ta h y d r o x y b u ty r a te
w as i s o l a t e d from r a t u r in e by B lu n d e r * s (1 9 3 8 ) p r o c e d u r e ; i t
was, f r e e d o f c a lc iu m and z i n c by th e u s e o f sod iu m c a r b o n a te .
She a c e t o a c e t a t e w as p r e p a r e d by c o ld d i l u t e a l k a l i n e h y d r o ly -
s i s o f e t h y l a c e t o a c e t a t e f o llo w e d by l y o p h i l i z a t i o n a c c o r d in g
to- th e p r o c e d u r e o f B ju n g g r e n (1 9 R 4 ); th e a c e t o a c e t a t e was
p r e p a r e d f r e s h f o r e a c h t e s t a s i t i s u n s t a b le . E ach p r e p a r a
t i o n was a d ju s t e d t o pH 7 .4 p r io r t o i t s u s e .
I n c u b a t io n m edia— Ih e in c u b a tio n m edia d id n o t f o llo w th e
sta n d a r d u s e o f R in ger* s s o l u t i o n s in c e a d d i t i o n s o f s u b s t r a t e
a t AIP w ould add e x tr a m e t a l l i c io n s t o th e m ed ia w h ich w ou ld
upset th e i o n i c b a la n c e o f th e R in ger* s. s o l u t i o n . M u scle i s
s e n s i t i v e t o c h a n g e s i n th e I o n ic b a la n c e and su ch c h a n g e s i n
v a l i d a t e th e e x p e r im e n ts w it h m u s c le . C o n s e q u e n tly t h e medium
o f e a c h v e s s e l w as a sse m b le d t o i t s f i n a l i s o t o n i c and i o n i c -
a l ly - b a la n e e d c o n c e n t r a t io n b y a d d i t io n s o f s t o c k s a l t s o l u t i o n s
t o th e v e s s e l , a f t e r t a k in g in t o e x a c t a c c o u n t t h e m e t a l l i c
i o n s d e r iv e d from t h e AEP and t h e s u b s t r a t e i n e a ch v e s s e l .
Our o b j e c t w as t o m a in ta in t h e b a la n c e d r a t i o o f I o n s w h ich
e x i s t in b lo o d seru m , (K aH K )-J-(C a) (M g )^ (1 5 4 x 5 ) ? ^ ( E .5 x , l ’ .J>).
Q 2he medium d i f f e r s from serum i n i t s h ig h e r p h o sp h a te c o n c e n
t r a t i o n due t o n eed f o r a p h o sp h a te b u f f e r w h ich h a s t h e s l i g h t
d is a d v a n ta g e o f c a u s in g th e s o l u t i o n o f c a lc iu m and m agnesium
t o p r c e e d s lo w ly and o n ly a f t e r s h a k in g . We f e e l t h a t our
22
p r e c a u t io n s w ith t h e in c u b a t io n m edia w ere e s s e n t i a l f o r t h e s e
d iap h ragm m u scle s t u d i e s . An exam ple o f t h e m ethod o f co m p o sin g
th e in c u b a t io n m ed ia i s g iv e n i n th e t a b l e b e lo w .
t y p i c a l d a ta s h e e t o f medium c o m p o s itio n
l e s t hem idiaph ragm C o n tr o l hem idiaph ragm
s u b s t r a t e 3 m g ./m l. 1 .0 0 m l. 1 .0 0 m l
s a l t m ix tu r e * 0 .2 5 0 .2 5
H a d 1 8 m g ./m l. 0 .0 1 .5 6
M /15 p h o sp h a te b u f f e r 0 . 3 0 0 .3 0
w a te r 0 . 78 0 .2 2
ATP 1 7 m g ./m l. ** 1 .0 0 0 .0
3 .3 3 m l. 3 .3 3 mi,
* S a l t m ix tu r e s o l u t i o n c o n s i s t s o f tw e n ty p a r t s o f 1 . 5 1
KC1, f i f t e e n p a r t s o f 1 . 1 M CaClg and t e n p a r t s o f 1 . 1 M
MgCl2 .
* * C o n ta in s 2 6 m g ./m l. o f HaCI by n e u t r a l i z a t i o n .
A n a ly s i s o f ch a n g e i n he t o n e b o d y s u b s t r a t e - - C h em ica l
a n a l y s i s o f d is a p p e a r a n c e (u p ta k e ) o f t h e k e to n e body s u b s t r a t e
i s n o t th e s im p le o p e r a tio n o f a n a ly z in g f o r th e c o n te n t o f a
s i n g l e s u b s ta n c e b e f o r e and a f t e r a p e r io d o f in c u b a t io n . A c e t o -
a c e t a t e may b e d e e a r b o x y la te d t o a c e t o n e or r e d u c e d t o h y d r o x y -
b u ty r a te and th e o n ly c o n s t a n t f r a c t i o n i n e a c h o f th e t h r e e
p o s s i b l e h o m o lo g o u s fo rm s i s t h e a c e to n e c o n t e n t . W h ile
£3
V an S ly k e r s (194QJ o r i g i n a l macro m ethod f o r t o t a l Ice to n e b o d ie s
i s b a se d on t h i s c o n s t a n t a c e t o n e f r a c t i o n , th e g r a v im e t r ic
f a c t o r s fo r e a ch o f the: k e to n e b o d ie s o b ta in e d by Van S ly k e
do n o t c h e c k c o n s i s t e n t l y w it h t h e i r a c e to n e c o n t e n t . IBhe
m icro m o d if ic a t io n by E d son (1 9 3 5 ) o f Van S ly k e 1s m acro p r o c e
dure h a s a p r e c i s i a n a c c o r d in g t o E dson o f a b o u t 1 0 $ .
We h ave b een a b le t o m o d ify Van S ly k e * s t o t a l k e to n e body
m ethod by a g r a v im e t r ic m ic r o p r o c e d u r e w h ich r e s u l t s i n g r a v i
m e t r ic f a c t o r s w h ich a r e more c o n s i s t e n t w it h t h e a c e to n e con
te n ts : o f the: v a r io u s k e to n e b o d ie s and w h ich h a s a p r e c i s i o n
o f a t l e a s t 4 $ and a r e c o v e r y f a c t o r o f 9 4 $ a t th e m ic r o con
c e n t r a t i o n s u s e d in th e e x p e r im e n ts . D e t a i l s fo llo w *
Wash a p p r o x im a te ly t h r e e m illig r a m s o f k e to n e body i n t o
a c o n ic a l c e n t r i f u g e tu b e ( 5 0 m l.) w ith a b o u t t h i r t y m i l l i
l i t e r s o f w a te r ; add: f i v e m i l l i l i t e r s o f t e n p e r c e n t co p p er
s u l f a t e s o l u t i o n and l e t s ta n d fo r a h a l f h o u r; add f i v e m il
l i l i t e r s o f t e n p e r c e n t c a lc iu m h y d r o x id e s u s p e n s io n and p la c e
th e co rk ed c e n t r if u g e tu b e in a r e f r i g e r a t o r o v e r n ig h t ; c e n t r i
fu g e t o p a c k h ard and c a r e f u l l y d e c a n t th e s u p e r n a ta n t f l u i d
in t o a r e f l u x f l a s k (1 5 0 m l.) f i t t e d w ith a ground g l a s s
J o in te d c o n d e n s e r ; add f i f t e e n m i l l i l i t e r s o f c o o l w a ter t o
th e p r e c i p i t a t e i n th e c e n t r i f u g e tu b e and m ix t o w ash; r e c e n -
t r i f u g e th e p r e c i p i t a t e in th e c e n t r if u g e tu b e an d - add th e
s u p e r n a ta n t f l u i d to th e r e s t i n th e r e f l u x f l a s k ; add s e v e n
t e e n m i l l i l i t e r s o f t e n p e r c e n t m er cu ric s u l f a t e and f i v e
£4
m i l l i l i t e r s o f f i f t y p e r c e n t s u l f u r i c a c id t o th e r e f l u x f l a s k
and a d j u s t t h e con d en ser* h e a t on a s lo w h o t p l a t e t o b a r e ly
m a in ta in b o i l i n g f o r one and a h a l f h o u r s; a s so o n a s th e
b o i l i n g com m ences in tr o d u c e tw o m i l l i l i t e r s o f f i v e p e r c e n t
p o ta s s iu m dich rorn ate i n t o th e r e flu x - f l a s k by d ro p p in g i t down
th e c o n d e n se r tu b e ; f i l t e r o f f th e p r e c i p i t a t e i n th e f l a s k
by s u c t io n th r o u g h a w e ig h e d , f i n e - p o r o s i t y , s i n t e r e d g l a s s
b ottom ed c r u c ib le and w e ig h a c c u r a t e l y a f t e r d r y in g f o r an
h ou r a t 10Q °C.
The m ethod h a s b een p r o v e n by r e p e a te d t r i a l s ; w ith k e to n e
v
b o d ie s i n th e p r e s e n c e o f g l u c o s e , p r o t e i n s and a d e n o s in e t r i -
p h o sp h a te and a p p e a r s t o be d e p e n d a b le . The m ain in n o v a t io n
i s th e le n g t h y p e r io d f o r t h e p r e c i p i t a t i o n o f i n t e r f e r i n g
s u b s ta n c e s and th e u s e o f th e e n t i r e r e c o v e r a b le sam ple f o r
a s s a y in s t e a d o f o n ly an a l i q u o t o f th e sa m p le , by u t i l i z i n g
a m icro p r o c e d u r e w h ich f r e e s th e e n t i r e sam ple of. i t s p r e
c i p i t a t e d im p u r it ie s i n p la c e o f th e p r e v io u s m ethod o f f i l t e r -
o f f an a l i q u o t . The f o l lo w in g t a b l e com p ares o u r g r a v im e t ic
f a c t o r s w ith Wan S l y k e ' s .
g r a v im e t r ic fa c to r s : b y t o t a l k e to n e bod y m ethods
compound t h e o r e t i c a l b a se d Wan S ly k e our m icro
pin 1 9 .3 f o r a c e to n e m acro m o d if ic a t io n
B -b e ta h y d ro x y b .u ty r .a te 1 0 .7 S . 4 5 1 0 .6 . $ 0 .3 *
a c e to n e (1 9 .3 1 8 0 .0 1 9 .£ t 0 .5
a c e t o a e s t a t e 1 0 .9 — 1 0 .6 t 0 . 3
as
The f ig u r e i n th e above t a b l e marked w ith t h e a s t e r i s k ,
i s n o t th e p r e c i s i o n o f th e m ethod* I t i s b a se d on th e l a r g e s t
d e v i a t io n o b ta in e d i n a s e r i e s o f t e s t s w it h pu re s u b s ta n c e s *
The p r e c i s i o n i s b e t t e r th a n th e fo u r p e r c e n t c la im e d f o r t h e
m eth od . D u p lic a t io n o f r e s u l t s in one s e r i e s o f c o n t r o l s i s
i l l u s t r a t e d b y t h e s e f i g u r e s : £ 9 * 9 , 30*0 and 2 9 * £ mg* The
c o n t r o l s c a r r ie d th r o u g h o u t th e e n t i r e p r o c ed u r e make th e
r e c o v e r y d a ta l e s s im p o r ta n t e x c e p t a s a v a l i d a t i o n o f th e
m ethod*
Our m icro m o d if ic a t io n o f th e t o t a l k e to n e body m ethod
e n a b le d u s t o o b t a in th e d is a p p e a r a n c e o f s u b s t r a t e k e to n e
b o d ie s w ith o u t r e g a r d t o p o s s i b l e c h a n g e s in t o o th e r h om ologou s
fo rm s s o l e l y on th e c o n s t a n t a c e to n e c o n t e n t and i n one a c c u r
a t e o p e r a t io n . D o s s e s o f s u b s t r a t e w ere assum ed t o b e u p ta k e
b y th e t i s s u e * To in s u r e c o n t r o l , two c o n t r o l v e s s e l s w ith
s u b s t r a t e but n o t i s s u e w ere c a r r ie d th r o u g h o u t th e in c u b a t io n
and a s s a y in g p r o c e d u r e s .
C h em ica l m eth od s fo r g ly c o g e n and l a b i l e p h o sp h a te a s s a y s -
The g ly c o g e n c o n t e n t o f t h e t i s s u e s exam in ed was o b ta in e d b y th e
m ethod o f G ood, Kramer and Som ogyi (1933'1* The d e p le te d r a t s
w e re f a s t e d fo r a w eek u n t i l p r a c t i c a l l y fre ® o f g ly c o g e n *
The l a b i l e p h o sp h a te was a s s a y e d i n a m o d ifie d way t o ob
t a i n th e t i s s u e c o n te n t e x i s t i n g a t th e tim e o f th e Warburg
t e s t s * Some l o s s e s i n l a b i l e p h o sp h a te from th e t i s s u e w ere
e x p e c te d t o r e s u l t due t o th e u n a v o id a b le h a n d lin g o f th d
2 6
t i s s u e s d u r in g t h e Warburg t e s t i n g ; th e h a n d lin g o f th e t i s s u e s
fo r a n a ly s is : d u p lic a t e d th e h a n d lin g i n th e t e s t i n g . Our mod
i f i e d p r o c ed u r e com bined f e a t u r e s o f th e m ethods g iv e n in
Wm.bre i t e t a l (1 9 € 6 | and i n p a p ers o f E is k e and Subbarow (1 9 2 5 r
1929} .
D e t a i l s o f t h e l a b i l e p h o sp h a te m ethod f o l l o w . E n t ir e
diap hragm s w ere q u ic k ly s t r ip p e d o u t and w e ig h e d . E ach w as
q u ic k ly im m ersed in t o f i f t e e n m i l l i l i t e r s o f c o ld f i v e p e r c e n t
t r i c h l o r o a c e t i c a c id c o n ta in e d i n a sm all, h o m o g e n iz sr , e n c l o s
ed by a d r y - ic e j a c k e t . f h e diaphragm w as q u ic k ly h o m o g en ised
and f i l t e r e d w ith an a d d i t io n a l w a sh in g o f t e n m i l l i l i t e r s o f
th e same c o ld a c id b y m eans o f f i l t e r p ap er on an i c e j a c k e t e d
f u n n e l; An added p r e c a u tio n a g a in s t h e a t in g w as th e u s e o f an
i c e j a c k e t fo r th e r e c e i v e r o f th e f i l t r a t e , f h e in o r g a n ic
p h o sp h a te w as ta k e n o u t o f an a l i q u o t o f th e f i l t r a t e a s th e
c a lc iu m s a l t f o llo w in g Timbre it * s p r o c e d u r e . fh e c r e a t in e p h o s
p h a te w as o b ta in e d a s th e a p p a ren t in o r g a n ic p h o sp h a te by p e r
m it t in g an a l i q u o t o f th e o r i g i n a l f i l t r a t e to warm up and
h y d r o ly z e th e p h o sp h a te o f th e c r e a t i n e . f h e " sev e n -m in u te"
l a b i l e p h o sp h a te w as done on an a l iq u o t o f th e o r i g i n a l f i l
t r a t e by h o t a c id h y d r o l y s i s and s u b t r a c t io n o f th e a p p a r e n t
in o r g a n ic p h o s p h a te , ! h e c r e a t in e p h o sp h a te was c a lc u l a t e d
s i m i l a r l y b y s u b t r a c t in g th e " tru e" in o r g a n ic p h o sp h a te from
th e "ap p aren t" in o r g a n ic p h o s p h a te . 1 he r e s u l t s a r e i n f a b l e
¥ w h ich a ls o c o n t a in s U m breit* s phos p h a te c o n te n t o f l i v e r .
l i v e r S lic e S tu d ie s
I h e l i v e r s l i a e t e s t s w ere done in th e Warburg a p p a r a tu s
i n a s im ila r manner t o th e p r e v io u s t e s t s w ith d ia p h ra g m s.
N orm al r a t s w ere u se d in s t e a d o f th e cu sto m a ry f a s t e d a n im a ls
due t o a b e l i e f t h a t p erh a p s l i v e r m ight r e t a i n more o f i t s
n o rm a l s m a ll c o n te n t o f l a b i l e p h o s p h a te , i f f e d . One s e r i e s
o f t e s t s w as made w ith th e same A1P a d d it io n and s u b s t r a t e co n
c e n t r a t io n a s w as u sed in t h e diaphragm t e s t s . A n o th er s e r i e s
c f t e s t s w ith l i v e r was done w ith o u t s u b s t r a t e t o o b t a in th e
en d o g en o u s o u tp u t o f k e to n e b o d i e s . A n o th er v a r i a t i o n w as a
s e r i e s i n w h ich th e s u b s t r a t e w as in c r e a s e d t h r e e f o l d i n an
e f f o r t t o s tu d y p o s s i b l e m ass a c t i o n e f f e c t s , w it h and w ith o u t
ATP a d d i t i o n . Iw o t o fo u r hundred m illig r a m s o f w e t l i v e r ,
w h ich had b een s l i c e d t o a u n ifo r m t h i c k n e s s o f 0 .4 mm. b y a
S t a d ie (1 9 4 4 ) m icro to m e, w ere em ployed in e a c h v e s s e l . S h e
o u tp u t and u p ta k e o f k e to n e b o d ie s o b ta in e d w ere sm a ll, b u t a
c a l c u l a t i o n e l i m i n a t e s th e p o s s i b i l i t y t h a t th e c h a n g e s o b s e r
ved a r e th e r e s u l t o f d i f f u s i o n due t o a s t a t i c s y s te m a c r o s s
a p erm ea b le membrane. I h e s e r e s u l t s a r e d is c u s s e d f o r w h at
t h e y a r e w o rth s i n c e th e number o f t e s t a , a lth o u g h c le a n c u t
i n r e s u l t , a r e few in a c t u a l num ber.
9
CHAPTER T
RESULTS
mRT>i i
E f f e c t o f U P on o x y g en co n su m p tio n o f r a t diap hragm
% 2
w it h o u t ATP
% 2
w ith ATP
one diaphragm
h a lv e d *
in hydr o x y t u t y r a t e ,
2 . 6 4 . 3
on e d iap hragm
h a lv e d *
in hyd r o x y b u ty r a t e ,
2 . 2 6 . 2
one diaphragm,
h a lv e d *
in a c e t o a c e t a t e ,
3 .0 3 . 5
on e d iap h ragm
h a lv e d *
in a c e t o a c e t a t e ,
4 . 4 5 .3
s i n g l e e n t i r e d iap h ragm , w ith
AIP added from s i d e arm 5*1 "before 8 . 3 a f t e r
o f s i n g l e f l a s k
3 . 5 a v . 5 . 5 air.
A v e r a g e i n c r e a s e in SL. b y ATP
2
* T h e se d iap h ragm s w ere h a lv e d and e a ch h a l f w as t e s t e d i n
a s e p a r a te f l a s k . T h e se p r e lim in a r y t e s t s p r o v id e d e x p e r
i e n c e w ith th e d e te r m in a tio n o f t h e s u b s t r a t e and w it h i t s
a p p r o x im a te o r d e r o f d is a p p e a r a n c e .
29
TAKHE. II
U t i l i z a t i o n o f 1 - b e t ah yd r o x y h u ty r a te and o x y g e n b y r a t d iap h ragm
' i
B a ta : te m p e r a tu r e 37°G , tim e 3§- t o 4§- h o u r s , 0.006M ATP when ad d ed ,
o x y g e n a tm o sp h e r e , 3 mg* b u ty r a ta a s f r e e a c id p e r f l a s h
diaphragm t i s s u e d ry wt*
msr.
a d d it io n s t o m edia
**02
n 4 * 4 *
Hh e t
n o r m a l, d iv id e d * 3 2 none 4 . 0 • 2 . 3
4 2 ATP 5 .4 2 .1
n o rm a l, d iv id e d * 4 2 none 4 . 5 3 .2
*
3 5 ATP 6 . 3 2 .7
n o r m a l, d iv id e d * 45' none 4 . 4 3 .0
4 1 ATP 6 . 2 2 .6
n o r m a l, e n t i r e
s m a ll diaphragm 4 6 none 5 .7 3 .1
n o r m a l, e n t i r e
s m a ll diaphragm 4 3 ATP 8 . 5 2 .2
A v e r a g e 2 . 6
* d iv id e d i n d i c a t e s t h a t t h e diaphragm w as h a lv e d and one h a l f s e r v e d
a s c o n t r o l f o r t h e oth er- h a l f , e a eh h a l f b e in g t e s t e d i n s e p a r a t e f l a s h
* * % cet i s d e f in e d a s th e number o f m icrogram s o f s u b s t r a t e ta h e n up by
a m illig r a m o f t i s s u e b y d ry w e ig h t p er h o u r; t h e m in u s s i g n i s im p lie d
Q 2A E L E I I I
30
U t i l i z a t i o n o f a c e t o a c e t a t e and o x y g en b y r a t diap hragm
B a ta : te m p e r a tu r e 3 7 °C , tim e 3§- t o 4-f- h o u r s , O.OQ6M AEP when a d d ed ,
oxygen, a tm o sp h e r e , ,3 mg. a c e t o a c e t a t a a s a c id p e r f l a s k
diap h ragm t i s s u e d r y w t. a d d i t io n s t o m edia
mg. -- '
% 2
a **
ICet
n o r m a l, d iv id e d * 41 0 .1 $ g lu c o s e 4 . 3 5 .4
37 5 .2 4 . 5
n o r m a l, d iv id e d * 1 35 0 .2 $ g lu c o s e 4 . 9 6 . 3
3 0 ( a c c i d e n t l y u p s e t}
n o r m a l, e n t i r e
sm a ll diaphragm 3 2 n on e 6 .5 8 . 3
f a s t e d fo u r d a y s, 30 0 .2 $ g lu c o s e 5 .1 4 . 6
d iv id e d * 24 none 5 .7 3 .6
f a s t e d fo u r d a y s , 30 n on e 5 .8 6 .2
d iv id e d * 27 AIP 1 0 .0 4 . 7
f a s t e d fo u r d a y s ,
e n t i r e diaphragm. 70 AIB' 6 .8 4 .2
f a s t e d s e v e n d a y s, 4 7 0. 2 $ g lu c o s e 3 .4 4 . 4
d iv id e d * 3 7 n on e 4 .7 5 .1
f a s t e d s e v e n d a y s, 4 2 none 4 .8 5 .6
d iv id e d * 5 3 AfP 5 .2 4 . 3
f a s t e d s e v e n d a y s , 3 2 ft'FP - . . .
— 5.-9—------ 4 v 2 ------------
d i v i d e d 1 t o th e r h a l f u se d fo r g ly c o g e n t e s t , n on e foun d)
A v e r a g e ............................. 5 .1
* d iv id e d i n d i c a t e s t h a t diaphragm w a s h a lv e d , e a c h h a l f s e r v in g a s c o n t r o l
fo r o th e r and t e s t e d In s e p a r a ta f l a s k s
i s d e f in e d a s th e number o f m icrogram s o f s u b s t r a t e ta k e n up b y a
m illig r a m o f t i s s u e b y d r y w e ig h t per' hou r; th e m inus s i g n i s im p lie d
t&b l e nr
G ly c o g e n c o n te n t
t i s s u e
u n fa ste d rat. diaphragm;
C 3 r a t s )
r a t diaphragm, 1 day f a s t
(2 r a ts )
r a t diap h ragm , 4 daw f a s t
£2 r a t s )
r a t d iap h ragm , 7 d ay f a s t
£ 2 r a t s )
s k e l e t a l g ly c o g e n o f n orm al r a t *
s k e l e t a l g ly c o g e n o f n orm al man * *
G ly co g en p e r c e n t
0.8
0 .1 S
0 .0 6
le s s ; th a n 0 .0 1
0 . 8 - 1 .0 5
0 . 4 - 0 .6
* G u e st, M. £1941)
* % o s c a t i , G. £ 1 008)
32
TABLE V
L a b ile p h o sp h a te c o n te n t
t i s s u e P mg-. '%* P ” irg. ° /o P mg* ° /o t o t a l m o la r it y 4"1 '
0 or 1 a t l a b i l e p h o sp h a te
________ • __________; _____________ ; ________________ t im e s \Q T* M
n orm al d iap hragm
(3 r a t s ) 4 2 * 0 21*3 33*7 181
1 d a y f a s t e d diaphragm
( 4 rats)? 4 4 * 8 1 6 * 0 26*5 135
n orm al r a t a b d o m in a l
m u scle * * * 1 7 .0 55*0 3 2 .0 293
d i a h e t i c r a t abdom--
i n a l m u scle * * * 2 2 .0 57*0 3 4 .0 3 0 0
f r a c t i o n s o f n o rm a l c r e a t i n e p h o sp h a te f r a c t i o n * . 2 0 .0
l i v e r * * * * AEP f r a c t i o n . . . ............................... 0 .5 6
ASP f r a c t i o n • • * • • • • • • « » . * • » » 0 *0
* P i s in o r g a n ic p h o sp h o r u s; P i s c r e a t in e p h o sp h o r u s; P „ , se v e n -m in u te
0 l a b i l e p h o sp h o ru s c r
^ ^ a l c u l a t e : d ^ t o t a l ^ o f ' l n b i r e “ p h o sp h a te s~ o 'f" o r e a tr h e and se v e n -m in u te exp ress
ad i n term s; o f 10~tW[> the; minimum l a b i l e p h o sp h a te f o r a c t i v a t i o n ; th e
l i v e r f r a c t i o n s a r e e x p r e s s e d s e p a r a t e l y f o r e a ch f r a c t i o n i n te r m s o f
a v a i l a b i l i t y a s l a b i l e p h o sp h a te i n t h a t t i s s u e *
** S o s k in , S . {1946} The t o t a l m o la r it y o f l a b i l e p h o sp h a te i s our
c a l c u l a t i o n b a sed on th e p u b lis h e d a n a l y t i c a l d a ta *
** TJmbreit e t a l {1 946} The f i g u r e s a r e our c a l c u l a t i o n s b a sed on c i t a t i o n .
The. ADP i s u n a v a ila b le a s l a b i l e p h o sp h a te in l i v e r *
TAE5E ¥ 1
l a b i l e p h o sp h a te e f f e o t on th e u p tak e o f k e to n e b o d ie s by l i v e r s l i c e s
number
Df t e s t s
w e t t i s s u e s u b s t r a t e p e r f l a s k
w e ig h t , mg>
ATP a v e r a g e ch an ge
m o la r it y o f s u b s t r a t e
i
m g /f la s k m g/100 i
t i s s i
4 2.00 none 0 .0 + 0 .3 3 + 0 .1 6 5
2 200 3 mg.
1 -b e ta h y d r o x y b u ty r a te
o
o
none none **
[ a 200 3 mg.
1 - b e t ahydr o x y b u ty r a te
0 .0 0 6 - 0 .2 2 - 0 .1 1
a 400 9 mg.
a c e t o a c e t a t e
0 .0 - 0 .8 3 - 0 .2 0 8
a 400 9 rag.
ac e t o a c e t a t e
O.0Q6 - 0 . 9 2 - 0 .2 3
* P lu s s ig n i n d i c a t e s g a in , in k e to n e b o d ie s i n f l a s k ; m inus s i g n , l o s s .
* * None m eans l e s s th a n 0 . 1 m g. p e r f l a s k . The la o k o f net: change may
be in t e r p r e t e d a s an e q u ilib r iu m , c o n d it io n .
CHAPTER VI
DISCUSSION OF RESULTS
-Two c le a r new f a c t a a r e e v id e n c e d i n th e d a ta o b ta in e d
from, th e Warburg: e x p e r im e n ts w ith r a t d iap h ragm s: (1 ) s t im u la
t i o n o f r e s p i r a t i o n o f m u sc le i s o b ta in e d b y a d d it io n o f ATP
t o th e in c u b a t io n m ed ia; (Z ) a c e t o a o e t a t e i s u t i l i z e d a p p r o x i
m a te ly t w ic e a s f a s t a s L -b e ta h y d r o x y b u ty r a te b y in c u b a te d
m u scle and t h e r e f o r e a p p e a r s t o be th e im m ed iate m e t a b o lit e
o f th e k e to n e bod y group* T h ere a l s o a p p e a r s to; b e an e f f e c t
o f l a b i l e p h o sp h a te com pounds upon th e k e to n e body m e ta b o lism
w it h in i n t a c t t i s s u e s ; i n a fe w e x p e r im e n ts w ith e x c i s e d r a t
l i v e r ; a d d it io n o f ATP t o th e m edia r e s u l t e d i n a s m a ll b u t
d e f i n i t e in c r e a s e o f u p ta k e o f k e to n e b o d ie s * T h is i s e s p e c i
a l l y i n d i c a t i v e , s i n c e th e same m ethod f a i l e d t o d e m o n str a te
a c o r r e s p o n d in g i n c r e a s e i n t h e s t e a d y u p ta k e o f k e to n e b o d ie s
b y r a t diaphragm s upon a s im ila r a d d it io n o f ATP t o t h e m ed ia .
I n o t h e r w o r d s, k e to n e b o d ie s ap p ea r t o be form ed , n o t u t i l i z e d ,
in t i s s u e su ch as; l i v e r w it h a lo w n orm al l a b i l e p h o sp h a te con
t e n t * H ow ever, la r g e r am ounts o f l a b i l e p h o s p h a te , e i t h e r
added t o l i v e r o r a s n o r m a lly p r e s e n t i n r a t m u s c le , a p p ea r t o
r e s u l t i n u t i l i z a t i o n o f k e to n e b o d ie s . The e v id e n c e may b e ,
a t l e a s t , t e n t a t i v e l y sum m arised by th e sta te m e n t t h a t th e
c o u r se o f k e to n e body m e ta b o lism w it h in i n t a c t t i s s u e s a p p e a r s_ _
t o b e a f u n c t io n o f i t s a v a i l a b l e l a b i l e p h o sp h a te c o n te n t*
The e x p e r im e n ta l e v id e n c e w i l l be d is c u s s e d in th e o r d e r i n
w h ich i t w as o b ta in e d and i t s r e l a t i o n t o th e above w i l l be
g i v e n in more d e t a i l .
The ATP s t i m u l a t i o n o f th e r e s p i r a t i o n o f diap hragm s i s
e v id e n c e d i n T a b le s I , IX and I I I b y th e marked in c r e a s e o f O-Qg*
The s t im u la t io n o f r e s p i r a t i o n was n o t accom p an ied by an i n
c r e a s e in u p ta k e o f k e to n e b o d ie s from th e m edia w it h added
ATP and t h e r e f o r e c a n n o t b e r e p o r te d a s c a u se d b y in c r e a s e d
k e to n e bod y m e ta b o lis m , B ie ls c h p v s k y and G reen (1945). r e p o r t
ed th at; ATP ca u ses; sh o c k t o i n t a c t a n im a ls and c o n t r a c t io n s .
o f e x c is e d m u sc le b u t i t w as n o t p o s s i b l e t o make o b s e r v a t io n s
o f c o n t r a c t i o n s o f t h e d ia p h ra g m s, w h ich w ere e n c lo s e d . T a b le
I I I d i s c l o s e s t h a t a d d i t io n s o f su g a r t o th e m edia c a u se d r e
d u c t io n s i n Q ,0g and a l s o t h a t g ly c o g e n -p o o r m u scle d id n o t
r e sp o n d t o ATP w it h l a r g e in c r e a s e s in The i n c r e a s e I n
r e s p i r a t i o n b y add ed ATP may p o s s i b l y be r e l a t e d t o I n c r e a s e d
g l y c o g e n o l y s i s ; t h e d e c r e a s e In r e s p i r a t i o n on a d d i t io n o f
s u g a r may p o s s i b l y be due t o d e p o s i t i o n o f g ly c o g e n b y t h e
mass; e f f e c t o f add ed su g a r w ith d e c r e a s e in g l y e o g e n o l y s i s ,
b u t t h i s i s s p e c u l a t i v e , S t a d ie and Zapp (1 9 4 ? ) r e p o r t e d an
I n c r e a s e i n o f d iap h ragm s w ith l a r g e i n c r e a s e s i n t h e i n
o r g a n ic p h o sp h a te c o n c e n t r a t io n o f t h e m e d ia . Our i n c r e a s e s
i n Q „ q w ere o b ta in e d by a d d i t i o n s o f v e r y s m a ll am ounts o f ATP,
I B
a s m a lle r o r d e r o f m a g n itu d e . We f e e l c e r t a i n t h a t th e e f f e c t
“ o b ta in e d w xtn ATP i s n o t t h e d i r e c t e f f e c t o f in o r g a n ic p h o s
p h a te o b ta in e d by S t a d ie and Z ap p . H ow ever,' we do n o t know
p r e c i s e l y how ATP a f f e c t s m u scle r e s p i r a t i o n o th e r th a n b y su c h
36
p o s s i b i l i t i e s a s c a u sin g , c o n t r a c t i o n s o r some o th e r a c c e l e r a
t i o n o f m e ta b o lism b e s i d e s t h a t o f k e to n e b o d ie s , w h ic h ap p ea r
t o be u n a ffe c te d *
f h e u t i l i z a t i o n o f a c e t o a c e t a t e a t t w ic e t h e r a t e f o r
L.-he t a h y d r o x y b u ty r a te i s e v id e n t from, t h e d a ta In f a b l e s I I
and I I I , o b ta in e d b y in c u b a t io n o f r a t diaphragm s* A s a b a s i s
fo r c o m p a riso n , we h a v e d e f in e d th e c o n s ta n t,' S L , a s t h e
number o f m icrogram s o f k e to n e body ta k e n up b y a m illig r a m
o f t i s s u e b y d r y w e ig h t , p er hour* f h e a v e r a g e f o r a c e t o -
a c e t a t e i s 5 . 1 , o r a b o u t t w ic e t h a t f o r k -b e ta h y d r o x y b u ty r a te
a t 2.-6 * I k e g r e a t e r u t i l i z a t i o n o f a c e t o a c e t a t e s u g g e s t s
t h a t i t may b e r e g a r d e d a s th e Im m ediate m e t a b o lit e o f t h e
k e to n e bod y group w ith b e ta h y d r o x y b u ty r a te a s a r e d u c t iv e
p r o d u c t, one s t e p away from u t i l i z a t i o n . Such an i n t e r p r e t a
t i o n o f t h e d a ta i s f u r t h e r c o r r o b o r a te d b y t h e f i n d i n g o f
K reb s and E g g le s t o n (194=5) who r e p o r te d t h a t u n d er a n a e r o b ic
c o n d i t i o n s a c e t o a c e t a t e i s r e d u c e d t o b e ta h y d r o x y b u ty r a te i n
k id n e y m in ce w h ich r e a d i l y o x i d i z e s a c e t o a c e t a t e a e r o b i c a l l y .
f h a e x p la n a t io n t h a t b e ta h y d r o x y b u ty r a te i s t h e l e s s u t i l i z a b l e
* . *
form may b® t h e b a s i s f o r th e f a c t t h a t I t c o n s t i t u t e s t h e
m ajor f r a c t i o n o f k e to n e b o d ie s In human u r in e d u r in g a k e t o s i s .
f h e a b s e n c e o f i n c r e a s e in u p ta k e o f k e to n e b o d ie s b y
^diaphragm s w ith added~E EF~to t h e m ed ia ap p eared " !.o show t h a t
AEP h a s n o e f f e c t on th e k e to n e body m e ta b o lis m o f I n t a c t t i s
s u e s . H ow ever, th e s t e a d y u p ta k e o f k e to n e b o d ie s may be an
27
optimum c o n d i t i o n w h ich c a n n o t he e x c e e d e d . f h e p o s s i b i l i t y
t h a t d iap h ragm s c o n t a in am oun ts o f £23? s u f f i c i e n t t o in s u r e
optim um u p ta k e due t o optim um a c t i v a t i o n I n e v e r y in s ta n c e ,*
w a s exam in ed b y a n a l y s i s o f th e p h o sp h a te d i s t r i b u t i o n i n
m u s c le . f h e r e s u l t s i n d i c a t e t h a t su ch e x c e s s e s o f l a b i l e
p h o sp h a te com pounds a r e p r e s e n t in th e m u s c le , l a s t i n g o f th e
r a t s , w h ich m ig h t be e x p e c te d t o r e d u c e t h e l a b i l e p h o sp h a te
c o n te n t o f th e m u sc le b y d e p l e t io n o f g ly c o g e n a s one p o s s i b l e
p r e c u r s o r , r e s u l t e d i n no ch a n g e in t h e u p ta k e o f k e to n e b o d ie s
b y t h e m u s c le , a s i s shown i n f a b l e I I I . A d d it io n o f £23? t o
th e m ed ia d id not. c a u s e a n y s i g n i f i c a n t ch a n g e i n u p ta k e o f
k e to n e b o d ie s b y e i t h e r t h e n orm al or t h e f a s t e d d ia p h ra g m s.
fh e l a c k o f e f f e c t o f added AIP on th e u p ta k e o f k e to n e
b o d ie s b y i n t a c t diap h ragm s r e q u ir e d an e x p la n a t io n and a n a ly
s e s o f t h e d iap h ragm s w ere u n d e r ta k e n t o s u p p ly a more c e r t a i n
a n sw e r . The l a b i l e p h o sp h a te d e t e r m in a t io n s i n f a b l e ¥ I n d i
c a t e t h a t t h e d iap h ragm s c o n t a in many t im e s th e minimum am ount
o f l a b i l e p h osp hate; r e q u ir e d f o r f a t t y a c id a c t i v a t i o n , e v en
a f t e r l o s s e s in c u r r e d b y h a n d lin g and s t a r v a t i o n , f h e l a r g e
e x c e s s e s o f l a b i l e p h o sp h a te p r e s e n t w it h in t h e d iap h ragm s may
e x p la in t h e f a c t t h a t A fP a d d it io n had no e f f e c t on t h e k e to n e
body u p ta k e , f h e s e e x p e r im e n ts , t h e n , t e l l u s n o t h in g c o n c lu
s i v e a s t o th e r o le ' o f A fP i n k e to n e m e ta b o lism o f i n t a c t
t i s s u e s .
f h e b a s i s f o r t h e minimum c o n c e n t r a t io n o f A fP r e q u ir e d
5 8
f o r k e to n e body a c t i v a t i o n a s a p r e r e q u i s i t e f o r i t s o x id a t io n
i s o b ta in e d from L e h n in g e r 's (1 9 4 5 ) hom ogen ate s tu d y on th e
a c t i v a t i o n o f f a t t y a c i d s . An a ssu m p tio n was made t h a t a lth o u g h
k e to n e b o d ie s m ig h t p resu m a b ly r e q u ir e m ore ATP th a n f a t t y
a c i d s , t h e a c t i v a t i o n am oun ts h a v e b e e n assum ed t o b e t h e
same t o e s t im a t e t h e o r d e r o f m a g n itu d e . L e h n in g e r fo u n d t h a t
- 4
an ATP c o n c e n t r a t io n o f 10 M was t h e minimum f o r a c t i v a t i o n .
The e q u iv a le n c e o f a l l t h e h ig h e n e r g y p h o sp h a te i n m u s c le
by m eans o f t h e i n t e r l o c k i n g sy s te m o f m u sc le enzym es h a s b een
d e m o n str a te d b y S z e n t.-G y o rg y i ( 1 9 4 7 ) . A l l o f t h e p h o sp h a te
o f t h e c r e a t in e and a l l b u t a v e r y s m a ll f r a c t i o n o f th e
" s e v e n -m in u te ” l a b i l e p h o sp h a te a r e e q u iv a le n t t o ATP i n
m u s c le . By s im p ly a d d in g t h e P c r and t h e P 7 o f t h e t a b l e s
and e x p r e s s in g t h e t o t a l i n ter m s o f t o t a l p h o sp h a te m o la r it y
tim e s 1 0 ”4 , one h a s a r e a d y b a s i s f o r t h e co m p a riso n o f t h e
t o t a l a v a i l a b l e l a b i l e p h o sp h a te c o n te n t o f a m u sc le t i s s u e
t o t h e minimum a c t i v a t i o n c o n c e n t r a t io n o f ATP.
The t o t a l l a b i l e p h o s p h a te , o b ta in e d in t h e ab o v e m anner,
i n d i c a t e s t h a t ou r n orm al d iap h ragm s had a n e t a v a i l a b l e con
t e n t o f a b o u t one hundred- and e ig h t y tim e s t h e minimum a c t i v a
t i o n c o n c e n tr a t iq n i n s p i t e o f l o s s e s in c u r r e d by t h e h a n d lin g
o f t h e t i s s u e s d u r in g t h e W arburg t e s t s . S o s k i n 's t o t a l l a b i l e
p h o sp h a te w h ich i s ' a l s o i n T a b le V snow s t h a t norm al and d i a -
b e t i c r a t ab d o m in a l m u s c le h a v e a b o u t t h r e e hu nd red t im e s t h e
minimum a c t i v a t i o n c o n c e n t r a t io n ; b u t t h i s l a r g e r t o t a l may be
39
th e r e s u l t o f a p r o c e d u r e w h ich a tte m p te d t o o b t a in t h e a b s o -
i l u t e am oun ts and a v o id e d h a n d lin g o f t h e t i s s u e . Our h a n d lin g
l o s s e s o f l a b i l e p h o sp h a te o f t h e d iap h ragm s a r e r e f l e c t e d
i n o u r r e s u l t s w h ich w ere o b ta in e d b y a p r o c e d u r e w h ich d e
l i b e r a t e l y in c u r r e d s i m i l a r l o s s e s in t h e a n a l y s e s a s in t h e
e x p e r im e n ts , b y i n d e n t i c a l h a n d lin g p r o c e d u r e s . !|b e f a s t e d
d ia p h r a g m s. d i s c l o s e d a s i m i l a r g r o s s e x c e s s o f l a b i l e p h o s
p h a te am ou n tin g t o a b o u t on e hu nd red and t h i r t y t im e s t h e
minimum a c t i v a t i o n c o n c e n tr a tio n * I t m ust be a ck n o w led g ed
th a t, one c a n n o t be- c e r t a i n o f th e e x a c t c o n c e n t r a t io n o f
l a b i l e p h o sp h a te w h ich i s c h e m ic a lly a c t i v e b y t h e t o t a l s
o b ta in e d from g r o s s a n a l y s e s o f t i s s u e s , s in c e n o t a l l o f
th e l a b i l e p h o sp h a te fou n d b y a n a l y s i s may be i n s o l u t i o n
b u t may be a d so r b e d t o a c to m y o s in a t t im e s or be m o m en ta rily
i n one or a n o th e r o f i t s h om ologu s fo r m s . H ow ever, th e v e r y
la r g e g ro ss: c o n t e n t o f m u scle ab ove th e minimum m akes one
c e r t a i n t h a t enough o f l a b i l e p h o sp h a te compounds a r e a lw a y s
p r e s e n t in r a t d iap h ragm s f o r optim um a c t i v a t i o n .
I h e f a c t t h a t a t h i r d o f th e t o t a l l a b i l e p h o sp h a te i s
l o s t on f a s t i n g a lo n g w ith l o s s o f c a rb o h y d r a te i s im p o r ta n t.
(She s i z e o f t h e l o s s w as l e s s th a n w as e x p e c te d but a t l e a s t
our a tte m p t w as in th e r i g h t d i r e c t i o n and a s im ila r a tte m p t
w it h another; t i s s u e may le a d t o l o s s e s o f l a b i l e p h o sp h a te
w h ich a r e s i g n i f i c a n t i f th e t i s s u e h a s a n o r m a lly lo w e r t o t a l .
I h s d e p l e t io n o f th e g ly c o g e n r e s e r v e i s i l l u s t r a t e d by
t y p i c a l diaphragm a n a ly s e s g iv e n in f a b l e I ¥ a lo n g w ith d a ta
from th e l i t e r a t u r e on t h e s k e l e t a l g ly c o g e n c o n t e n t o f man
and t h e r a t . A. co m p a riso n o f th e s k e l e t a l g ly c o g e n o f th e
r a t t o th e l e s s e r s k e l e t a l g ly c o g e n o f man h a s l e d t o t h e s p e c
u l a t i o n t h a t p e r h a p s t h e s p e c i e s . d i f f e r e n c e in m u scle g ly c o g e n
may he r e l a t e d t o an in h e r e n t s t r u c t u r a l d if f e r e n c e a s t o
g ly c o g e n c a p a c it y or. t o a s p e c i e s d i f f e r e n c e in b io c h e m ic a l
t r a i t w h ich may p o s s i b l y r e s u l t in a g r e a t e r AfP h o ld in g
c a p a c it y by r a t m u s c le . f h i s i s m ere s p e c u l a t i o n , y e t a r a t
h a s t w ic e a s much m u scle g ly c o g e n a s a man and i s n o t s u b j e c t
t o a f a s t i n g k e t o s i s a s i s man. W h ile our t e s t s h ave demon
s t r a t e d t h a t r a t m u scle lo .s e a an i n s i g n i f i c a n t p a r t o f i t s
l a b i l e p h o sp h a te on f a s t i n g , our t e s t s h ave n o t e x c lu d e d t h e
p o s s i b i l i t y t h a t o th e r s p e c i e s may l o s e a more s i g n i f i c a n t
p o r t io n o f m uscle l a b i l e p h o sp h a te and h e n c e th e f a s t i n g
k e t o s i s . Our r e s u l t s m e r e ly c o n fir m t h a t a r a t d o e s n o t h a v e
a f a s t i n g k e t o s i s , b u t t h i s i s n o t new .
H ow ever, th e l e s s o f l a b i l e p h o sp h a te o b ta in e d in f a s t e d
d iap hragm h a s more s i g n i f i c a n c e when i t i s c o n t r a s t e d w it h
th e f a c t t h a t S o s k in d id n o t o b s e r v e a l o s s o f l a b i l e p h o sp h a te
b y a b d o m in a l r a t m u scle d u r in g d i a b e t e s , f a b l e ¥ l i s t s t h e
same v a l u e s fo r n orm al and fo r d i a b e t i c m u scle a s r e p o r te d
b y S o s k in ' f 1916TY aitn ou gh T a 'p r o c e d u r a l h i f f e r e h c a i n “m ethod
may a c c o u n t fo r d i f f e r e n t o v e r a l l v a l u e s i n S osk in * s f i g u r e s
and our f i g u r e s , lo s s - e s in l a b i l e , p h o sp h a te sh o u ld be e v id e n t
41
b y e ith e r , method, when t h e y occu r * S o s k in h a s in t e r p r e t e d t h e
f a c t t h a t n o l o s s w as o b s e r v e d d u r in g d i a b e t e s t o mean t h a t
l a b i l e p h o sp h a te c a n n o t be a f a c t o r in a d i a b e t i c k e t o s i s .
H ow ever, our r e s u l t s i n d i c a t e a l o s s i n l a b i l e p h o s p h a te , e v e n
w ith o u t a k e t o s i s . f h e Judgem ent o f t h i s im p lie d d is c r e p a n c y
m ust be p o stp o n e d u n t i l d i r e c t l y com parable, a n a ly s e s a r e ob
t a in e d d u r in g b o th c o n d i t i o n s by th e u se o f i d e n t i c a l p r o c e d u r e .
We f e e l t h a t ou r o b s e r v e d l o s s o f l a b i l e p h o sp h a te i s v a lid *
I n s p i t e o f th e in c o n c lu s i v e n a tu r e o f th e diaphragm
t e s t s , we rem ain ed c o n v in c e d t h a t v a r i a t i o n i n c o n t e n t o f
l a b i l e p h o sp h a te may b e a f a c t o r in k e to n e bod y m e ta b o lis m .
B e fo r e g o in g on t o f h e r e s u l t s o f our t e s t s w ith r a t l i v e r
s l i c e s , i t may be w e l l t o g i v e some o f th e r e a s o n s f o r ou r
c o n tin u e d e f f o r t s .
A . r e v ie w o f th e s i m i l a r i t i e s o f s t a r v a t i o n and d i a b e t e s
in man w i l l i n d i c a t e t h a t b o th c o n d i t i o n s r e s u l t in a k e t o s i s ,
l o s s o f gL y co g en and o f e s t e r i f i e d b lo o d p h o sp h a te and a r i s e
in in o r g a n ic b lo o d p h o s p h a te . Food to th e s t a r v e d and I n s u l i n
t o t h e d i a b e t i c c h e c k th e k e t o s i s w it h a d e p o s it o f g ly c o g e n
and a r e v e r s e s h i f t o f b lo o d p h o sp h a te from t h e in o r g a n ic t o
th e e s t e r i f i e d ? - fh e a c t i v a t i o n b y i n s u l i n o f h e x o k ln a s e In
f a c i l i t a t i n g t h e p h o s p h o r y la tio n and u t i l i s a t i o n o f g lu c o s e
_ h a s _been^bem e: n ^ stra te3 _by^the~_C or:ls^tl94'6} „ fh e - u t i l i z a t i o n “
o f g lu c o s e i s a c c e p t a b le t o a l l a u t h o r i t i e s a s t h e b e s t m eans
o f c h e c k in g a k e t o s i s ; t h e i s s u e i s how d o e s t h e u t i l i s a t i o n
4£
o f g lu c o s e a f f e c t t h e abn orm al m e ta b o lism o f k e to n e b o d ie s ?
S o s k in b e l i e v e s th a t " 'liver- p r e f e r s c a r b o h y d r a te when i t i s
p r e s e n t" ; we f e e l t h a t c a r b o h y d r a te can e a s i l y s u p p ly a d d i t i o n
a l l a b i l e p h o sp h a te w h ich may be e f f e c t i v e i n c e r t a i n s p e c i e s
o r p e r h a p s i n a n o th e r r a t t i s s u e o th e r th a n r a t m u s c le .
B o b b itt and D e u e l (1 9 4 £ ) r e p o r te d an in c r e a s e i n k e to n e
body u p ta k e b y p a r t i a l l y s t a r v e d r a t l i v e r s l i c e s in c u b a te d
i n g l y c o g e n - r ic h m ed ia . A p o s s i b l e m echanism f o r th e i n c r e a s
ed u p ta k e o f k e to n e b o d ie s b y g ly c o g e n e n r ic h m e n t o f th e
liv e r * s m ed ia i s by g l y c o g e n o l y s i s and f u r t h e r d is m u ta t io n
o f t h e g ly c o g e n t o p r o v id e A fP i n s i g n i f i c a n t e x t r a a m o u n ts.
f h e s t r i k i n g f a c t t h a t l i v e r a t m o st h a s o n ly a b o u t a
f i f t e e n t h o f t h e n orm al am ount o f a v a i l a b l e l a b i l e p h o sp h a te
i n m u scle i s e v id e n t in f a b l e V . More s i g n i f i c a n t i s t h e
f a c t t h a t t h e lo w c o n te n t o f l a b i l e p h o sp h a te in l i v e r c o in
c i d e s w ith i t s p r o d u c tio n o f k e to n e b o d ie s ; w h ile th e h ig h e r
l a b i l e p h o sp h a te c o n t e n t o f m u scle i s accom p an ied b y o x i d a t i v e
u t i l i z a t i o n o f th e k e to n e b o d ie s in t h e r a t . f h e a c t u a l con
t e n t o f r a t l i v e r i n te r m s o f i t s a v a i l a b l e l a b i l e p h o sp h a te
may be c a lc u l a t e d from th e a n a l y s i s o f th e p h o sp h a te d i s t r i
b u t io n i n l i v e r by TJmbreit e t a l ( 1 9 4 6 ) . f h e ADP c o n t e n t o f
l i v e r i s c o n s id e r a b le b u t i s u n a v a ila b le f o r p h o s p h o r y la tio n
m yok in ase in t h a t o r g a n , a s t h e w ork o f G o lo w ic k and K a lc k a r
(19431 sh o w s. (G o lo w ic k and K a lc k a r found m y o k in a se i n m u sc le
43
and In o t h e r t i s s u e s . ) L iv e r ALP can t h e r e f o r e o n ly a o t a s
an a c c e p t o r r a t h e r th a n a s a d on or o f l a b i l e p h o s p h a te . L iv e r
h a s a c o n te n t o f A fP w h ich I s a b o u t h a l f o f minimum f o r t h e
a c t i v a t i o n o f f a t t y a c M o x id a t io n a s i s a p p a r e n t from, f a b l e
¥ . f h e c r e a t i n e p h o sp h a te f r a c t i o n o f l i v e r , w h ic h i s u n c e r
t a i n a s t o I t s a v a i l a b i l i t y a s a p r e c u r s o r o f A fP ~ e q u lv a le n t
p h o sp h a te i n t h a t organ b u t i s a p o s s i b l e p r e c u r s o r , i s o n ly
a b o u t tw e n ty t im e s t h e minimum c o n c e n t r a t io n o f l a b i l e p h o s
p h a te f o r a c t i v a t i o n , a m eager am ount when c o n t r a s t e d w ith
th e l a r g e t o t a l s o f l a b i l e p h o sp h a te a v a i l a b l e i n n orm al r a t
m u s c le . One may s p e c u la t e a s t o th e r e a s o n f o r t h e s m a ll
am ount o f l i v e r l a b i l e p h o sp h a te a s due t o t h e u s e o f l a b i l e ,
p h o sp h a te in l i v e r f o r d e p o s i t i o n o f g ly c o g e n w h en ever th e
s t o r e o f g ly c o g e n f a l l s b e lo w i t s c a p a c it y and a l s o by th e
A fP r e q u ir e m e n ts o f th e su n d ry co m p etin g s y n t h e s e s known t o
ta k e p la c e i n t h e l i v e r . , f h e t o t a l o f a v a i l a b l e l a b i l e p h o s
p h a te i s so lo w in l i v e r t h a t a n y v a r i a t i o n in i t s t o t a l
s im ila r ' t o th e l o s s b y f a s t i n g in r a t diap hragm may p o s s i b l y
be s i g n i f i c a n t i n t h e k e t o n e body m e ta b o lism of: th e i n t a c t
a n im a l, f h i s s u g g e s te d t h a t l i v e r s l i c e s , m ight be u se d t o
d e m o n str a te th e e f f e c t o f added A fP upon k e t o n e body m etab o
l i s m b y th e same in v i t r o m ethod p r e v io u s l y t r i e d on d iap h ragm .
f h e -r a s u its -o f-'-o u r - t e s t s -w ith -n o r m a l - li v e r al-i-oe s -rP-r e --------
g iv e n in f a b l e ¥ 1 . f h e r e s u l t s a r e in t e r p r e t e d f o r w hat t h e y
are w o r th p e n d in g a d d i t io n a l c o n fir m a tio n s i n c e t h e y a r e few
*
44
In number but r a th e r c le a n ou t * I t s h o u ld b e n o t ed, t h a t ou r
u s e o f n orm al l i v e r s i l o e s , w h ich was done to r e t a i n a s much
o f the; norm al l a b i l e p h o sp h a te a s p o s s i b l e I f f a s t i n g h a s a n y
p a s s i b l e e f f e c t , i s a d e p a r tu r e from t h e u s u a l u s e o f f a s t e d
l i v e r s l i c e s by o th e r e x p e r im e n te r s*
The f i r s t s e r ie s : o f t e s t s ( I ) w ith r a t l i v e r s l i c e s was
done in i s o t o n i c m edia w it h o u t any s u b s t r a t e t o o b t a in th e
en d ogen ou s o u tp u t o f k e to n e b o d ie s and i t r e s u l t e d i n a s m a ll
b u t m ea su ra b le o u tp u t o f k e to n e b o d ies, s im ila r t o th e o u tp u t
o b ta in e d b y Q u a s te l (19331 and D e u e l (1942} who u se d d i f f e r e n t
m eth ods f o r a s s a y * The n e x t s e r i e s o f t e s t s ( I I and I I I ) w as
done u s in g th e sam e s u b s t r a t e o f L -b e t ahydr o x y b u ty r a te and
th e same ATP a d d i t io n t o h a l f o f th e f l a s k s a s in t h e p r e v io u s
diaphragm t e s t s ; t h e s e l i v e r t e s t s r e s u l t e d i n a s m a ll d e f i n
i t e u p ta k e o f k e to n e b o d ie;s by t h o s e t o w h ich ATP had b e e n
added but n o n e t change i n k e to n e body c o n c e n tr a t io n was
foun d , i n t h e f l a s k s w ith o u t added ATP. A n o th e r s e r i e s o f
l i v e r t e s t s , In w h ich a c e t o a c e t a t e was u sed a t a c o n c e n t r a t io n
t h r e e tim e s t h e u s u a l o n e , r e s u l t e d in a h ig h e r u p tak e th a n
w ith L -b e ta h y d r o x y b u ty r a te . The h ig h e r u p ta k e was e v id e n t i n
b oth th e p r e s e n c e and t h e a b s e n c e o f added ATP b u t ATP p ro d u ced
a s m a ll in c r e a s e in u p ta k e .
I t a p p e a r s t h a t th e e f f e c t o f ATP upon th e ~ k e to n e tdcty
m e ta b o lism o f i n t a c t t i s s u e s h a s b een d e m o n stra te d i n th e
ab ove e x p e r im e n t s . The f a c t t h a t ATP a d d it io n to l i v e r , w h ic h
45
h a s a lo w l a b i l e p h o sp h a te c o n t e n t , c a u se d an added u p ta k e o r
u t i l i z a t i o n o f k e to n e b o d ie s , w h ile th e same m ethod f a i l e d t o
p r o d u ce an e f f e c t upon th e k e to n e bod y u p ta k e b y d iap h ragm ,
w ith a h ig h c o n t e n t o f l a b i l e p h o s p h a te , p o i n t s t o a r o l e o f
A 3TP in th e r e g u l a t i o n o f th e k e to n e bod y m e ta b o lism o f v a r io u s
i n t a c t t i s s u e s . A d i r e c t i n t e r p r e t a t i o n o f t h e s e few r e s u l t s
may w e l l be sum m arized by th e t e n t a t i v e s ta te m e n t t h a t th e
c o u r s e o f k e to n e b od y m e ta b o lism w it h in i n t a c t t i s s u e s a p p e a r s
t o be a f u n c t io n o f t h e i r a v a i l a b l e l a b i l e p h o sp h a te c o n t e n t s ,
b u t t h i s i s a som ew hat h a s t y c o n c lu s io n i n v ie w o f t h e number
o f e x p e r im e n ts and fu r t h e r c o r r o b o r a tio n i s n e e d e d .
f h e m echanism by w h ich AIP a p p e a r s t o o b t a in add ed u t i l i
z a t i o n o f k e to n e bo d i e s i n our l i v e r s l i c e e x p e r im e n ts i s
m e r e ly s u g g e s te d a s a w o rk in g h y p o t h e s is i n t h e f o l l o w i n g .
fh e f a c t t h a t , a t th e m edian c o n c e n t r a t io n o f s u b s t r a t e
w ith o u t added A fP ( I I ) , no n e t change i n k e to n e bod y c o n c en
t r a t i o n r e s u l t e d , s u g g e s t s t h a t t h i s i s e v id e n c e f o r an e q u i
lib r iu m b etw een th e k e to n e bod y and i t s u t i l i z a b l e in te r m e d i
a t e s . f h e l i v e r had an o u tp u t o f k e to n e b o d ie s ( I ) i n t h e
a b s e n c e o f a k e to n e b od y s u b s t r a t e b u t had no n e t o u tp u t In
t h e p r e s e n c e o f a k e to n e body s u b s t r a t e . A d d it io n o f A IP t o
th e k e to n e bod y s u b s t r a t e ( I I I ) s h i f t e d the. d i r e c t i o n o f th e
e q u ilib r iu m t o c a u se a m ea su ra b le u p ta k e o f k e to n e b o d ie s fi
d e m o n s tr a tin g t h a t A fP i s a f a c t o r in t h e e q u ilib r iu m , f h e
m ass e f f e c t o f a la r g e r c o n c e n tr a t io n o f s u b s t r a t e i s s e e n
46
(I ¥ ) I n th e g r e a t e r u p ta k e o f keton e, t o d i e s a t th e lo w l a b i l e
p h o sp h a te c o n t e n t o f n orm al l i v e r w ith o u t added A IP ; h u t t h e
m ass e f f e c t o f th e I n c r e a s e d s u b s t r a t e I s en h an ced (V) b y
a d d it io n o f AIP t o th e m edia*
f h e r e a c t i v e tw o ca rb o n in t e r m e d ia t e o f t h e l i t e r a t u r e
app>ears t o b e d i r e c t l y r e l a t e d t o ATP in th e a b o v e t e s t s and
we h a v e ta k e n t h e l i b e r t y o f p o s t u l a t i n g a c e t y l p h o sp h a te
a s one p o s s i b l e in t e r m e d ia t e b y th e f o llo w in g p o s s i b l e e x p r e s s
io n o f th e o b s e r v e d e q u ilib r iu m :
GH3COGH2G O O K + Z ATP ==== a ADP + Z CH3C00P03H2 .
I n t h i s e x p r e s s io n , t h e r o l e o f a h ig h c o n c e n t r a t io n o f ATP
upon k e to n e body m e ta b o lism i s l i k e t h a t o f a v a lv e w h ich
k e e p s th e k e to n e b o d ie s a t a lo w c o n c e n t r a t io n by e i t h e r
p r o m o tin g t h e fo r m a tio n o f a m ore r e a c t i v e and u t i l i z a b l e
tw o ca rb o n in t e r m e d ia t e or p r e v e n t s th e fo r m a tio n o f k e to n e
b o d ie s from t h e tw o ca rb o n in t e r m e d ia t e , b u t one c a n n o t d i s
t i n g u i s h b etw een e i t h e r f u n c t io n in t h e e q u ilib r iu m .
One can f in d som e fu r t h e r a p p a r e n t J u s t i f i c a t i o n f o r
a c e t y l p h o sp h a te a s t h e m ost p r o b a b le in t e r m e d ia t e b y an
e x a m in a tio n o f th e a c e t a t e t o a c e t o a c e t a t e c o n d e n s a tio n and
e q u ilib r iu m w h ich we h a v e r e f e r r e d t o p r e v io u s ly in our r e v ie w
o f ~ t h e ~ llt e r a t u r e T “ G o u sld e2 r ”o u r p o s t u la t e d e x p r e s s io n f o r th e
v e r i f i e d c e l l u l a r c o n d e n s a tio n o f a c e t a t e t o a c e t o a c e t a t e . '
47
2 CH3 COOH + 2 ATP = 2 CHSG0 0 P 0 3 H2 + 2 ALP s CH3C0 CH2C0 0 H + 2 ATP
I I I i n
The e q u a tio n from I t o I I i s t h e e x a c t e x p r e s s io n o f t h e e q u i
lib r iu m w h ich Lipmann fo u n d i n r a t l i v e r h o m o g e n a te s« w h ich
d id n o t r e s u l t i n t h e o v e r a l l c o n d e n s a tio n o f a c e t a t e t o a e e t o -
a o e t a t e , o b ta in e d by c e l l u l a r c o n d it io n s i n e x p e r im e n ts b y o th e r s #
The f a c t t h a t Lipmann c a n n o t o b t a in t h e o v e r a l l c o n d e n s a tio n t o
a c e t o a c e t a t e i n a hom ogen ate i s n o t an argum ent a g a i n s t t h e
p o s s i b i l i t y o f a c e t y l p h o sp h a te b e in g th e a c t u a l in t e r m e d ia t e
a s much a s i t , r a t h e r , may b e t h e e x p la n a t io n f o r L ip m an n 's
s u c c e s s i n t r a p p in g th e e l u s i v e in t e r m e d ia t e u n d er th e g iv e n
c o n d i t i o n s . Our e x p e r im e n ts a p p ea r t o be r e l a t e d t o t h e e x
p r e s s i o n from I I t o I I I by ou r u s e o f s p e c i a l c o n d i t i o n s on
i n t a c t C e l l s o f t h e same t i s s u e u s e d b y b o th Lipmann and L oeb .
T h a tt a c e t y l p h o sp h a te - i s th e p r o b a b le a c t i v a t e d s t a t e i s
e v id e n t from t h e e n e r g y r e l a t i o n s w h ich h a v e b e e n e s t im a t e d
by Lipm ann t o b e su c h t h a t t h e f r e e e n e r g y o f two m o le s o f
a c e t a t e i s a b o u t 12 K g. c a l . l e s s th a n a m o le o f a c e t o a c e t a t e ,
b u t b o th a r e l e s s th a n t h e f r e e e n e r g y o f two m o le s o f a c e t y l
p h o s p h a te . H ow ever, t h e s e e q u a tio n s a r e n o t p r o v e n , b u t a r e
m e r e ly p l a u s i b l e e x p la n a t io n s o f t h e e f f e c t w h ich we h ave
o b ta in e d on i n t a c t l i v e r s l i c e s by ATP.
GB&PTER ¥ 1 1
SSJM M & SY A im COEdtUSIQHS
A h y p o t h e s is h a s b e e n s t a t e d t h a t c a r b o h y d r a te s may p o s
s i b l y e f f e c t t h e m e ta b o lism o f f a t s b y t h e i r u n iq u e p r o p e r ty
o f a f f o r d i n g l a b i l e p h o sp h a te by a n a e r o b ic g l y c o l y s i s i n ad
d i t i o n t o th e o x id a t iv e g e n e r a t io n * o f l a b i l e p h o sp h a te w h ich
c a r b o h y d r a te s p r o v id e in common w ith o th e r m e t a b o lit e s * A
m ethod f o r t h e in v iv o t e s t i n g o f th e h y p o t h e s is h a s b een
o u t lin e d *
A. s t u d y o f t h e e f f e c t o f l a b i l e p h o sp h a te compounds upon
the. ice to n e bod y m e ta b o lis m o f e x c i s e d r a t d iap h ragm s and l i v e r
h a s b een u n d e r ta k e n in an e f f o r t t o e x te n d th e hom ogenate
s t u d i e s o f f a t t y a c id a c t i v a t i o n b y l a b i l e p h o sp h a te com pounds,
r e c e n t l y r e p o r t e d b y h e h n in g e r * The m eth od s u se d w ere:
W arburg in c u b a t io n o f th e t i s s u e s i n k e to n e body s u b s t r a t e s
w it h a d d it io n o f ATP to; th e t e s t v e s s e l s and m easu rem en ts o f
th e u p ta k e o f s u b s t r a t e and o x y g e n a s a f f e c t e d b y t h e a d d i t io n
o f ATP* C h em ica l a s s a y s w ere made o f t h e t e s t e d t i s s u e s t o
to i a s c e r t a i n th e l a b i l e p h o sp h a te and g ly c o g e n c o n t e n t s i n an
e f f o r t t o r e l a t e t h e i r p o s s i b l e c o n n e c tio n t o th e a c t i v i t y o f
t h e t i s s u e s *
Two new f a c t s r e s u l t from th e s tu d y o f t h e diap h ragm s
a lo n e : ~(T ) adoeoTAEP c a u se d in c r e a s e d r e s p i r a t i o n ; (2 ) a c e t o
a c e t a t e was. u t i l i z e d t w ic e a s f a s t a s l- b e t a h y d r o x y b u t y r a t e
and may, i n c o n ju n c tio n w ith o th e r e v id e n c e , be r e g a r d e d a s
49
th e im m ed ia te m e t a b o lit e o f the; k e to n e body g r o u p .
The f a c t t h a t d iap h ragm d id n o t in c r e a s e i t s u p ta k e o f
k e to n e b o d ie s upon a d d it io n o f ATP t o th e m ed ia , b u t t h a t
l i v e r s l i c e s d id h a v e a n in c r e a s e d u p ta k e o f k e to n e b o d ie s
upon sim iX a r a d d i t io n o f ATP - a p p e a r s t o p o in t to; t h e p o s
s i b i l i t y t h a t th e c o u r se o f k e to n e bod y m e ta b o lism w it h in
i n t a c t t i s s u e s i s a f u n c t io n o f th e a v a i l a b l e r e s e r v e o f
l a b i l e p h o s p h a te . T h e se o b s e r v a t io n s o f l i v e r s l i c e s w ere
r a th e r c le a n c u t b u t few i n number and a n y d e f i n i t e c o n c lu s
ion s: m ight be p o stp o n e d u n t i l f u r t h e r c o r r o b o r a tio n * The
e f f e c t o f a c t i v a t i o n o f k e to n e body m e ta b o lism by add ed ATP
h a s , h o w e v e r , b een o b s e r v e d in i n t a c t t i s s u e s l i c e s .
A new c o m p u ta tio n o f th e a v a i l a b l e l a b i l e p h o sp h a te f o r
a c t i v a t i o n i n th e t i s s u e s d i s c l o s e s t h a t m u sc le t i s s u e w h ic h
o x i d i z e s k e to n e b o d ie s h a s a la r g e e x c e s s o f l a b i l e p h o s p h a te ,
w h ile l i v e r w h ich d o e s n o t u t i l i z e k e to n e b o d ie s h a s a s m a ll
minimum o f l a b i l e p h o s p h a te . 2*his a p p ea r s t o c o r r o b o r a te th e
4
a b o v e g e n e r a l i z a t i o n .
BIBLIOGRAPHY
B ie ls c h o v s k y , M. and H . N . G reen , S h ock p r o d u c in g f a c t o r s
from s t r i a t e d m u s c le . N a tu re 1 5 6 :1 1 7 , 1945
B o b b it t , B . G. and H. J . D e u e l. The e f f e c t o f g ly c o g e n on
t h e o x id a t io n o f b u t y r ic a c id by r a t l i v e r s l i c e s .
J . B i o l . Chem. 1 4 5 : 1 , 1942
B u ch anan , J . M. and A . B . H a s t in g s . The u s e o f i s o t o p i c a l l y
m arked ca rb o n i n t h e s tu d y o f in te r m e d ia r y m e ta b o lis m .
P h y s. R e v . 2 6 : 1 2 1 , 1946
B u ch anan , J . M ., VI, Sakam i and S . G u r in . S tu d y o f t h e m echan
ism o f f a t t y a c id o x i d a t i o n . J . B i o l . Chem. 1 6 9 :4 1 1 , 1946
B uchanan, J . M ., VI, S ak am i, S . G urin and D. V /ils o n . In ter m ed
i a t e s i n t h e b i o l o g i c a l o x id a t io n o f i s o t o p i c a c e t o a c e t a t e .
J . B i o l . Chem. 1 6 £ : 4 0 3 » 1946
B lu n d e n , H. I s o l a t i o n o f b e ta h y d r o x y b u ty r ic a c i d . P r o c . S o c .
E x p . B i o l . 3 8 :4 6 6 , 1 9 3 8
C e r v e l lo , C. and F . G ir g e n t i. Q u a l i t a t i v e r und q u a n t i t a t i v e r
N ach w eis d e s A c e to n s . A r ch . E xp. P a th , und P h . 7 5 :1 5 5 , 1 9 1 4
C o lo w ic k , S . and M. K a lc k a r , The r o l e o f m y o k in a se i n t r a n s -
p h o s p h o r y la tio n . J . B i o l . Chem. 1 4 8 :1 1 7 , 1943
C o r i, C ., VI, P r i c e and S . C o lo w ic k . The e f f e c t o f a n t e r i o r
p i t u i t a r y e x t r a c t and o f i n s u l i n on t h e h e x o k in a s e
r e a c t i o n . J . B i o l . Chem. 1 6 0 :6 3 5 , 1945
C o r i, C ., VI, P r i o e , W. S l e i n , S . C o lo w ick and G. T . C o r i.
The e f f e c t o f a d r e n a l c o r t e x on t h e h e x o k in a s e r e a c t i o n .
F e d . P r o c . 5 : 1 5 0 , 1946
D e u e l, H. I . , L . H allm an and S . M urray. The r e l a t i o n o f f a t t y
l i v e r s t o f a s t i n g k e to n u r ia i n r a t s . I . B i o l . Chem. 1 1 9 : 1 .
1937
D e u e l, H. J . , L . H allm an and S . M urray. K e t o l y s i s and a n t i -
k e t o g e n e s is a s an e x p la n a t io n f o r t h e a c t i o n o f g lu c o s e
and c a r b o h y d r a te i n t e r m e d ia t e s . J . B io . Chem. 1 2 4 :5 8 5 , 1938
E d so n , N . L . I n f lu e n c e o f ammonium c h lo r id e on k e to n e body
fo r m a tio n i n l i v e r . B ioch em . J . 2 9 :2 0 8 2 , 1935
51
Eds on r H. 1 . and B . F . B e l o i r . M e ta b o lism o f k e to n e b o d ie s .
B io ch em . «F. j3B :2023, 1 9 3 6
F i s k e , G. H. and Y . Subbarow . C o lo r im e t r ic d e te r m in a tio n o f
p h o sp h o r u s. J . B i o l . Chem. 6.6: 1 1 1 . 1 9 2 5
F is k e ., C. E . and Y . Sub b a r e P h o s p h o c r e a t i n e . J. B i o l . Chem.
S i r 6 2 9 , 1 9 2 9
K o e n ig , M. and H . S v a r z . I s o l a t i o n o f a d e n o s in e t r ip h o s p h a t e .
A r c h . B io . _6: 2 6 9 , 1945
K r e b s, H* A . and B . Y. E g g le s t o n . M eta b o lism o f a c e t o a c e t a t e
i n a n im a l t i s s u e . Biochem * A. 3 9 r4 0 8 . 1945
B e h n in g e r , A . L . ih e a o id s p l i t t i n g r e a c t i o n o f a c e t o a c e t i e
a c id and e n zy m ic fo r m a tio n o f a c e t i c a c id from a c e t o a c e t a t e .
J . B i o l . Chem. 1 4 3 : 1 4 7 . 1942
B e h n in g e r , A . L . B iv e r h o m o g en a tes and f a t t y a c id o x i d a t i o n .
J . B i o l . Chem. 1 5 Y :3 6 5 , 1 9 4 5
B e h n in g e r , A .,\B . A c t i v a t i o n o f f a t t y a c id o x i d a t i o n .
J . B i o l . Chem. 1 6 1 : 4 3 1 . 1 9 4 5
B e h n in g e r , A . L . F attty a c id o x id a t io n and K reb s c y c l e .
J . B i o l . Chem. 1 6 4 : 2 9 1 . 1945
B e h n in g e r , A . B . O x id a tio n o f h ig h e r f a t t y a c i d s .
J". B i o l . Chem. 1 6 5 : 1 5 1 . 1 9 4 6
B ipm an a, f . S y n t h e s i s o f a c e t y l p h o s p h a te . 1 . B i o l . Chem.
1 5 5 :6 5 . 1 9 4 4
lip m a n n , F . M icrom ethod for: th e d e te r m in a tio n o f a c e t y l
p h o s p h a te . JY B i o l . Chem. 1 5 9 : 2 1 . 1945
lip m a n n , F . D e t e c t io n o f a c t i v a t e d c a r b o x y l g r o u p s w it h h y -
d r o x y la m in e a s i n t e r c e p t o r . < 3 Y B i o l . Chem. 1 6 1 : 4 1 5 , 1 9 4 5
B ju n g g r e n , G . B a r s t e l l u n v o n A c e t e s s lg s a u r e lo s u n g e n . B io .
Z e i t . 1 45:42£;. 1 9 2 4
B oeb, A . TTber der Y erh a lten der E ss.igsau re b e i k u n s tlic h e r
Bur eh b lu t tang-de r-Babexrr BfoT~i5:eitr^4 7riI:8~r~lS12~J------------------
M ed es, G ., H. F . F lo y d and S . W e in h o u se . K eton e b o d ie s a s
in t e r m e d ia t e s o f a c e t a t e o x i d a t i o n . J . B i o l . Chem. 1 6 2 : 1 .
1 9 4 6
5 2
MlrsiIcy, I . A . S t u d i e s on n orm al and d e p a n c r e a tiz e d d u c t s .
Am. J . P h y s . 15 5 ; 22-3. 1 9 4 1
M o reh o u se, M. and H. J . B e u e l. M eta b o lism o f a lp h a , b e ta and
gamma d e u t e r o c a p r o ic a c i d s . P r o c . S o c . E x p . B i o l . 4 5 : 9 6 ,
1941
Q u a s ta l, J . H. and A . W h e a tle y , O x id a tio n o f f a t t y a c i d s i n
l i v e r . B ioeh en u JT. 2 7 :1 7 5 2 , 1935
S c h o e n h eim e r , B . and P . R it t e n b e r g . She c o n v e r s io n o f s t e a r i c
a c id i n t o p a l m i t i c a c id i n th e o r g a n ism . J . B i o l . Chem.
1 2 0 :1 5 5 , 1 9 3 7
( .S ?h ap iro, I . C om p arative g L y c o g e n io and k e t o l y t i c a c t i o n o f
g lu c o s e and c a r b o h y d r a te in t e r m e d ia t e s . J . B i o l . Chem.
1 0 8 : 5 7 5 . 1935
S t a d i e , W. C. M icrotom e fo r p r e p a r a tio n o f t i s s u e s l i c e s .
J . B i o l . Chem. 1 5 4 :6 8 7 , 1944
S t a d i e , W. 0 . and J . E . Z app. E f f e c t o f I n s u li n upon t h e
s y n t h e s i s o f gL ycogen by r a t d iap hragm i n v i t r o . .
J . B i o l . Chem. 1 7 0 : 5 5 . 1 9 4 ?
S w e n se id , M ., R . B a r n e s, A . Hemingway and A . H le r . F o rm a tio n
o f a c e t o n e b o -d ies from a c e t i c a c i d . JT . B i o l . Chem. 1 4 2 :
4 7 , 1 9 4 2
S a e n t-G y r a g y i, A . The C h em istry o f M u scu la r C o n tr a c tio n 1947
S o s lc in , S . C a rb o h y d ra te M etab olsira 1946
U m b reit e t a l M anom etrie M ethods 1946
¥ a n S ly fc e , P . P e te r m in a tio n o f a c e t o n e b o d i e s . «T . B i o l . Chem.
8 5 : 4 1 5 , 1 9 4 0
W ein h o u se, S . , G. M edes and H . F lo y d . K eto n e s y n t h e s i s from,
f a t t y a c i d s w ith i s o t o p i c carb on w ith t r a c e r . J . B i o l .
Chem. 1 5 7 : 3 5 . 1 9 4 4
W e in h o u s e , S . , G. M ed es, E* F lo y d and I*. H ada. I n t e r m e d ia t e s
Wood, H. S . F ix a t i o n o f ca rb o n d i o x i d e . P h y s . R e v . 2 6 : 2 3 6 ,
1 9 4 6
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Asset Metadata

Creator Dulberg, Jasper (author)

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

School Graduate School

Degree Master of Science

Degree Program Biochemistry

Degree Conferral Date 1948-08

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)

Advisor [illegible] (committee chair), [illegible] (committee member), Deuel, H.J. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-776027

Unique identifier UC11348216

Identifier EP41290.pdf (filename),usctheses-c17-776027 (legacy record id)

Legacy Identifier EP41290.pdf

Dmrecord 776027

Document Type Thesis

Rights Dulberg, Jasper

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