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Cholinesterase levels in the sera of rabbits with livers experimentally poisoned by carbon tetrachloride

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Cholinesterase levels in the sera of rabbits with livers experimentally poisoned by carbon tetrachloride

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Content CHOLINESTERASE LEVELS IN THE SERA OF
RABBITS WITH LIVERS EXPERIMENTALLY
POISONED BY CARBON TETRACHLORIDE
A T h e s is
P r e s e n t e d t o
t h e F a c u l t y o f t h e D e p a rtm e n t o f B io c h e m is tr y
t h e 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 i a
I n P a r t i a l F u l f i l l m e n t
o f t h e R e q u ire m e n ts f o r t h e D e g ree
M a s te r o f S c ie n c e
A n i t a A l t a S u ra n
Ju n e 1949
UMI Number: EP41296
All rights reserved
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fcio H i S i y
J ’ A t'j thesis, written by
. A n ita .. Al.t.a..Sur.arL.
"Bg'an"
Da/e......J.uiua-.19.4.9..
under the guidance of hBT.... Faculty Committee, 1 ^
a,nd 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 for the degree of
M a s te r o f S c ie n c e
E .S .B o g a rd u s
Faculty C om m ittee"
L ' - ' Y w J L S L
Chairman
TABLE OP CONTENTS
CHAPTER PAGE
I . INTRODUCTION................................................ ......................................... 1
I I . HISTORICAL REVIEW .......................... . . . . . . . . . . 3
P r o p e r t i e s o f t h e c h o l i n e s t e r a s e s . . . . . . . 3
H i s t o l o g y o f l i v e r p o is o n e d b y
c a rb o n t e t r a c h l o r i d e .. .. . . . 6
C h o l i n e s t e r a s e i n l i v e r damage • .................................... 9
C u r r e n t s t a t u s o f t h e p ro b le m . .................................... 11
I I I . EXPERIMENTAL WORK . . . . . . . . . . . . . . . . 14
M ethods • • • • • • • • • • . • • • • • • • « • 14
F e e d i n g s .................................................. 14
M e asu rem en ts • • • . . . . • • • • • • • • . . 14
H i s t o l o g i c a l t e c h n i q u e s . • ••••••• •• • IV
H i s t o l o g i c a l c r i t e r i a o f l i v e r d am ag e. • • « • 19
R e s u l t s ..............................................................* ............................. 19
S t a b i l i t y o f t h e enzyme. . . . . . .......................... 19
N orm al r a n g e o f v a lu e s . • • • • • • • • • • • 19
C o n s ta n c y o f v a lu e s o v e r a lo n g p e r i o d . . . . 21
E f f e c t s o f a s i n g l e d o s e o f c a rb o n
t e t r a c h l o r i d e .............................................. 21
E f f e c t s o f s e v e r a l s m a ll d o s e s o f
c a rb o n t e t r a c h l o r i d e ........................................................ 21
iv
CHAPTER PAGE
E f f e c t s o f c o n tin u o u s s m a ll d o s e s o f
c a rb o n t e t r a c h l o r i d e ..................... . . . . . . . . 27
I n h i b i t i o n b y e s e r i n e . ..............................• • • • 38
IV . DISCUSSION OF RESULTS......................... 39
BIBLIOGRAPHY..........................................................................................................
LIST OP TABLES
TABLE PAGE
I . V a lu e s f o r 20 M in u te H y d r o l y s i s o f A c e t y l c h o l i n e
an d B e n z o y lc h o lin e b y N orm al R a b b it S e r a • • . . 20
I I , H y d r o l y s i s o f A c e t y l c h o l i n e b y Norm al R a b b it
S e r a o v e r a P r o lo n g e d P e r i o d 22
I I I , H i s t o l o g y o f R a b b it L i v e r s Damaged by a S i n g l e
D ose o f 0 ,4 m g/kg C arbon T e t r a c h l o r i d e ..................... 25
IV , H i s t o l o g y o f R a b b it L i v e r s Damaged by Th r e e .. .
0 ,1 m g/kg D o se s o f C arbo n T e t r a c h l o r i d e ..................... .28
V, H i s t o l o g y o f R a b b it L i v e r s Damaged by
C o n tin u o u s S m a ll D oses o f C arb on T e t r a c h l o r i d e • 36
A
V I , A C o m p ariso n o f t h e E f f e c t s o f 2 x 15 M E s e r i n e
on R a b b it and Human Serum ............................................... * 38
LIST OF FIGURES
FIGURE PAGE
1 . T im e - a c t i o n c u r v e s f o r A c e t y l c h o l i n e and B e n z o y l-
c h o l i n e H y d ro ly s e s b y t h e S e r a o f Norm al R a b b its
and R a b b i t s P o is o n e d by O arbon T e t r a c h l o r i d e . . . 16
2 . E n d o g en eo u s A b s o r p tio n o r E v o l u t i o n o f Gas when
B u f f e r a n d A c e t y l c h o l i n e o r B e n z o y lc h o lin e a r e
M ixed ................................... 18
3 a . H y d r o l y s i s o f A c e t y l c h o l i n e b y S e r a o f R a b b it s
f o l l o w i n g a S i n g l e Dose o f 0 .4 m l/k g C arb o n
T e t r a c h l o r i d e * . . . . . . . . . . ....................... •• 23
3 b . H y d r o l y s i s o f A c e t y l c h o l i n e by S e r a o f R a b b it s
f o l l o w i n g a S i n g l e Dose o f 0 .4 m l/k g C arbon
. T e t r a c h l o r i d e . • 24
4 . E f f e c t o f t h r e e s m a ll d o s e s o f 0 .1 m l/k g C arbon
T e t r a c h l o r i d e on A b i l i t y o f R a b b it S e r a t o
H y d ro ly z e A c e t y l c h o l i n e . • • • • • • ....................... . 26
5 a . E f f e c t o f C o n tin u o u s S m a ll D oses o f C arb on T e t r a
c h l o r i d e on A b i l i t y o f Rab b i t S e r a To H y d ro ly z e
A c e t y l c h o l i n e and B e n z o y lc h o lin e . . . . . . . . 29
5 b . E f f e c t o f C o n tin u o u s S m a ll D o ses o f C arb on T e t r a
c h l o r i d e on A b i l i t y o f R a b b it S e r a t o H y d ro ly z e
A c e t y l c h o l i n e and B e n z o y lc h o lin e • • . . . . • • 3 0
FIGURE PAGE
5 c . E f f e c t o f c o n tin u o u s s m a ll d o s e s o f C arbon
T e t r a c h l o r i d e on A b i l i t y o f R a b b it S e r a t o
H y d ro ly z e A c e t y l c h o l i n e and B e n z o y lc h o lin e . . . 31
5 d . E f f e c t o f c o n tin u o u s s m a ll d o s e s o f C arb on
T e t r a c h l o r i d e on A b i l i t y o f R a b b it S e ra t o
H y d ro ly z e A c e t y l c h o l i n e and B e n z o y lc h o lin e . . . 32
5 e . E f f e c t o f c o n tin u o u s s m a ll d o s e s o f C arbon
T e t r a c h l o r i d e on A b i l i t y o f R a b b it S e r a t o
H y d ro ly z e A c e t y l c h o l i n e an d B e n z o y lc h o lin e . . . 33
5 f . E f f e c t o f c o n tin u o u s s m a ll d o s e s o f C arbo n
T e t r a c h l o r i d e on A b i l i t y o f R a b b it S e ra t o
H y d ro ly z e A c e t y l c h o l i n e an d B e n z o y lc h o lin e . . . 34
5 g . E f f e c t o f c o n tin u o u s s m a ll d o s e s o f C arbon
T e t r a c h l o r i d e ofi A b i l i t y o f R a b b it S e ra t o
H y d ro ly z e A c e t y l c h o l i n e an d B e n z o y lc h o lin e . . . 35
CHAPTER I
INTRODUCTION
The p r im a r y p u r p o s e o f t h i s i n v e s t i g a t i o n was t o
d e te r m in e th e c a u s e o f t h e d i f f e r e n c e i n b e h a v i o r b e tw e e n
serum c h o l i n e s t e r a s e i n l i v e r damage i n d i s e a s e d hum ans
and i n r a b b i t s w i t h e x p e r i m e n t a l l y c a u s e d l i v e r dam age.
A s e c o n d a r y o b j e c t i v e w as t h e f u r t h e r e l u c i d a t i o n o f t h e
n a t u r e o f r a b b i t c h o l i n e s t e r a s e . Num erous o b s e r v e r s hav e
n o te d t h a t i n human l i v e r dam age, p a r t i c u l a r l y c i r r h o s i s ,
serum c h o l i n e s t e r a s e v a l u e s a r e d e p r e s s e d b e lo w n o rm a l
l e v e l s . R e c e n t w ork on v a r i o u s s p e c i e s o f a n im a ls i n
w h ic h l i v e r damage h a s b e e n p ro d u c e d b y p o i s o n i n g w i t h
c a rb o n t e t r a c h l o r i d e h a s n o t p ro d u c e d c o r r e s p o n d i n g r e
s u l t s .
I t a p p e a re d t h a t t h e r e m ig h t be two p o s s i b l e
e x p l a n a t i o n s f o r t h e s e a p p a r e n t l y c o n t r a d i c t o r y phenom ena;
f i r s t , t h a t t h e e x p e r i m e n t a l a n im a ls w e re n o t b r o u g h t t o
a c i r r h o t i c s t a t e s i n c e t h e r e p o r t e d w ork w as o f s h o r t
d u r a t i o n , and s e c o n d , t h a t t h e enzyme s y s te m s o f t h e
s p e c i e s m ig h t be d i f f e r e n t .
The e x p e r i m e n t a l p a r t o f t h i s w o rk w as p la n n e d
a c c o r d i n g l y . R a b b i t s w e re f e d c a rb o n t e t r a c h l o r i d e and
t h e c o u r s e o f l i v e r damage w as fo llo w e d b y serum c h o l i n
e s t e r a s e d e t e r m i n a t i o n s and h i s t o l o g i c a l e x a m in a tio n —
2
a t a u to p s y o r b i o p s y — o f t h e l i v e r s . Normal r a b b i t seru m
was com pared t o n o rm al human seru m w i t h r e g a r d t o s u b s t r a t e
s p e c i f i c i t y a n d b e h a v i o r i n th e p r e s e n c e o f i n h i b i t o r s .
The b a la n c e o f t h i s p a p e r i s p r e s e n t e d a s f o l l o w s :
F i r s t , a n h i s t o r i c a l r e v ie w d e a l in g w i t h th e p r o p e r t i e s of
c h o l i n e s t e r a s e , th e h i s t o l o g y o f l i v e r s damaged by c a rb o n
t e t r a c h l o r i d e , c h o l i n e s t e r a s e i n l i v e r damage and a s t a t e
ment o f t h e c u r r e n t s t a t e o f t h e p r o b le m . F o l l o w i n g , i s a
s e c t i o n d e a l i n g w i t h e x p e r im e n ta l m eth o d s and r e s u l t s , t h e n
a sum m ation o f r e s u l t s , f o llo w e d b y t h e b i b l i o g r a p h y *
CHAPTER II
HISTORICAL REVIEW
The p i o n e e r w ork o f D a le on a c e t y l c h o l i n e i n 1914
s u g g e s te d t h a t a n enzym e was p r o b a b ly in v o lv e d i n c o n t r o l l
in g th e am ount o f t h i s s u b s ta n c e i n t h e c i r c u l a t i o n . I n
1926 Loew i fo u n d t h a t a c e t y l c h o l i n e a s w e l l a s t h e s u b s ta n c e
l i b e r a t e d on v a g u s s t i m u l a t i o n w ere b o t h i n a c t i v a t e d by
i n c u b a t i o n w ith h e a r t e x t r a c t . H e a te d o r i r r a d i a t e d h e a r t
e x t r a c t h a d no su c h e f f e c t . F u r t h e r w ork by L oew i and
N a v r a t i l on t h e e f f e c t o f p h y s o a tig m in e on h e a r t l e d t o
t h e c o n c l u s i o n t h a t t h e e f f e c t o f t i l l s a l k a l o i d was t h e
i n h i b i t i o n o f t h e a g e n t w hich d e s t r o y e d a c e t y l c h o l i n e ,
G a le h r and P l a t t n e r fo u n d t h e a c e t y l c h o l i n e d e s t r o y i n g
a g e n t i n t h e b l o o d o f c e r t a i n s p e c i e s , E n g l e h a r t an d Loewi
i n 1930 an d M a t th e s , i n th e same y e a r , p ro v e d t h e enzym ic
n a t u r e o f t h i s a g e n t . ( G e n e r a l r e f e r e n c e s t o e a r l y h i s t o r y
4 , 2 7 , 3 2 .)
I . PROPERTIES OF THE CHOLINESTERASES
The e a r l y c h a r a c t e r i z a t i o n o f t h e enzyme s y s te m s
in v o lv e d i n c h o li n e m e ta b o lis m may be f o u n d i n t h e w ork o f
S ted m an a n d c o - w o r k e r s . They s t u d i e d th e i n h i b i t o r y a c t i o n
o f c e r t a i n p h y s o a tig m in e a n a lo g u e s on l i v e r e s t e r a s e and
4
g u in e a p i g serum e s t e r a s e * ( 3 1 ) .
U sin g a c o n ti n u o u s t i t r a t i o n m e th o d , S ted m an , S te d m a n ,
an d E a s s o n i n v e s t i g a t e d th e a c t i o n o f h o r s e serum on a c e t y l
c h o l i n e , m e th y l b u t y r a t e , a n d t r i b u t y r i n , a n d fo u n d t h a t
t h e l a t t e r tw o e s t e r s w ere h y d r o ly z e d a t a much lo w e r r a t e
th a n th e a c e t y l c h o l i n e (3 4 )* The serum enzyme h y d r o ly z in g
a c e t y l c h o l i n e t h e y c a l l e d " c h o l i n e s t e r a s e ” , and t h e y c o n
c lu d e d t h a t t h e r e m l g i t be t h r e e enzym es in v o lv e d i n th e
p r e v i o u s l y a c c e p t e d ”e s t e r a s e M *
S te d m a n , S te d m a n , and W h ite (3 5 ) r e p o r t e d a n im p ro v ed
t i t r a t i o n m ethod i n a s t u d y o f t h e h y d r o l y s i s o f th e t h r e e
e s t e r s b y t h e s e r a o f v a r i o u s s p e c i e s . They fo u n d a c e t y l
c h o l i n e s t e r a s e i n v a r y in g a m o u n ts, an d no c o n s t a n t c o rre * .
l a t i o n b e tw e e n t h i s enzyme a n d th e common e s t e r a s e (m e th y l
b u t y r a s e ) . The n o t e d t h a t , w h ile t h e r e w ere p r o b a b ly t h r e e
s e p a r a t e enzym es i n v o l v e d , t h e p o s s i b i l i t y o f c h o l i n e s t e r a s e
a t t a c k i n g sim p le e a t e r s h a d n o t b e e n e x c lu d e d .
I n 1935 Stedm an an d S tedm an d e s c r i b e d a m an o m etric
d e t e r m i n a t i o n o f c h o l i n e s t e r a s e u s i n g a B a n c r o f t a p p a r a t u s ,
an d d e m o n s tr a te d a c e t y l c h o l i n e s t e r a s e i n r e d b lo o d c e l l s
( 3 3 ) . E a s s o n and Stedm an (1 1 ) b y m eans o f i n h i b i t i o n and
m ixed s u b s t r a t e s t u d i e s f u r t h e r e d t h e d i f f e r e n t i a t i o n o f
" e s t e r a s e " i n t o c h o l i n e s t e r a s e a n d m e th y l b u t y r a s e , and
fo u n d o n ly th e f o r m e r i n human se ru m .
A l l e s a n d Hawes (2 ) i n t r o d u c e d t h e g l a s s e l e c t r o d e
c o n ti n u o u s t i t r a t i o n m ethod f o r c h o l i n e s t e r a s e d e t e r m i n a t i o n
a n d d e m o n s tr a te d a n o t a b l e q u a l i t a t i v e d i f f e r e n c e b e tw e e n
t h e c h o l i n e s t e r a s e s o f r e d c e l l s and se ru m . The enzym es
h e h a v e d d i f f e r e n t l y w i t h r e g a r d to a c e t y l c h o l i n e d e r i v a
t i v e s , s u b s t r a t e c o n c e n t r a t i o n s , pH a c t i v i t y c u r v e s , a n d
s a l t c o n c e n t r a t i o n .
M endel and Hudney (2 4 ) d e m o n s tr a te d t h e p r e s e n c e
o f two d i f f e r e n t c h o l i n e s t e r a s e s w h ic h th e y d e s i g n a t e d a s
" t r u e " an d "pseudo" c h o l i n e s t e r a s e s , th e f o r m e r b e in g
s p e c i f i c f o r c h o li n e e s t e r s and th e l a t t e r b e in g a b le t o
s p l i t c h o l i n e e s t e r s a s w e l l a s o t h e r e s t e r s . I t was
d e m o n s tr a te d t h a t A lle s * c e l l and serum c h o l i n e s t e r a s e s
w e re r e s p e c t i v e l y t r u e c h o l i n e s t e r a s e an d p se u d o c h o l i n —
e s t e r a s e . I t w as fo u n d t h a t a t h ig h s u b s t r a t e c o n c e n t r a t i o n s
th e a c t i o n o f th e p s e u d o - c h o l i n e s t e r a s e p re d o m in a te d w h ile
th e t r u e c h o l i n e s t e r a s e w as m ore a c t i v e a t low a c e t y l c h o l i n e
c o n c e n t r a t i o n s .
A more c o n c r e t e d e m o n s tr a ti o n o f the p r e s e n c e o f two
enzym es c a p a b le o f h y d r o l y z i n g a c e t y l c h o l i n e was made b y
M e n d el, M u n d e ll, and R udney ( 2 3 ) . I t was fo u n d t h a t t h e
t r u e c h o l i n e s t e r a s e h y d r o ly z e d a c e t y l ■ ^ g -m e th y lc h o lin e
w h ile p s e u d o - c h o l i n e s t e r a s e h a d n o e f f e c t on i t , a n d t h a t
t h e p s e u d o - c h o l i n e s t e r a s e a lo n e c o u ld h y d r o ly z e b e n z o y l
c h o l i n e . F o r t u n a t e l y , n e i t h e r of t h e s e e s t e r s w ere a t t a c k e d
6
toy t h e common e s t e r a s e . I t w as a l s o n o t e d t h a t t h e d i s t r i
b u t i o n o f t h e c h o l i n e s t e r a s e d i f f e r e d i n t h e p lasm a o f
v a r i o u s s p e d i e s . The t r u e c h o l i n e s t e r a s e was p r e s e n t i n
p la sm a o f . a l l s p e c ie s * w h ile t h e p s e u d o - c h o l i n e s t e r a s e v a r i e d
fro m a c o m p le te a b s e n c e t o p re d o m in a n c e i n t h e v a r i o u s
s p e c i e s i n v e s t i g a t e d .
The s i t u a t i o n was f u r t h e r c o m p lic a te d toy Saw yer (>30)
who c o n c lu d e d t h a t t h e r e w a s, i n th e l i v e r s o f t h e g u in e a
p i g and r a b b i t , a n enzyme c a p a b le o f h y d r o l y z i n g b e n z o y l
c h o lin e b u t n o t a c e t y l c h o l i n e . T h is he te r m e d ’’b e n z o y l
c h o l i n e s t e r a s e . ”
H aw kins an d G u n te r (1 9 ) d e t a i l e d a m ethod u s i n g t h e
i n h i b i t o r e s e r i n e t o d e te r m in e w h e th e r th e y w e re d e a l i n g
w ith p s e u d o - c h o l i n e s t e r a s e o r b e n z o y l c h o l l n e s t e r a s e .
F u r t h e r d i f f e r e n t i a t i o n o f t h e c h o l i n e s t e r a s e s was
made b y H aw kins and M en d el, (2 0 ) u s in g th e i n h i b i t o r
d i l s o p r o p y l f l u o r o p h o s p h a t e . DFP a t low c o n c e n t r a t i o n s
w as fou nd t o s p e c i f i c a l l y i n h i b i t t h e p s e u d o - c h o l i n e s t e r a s e .
I I . HISTOLOGY OF LIVER POISONED BY CARBON TETRACHLORIDE.
The c l a s s i c w ork i n t h i s f i e l d was t h e s tu d y made b y
Cam erdn i n 1936 (9)>, v«ho f o llo w e d t h e c o u r s e o f l i v e r damage
i n t h e r a t . I n t h i s p a p e r t h e w ork o f some e a r l y i n v e s t i g a t o r s
w ith r e g a r d to t h e r a b b i t was re v ie w e d ( 1 9 , 2 8 , 3 8 ) , w h ic h
f i t i n w e l l w ith C am ero n ’ s f i n d i n g s . The s t u d i e s by s e v e r a l
7
o t h e r w o r k e rs i n v a r i o u s s p e c i e s w ere a l s o i n g e n e r a l a g r e e -
tnent w i t h Cam eron ( 7 , 1 0 , 1 4 , 1 5 , 1 6 , 3 7 ) . V a r i a t i o n s among
d i f f e r e n t s p e c i e s a r e e n c o u n te r e d w ith r e g a r d to am ount o f
l e t h a l d o s e , t o l e r a n c e , tim e f o r n e c r o s i s to a p p e a r , an d
l e n g t h o f r e c o v e r y p e r i o d . The h i s t o l o g i c a l p i c t u r e , a s
i n d i c a t e d a b o v e , i s f a i r l y c o n s t a n t . T h e r e f o r e , a s u r v e y
o f C a m e ro n 's w ork w i l l s e r v e t o o u t l i n e t h e t y p i c a l h i s t o
l o g i c a l c h a n g e s i n a l i v e r dam aged b y c a rb o n t e t r a c h l o r i d e .
One d a y a f t e r a s i n g l e d o se o f c a rb o n t e t r a c h l o r i d e ,
0 .0 1 6 - 0 .0 3 3 gram s p e r k i l o o f body w e i g h t , t h e l o b u l a r
p a t t e r n o f th e l i v e r a p p e a re d e x a g g e r a t e d . The c e l l s n e a r
t h e c e n t r a l v e i n s t a i n e d h o m o g e n e o u sly , b u t w ere d i f f e r e n t
f r o m t h e p e r i p h e r a l c e l l s and o f t e n showed p y k n o t ic o r
d e g e n e r a t e d n u c l e i . T hese c e l l s w e re b o r d e r e d b y e n l a r g e d
t h r e a d l i k e s t r u c t u r e s . N um erous m it o s e s w ere o b s e rv e d #
T h ere w as c o n g e s t i o n o f c e n t r a l a n d m id - z o n a l a r e a s , h e m o rr
h a g e , and o e l l u l a r i n f i l t r a t i o n among t h e n e c r o t i c c e l l s b y
ly m p h o c y te s an d h i s t o c y t e s .
T h ree d a y s a f t e r th e i n j e c t i o n , t h e l o b u l a r c e l l s
w ere g o n e , l e a v i n g o n ly m esenchym al s tro m a . T h e re w as some
ly m p h o c y tic i n f i l t r a t i o n . The c e l l s o f t h e m id - z o n a l r e g i o n
showed f a t t y i n f i l t r a t i o n . No g ly c o g e n was o b s e r v e d . T h ere
was a l a r g e num ber of m i t o s e s . I n t h e p o r t a l t r a c t s t h e r e
w ere ly m p h o c y te s , young f i b r o b l a s t s , an d some p o ly m o rp h s .
8
I n f i v e d a y s t h e n e c r o t i c c e l l s w e re gone w h ile some
ly m p h o c y te s r e m a in e d . T h ere w e re v a ry few c e l l s i n t h e p o r t a l
t r a c t s . T h e re w ere o n ly few m i t o s e s . The K u p fe r c e l l s w ere
p r o m in e n t . >
F o u r t e e n d a y s a f t e r t h e i n j e c t i o n t h e l i v e r a p p e a r e d
n o r m a l.
The e f f e c t s o f m u l t i p l e d o s e s w ere q u i t e s i m i l a r t o
t h e ab ove d e s c r i p t i o n . H ow ever, w i t h an i n c r e a s e d num ber
o f i n j e c t i o n s a t tw ic e w e e k ly i n t e r v a l s , t h e p i c t u r e b e g a n
t o re s e m b le p o r t a l c i r r h o s i s i n w h ich t h e f i b r o s i s s p r e a d s
o u tw a rd fro m t h e c e n t r a l v e i n s . The p o r t a l c a n a l s i n c r e a s e d
i n s i z e and c o n t a i n e d f i b r o b l a s t s and f i b r o u s t i s s u e . The
r e t i c u l a r f i b e r s t h i c k e n e d and i n c r e a s e d . The c e l l s a t t h e
c e n t e r o f th e lo b u le a ro u n d t h e f i b r o s i s showed d e g e n e r a t i o n
an d n e c r o s i s . Few m it o s e s w ere o b s e r v e d .
C an taro w (1 0 ) made a s i g n i f i c a n t a d d i t i o n t o t h i s
p i c t u r e . By m eans o f th e i n j e c t i o n o f I n d i a in k p r i o r t o
s a c r i f i c i n g h i s a n i m a l s , he was a b le to s t u d y t h e a c t i v i t y
o f th e K u p fe r c e l l s , an d he fo u n d t h a t im m e d ia te ly a f t e r t h e
i n j u r y t h e s e c e l l s d i d n o t f u n c t i o n .
A sh b u rn (3 ) p e r f u s e d a f r e s h l y i s o l a t e d l i v e r w i t h a
g e l a t i n o u s c h a r c o a l m ass and fo u n d t h a t i n c a r b o n t e t r a c h l o r i d e
damage th e f i b r o u s t i s s u e e m a n ated fro m a b o u t c e n t r a l v e i n s
r a t h e r th a n a lo n g th e p o r t a l a r e a s a s d e s c r i b e d by s e v e r a l
o t h e r w o rk e n s . He c o n c lu d e d t h a t t h e f i b r o u s t i s s u e a p p e a r s
i n th e same a r e a a s t h e damage — a b o u t t h e c e n t r o l o b u l a r
h e p a t i c v e i n s .
R o sin (2 9 ) r e l a t e d h i s t o l o g i c a l c h a n g e s i n l i v e r to
m e t a b o l i c d i s t u r b a n c e . H e p a tic c e l l s i n t h e c e n t r a l p a r t
o f t h e l o b u le w hich s t a i n e d i n t e n s e l y on a h i g h p r o t e i n
d i e t and d i s a p p e a r e d i n p r o t e i n d & f ic ie n c y w ere c a u s e d t o
d i s a p p e a r w i t h i n one h o u r a f t e r c a r b o n t e t r a c h l o r i d e adm in
i s t r a t i o n ,
I I I . CHOLINESTERASE IN LIVER DAMAGE
As t h e kno w led ge o f th e p h y s i o l o g i c a l f u n c t i o n o f
a c e t y l c h o l i n e h a s grow n , a n d b e c a u s e o f I t s w id e s p r e a d
d i s t r i b u t i o n i n t i s s u e s , many a t t e m p t s h a v e b e e n made t o
c o r r e l a t e c h o l i n e s t e r a s e v a lu e s w i t h p a t h o l o g i c a l c o n d i t i o n s .
Serum c h o l i n e s t e r a s e l e v e l s h a v e b e e n r e p o r t e d a s b e i n g
d e p r e s s e d b elo w n o rm a l I n human l i v e r dam age, p a r t i c u l a r l y
c i r r h o s i s . ( 1 , 26, 8 , 2 1 ) .
W e s c o e .n o te d low v a l u e s i n l i v e r dam age b u t no c o r r e
l a t i o n o f l i v e r f u n c t i o n t o r e g e n e r a t i o n o f enzyme ( 3 9 ) ,
More r e c e n t l y , w ork h a s b e g u n on a n i m a l s . I t w as
c o n s i d e r e d b y s e v e r a l w o rk e rs t h a t i f , in d e e d , c h o l i n e s t e r
a s e w ere a n enzyme s y n t h e s i z e d by t h e l i v e r , t h e n i t w ould
o f f e r a u s e f u l t o o l f o r t h e s tu d y o f b o th enzyme a n d p r o t e i n
s y n t h e s i s by t h a t o r g a n .
10
S t e e n s h o l t (3 6 ) a d m i n i s t e r e d c a r b o n t e t r a c h l o r i d e
o r a l l y to d o g s . 2 .5 m l. p e r d a y f o r a week g a v e a f a i r l y
s h a r p r i s e i n serum c h o l i n e s t e r a s e v a l u e s f o llo w e d b y a
slow r e t u r n t o t h e n o rm al l e v e l . A s i n g l e l a r g e d o s e of
8 m l. g av e a s i m i l a r c u r v e . I n t e r r u p t e d d o s e s g av e t h e
same t y p e b ro k e n c u r v e . B i l i r u b i n v a l u e s c l o s e l y p a r a l l e d
th o s e o f t h e enzyme •
B r a u e r an d R oot ( 5 ) f e d c a r b o n t e t r a c h l o r i d e t o r a t s .
I n g ro w in g a n im a ls c h o l i n e s t e r a s e v a l u e s i n c r e a s e d and i n
m a tu re a n i m a l s , a f t e r a s h o r t l a t e n t p e r i o d , t h e v a lu e s
f e l l . I n d o g s t h e r e w as an i n i t i a l r i s e , th e n a slow f a l l .
S e v e re l i v e r damage was n o te d a n d a c o r r e l a t i o n t o l i v e r
f u n c t i o n was d ra w n . I n a l a t e r p a p e r (6 ) a more e x t e n s i v e
s tu d y was made w i t h d o g s f e d c a r b o n t e t r a c h l o r i d e . A r o u g h
r e l a ti o n s h i p > b e tw ee n l i v e r c h o l i n e s t e r a s e an d p la sm a c h o l i n
e s t e r a s e l e v e l w as d ra w n . I t w as c o n c lu d e d t h a t t h e r e w as
a l i v e r - p l a s m a c h o l i n e s t e r a s e e q u i l i b r i u m , t h a t 25# o f t h e
c i r c u l a t i n g enzyme c o u ld b e r e p l a c e d fro m t h e l i v e r , and
i n c a rb o n t e t r a c h l o r i d e l i v e r I n j u r y t h e enzyme s p i l l s i n t o
t h e p lasm a fro m th e dam aged l i v e r .
E l l i s (1 3 ) c h o se t h e r a b b i t and r a t a s e x p e r im e n ta l
a n im a l s . I n t r a p e r i t o n e a l i n j e c t i o n o f 0 . 0 5 - 0 . 5 p e r k i l o
o f c a rb o n t e t r a c h l o r i d e f o r up t o tw e lv e d a y s g av e c h a n g e s
i n serum a n d l i v e r c h o l i n e s t e r a s e w hich w e re n o t c o n s i d e r e d
11
t o d i f f e r s i g n i f i c a n t l y fro m n o rm a l i n t h e r a b b i t a lt h o u g h
a m arked h i s t o l o g i c a l change w as n o t e d . I n t h e r a t t h e
l e v e l s w ere d e p r e s s e d . T h is d i f f e r e n c e i s a t t r i b u t e d to
s p e c i e s d i f f e r e n c e s . C o m p ariso n o f the h y d r o l y s i s r a t e s
o f b e n z o y l c h o l i n e an d a c e t y l ^ - m e t h y l c h o l i n e f o r t h e two
s p e c i e s y i e l d e d d i s t i n c t l y d i f f e r e n t r a t i o s . T h is strength
e n ed th e a b o v e a s s u m p tio n and i n d i c a t e d t h a t i n t h e s e two
a n im a ls t h e p r o p o r t i o n s o f s p e c i f i c and n o n - s p e c i f i c c h o l i n
e s t e r a s e d i f f e r .
IV . CURRENT STATUS OP THE PROBLEM
I t . w i l l b e n o te d t h a t i n t h e l i t e r a t u r e c i t e d t h e r e
i s g e n e r a l a g re e m e n t among a l l th e c l i n i c a l o b s e r v e r s t h a t
i n human l i v e r damage serum c h o l i n e s t e r a s e v a lu e s f a l l
( 1 , 8 , 2 6 ) . I n e x p e r i m e n t a l a n i m a l s , h o w e v e r, t h e p i c t u r e
I s m ore c o n fu s e d w i t h a r i s e r e p o r t e d I n t h e dog ( 6 ) , a
f a l l i n t h e r a t ( 5 , 6 ) , a n d no ch an g e i n t h e r a b b i t ( 1 3 ) .
T h a t s p e c i e s d i f f e r e n c e s e x i s t w i t h r e g a r d t o t h e
d i s t r i b u t i o n o f c h o l i n e s t e r a s e and t h e r a t i o o f t h e s p e c i f i c
a n d n o n - s p e c i f i c enzym es h a s b e e n d e m o n s tr a te d ( 1 3 , 2 0 , 2 5 ,
2 9 ) . A c c o rd in g t o M endel (25')'*r a b b i t serum h a s a s u b s t a n
t i a l am ount o f t h e t r u e o r s p e c i f i c c h o l i n e s t e r a s e , w h ile
human seiu m c o n t a i n s p r e d o m in a n tly t h e n o n - s p e c i f i c
p s e u d o c h o l i n e s t e r a s e •
12
I t w ould a p p e a r n e c e s s a r y , t h e n , i n a n y c o m p a ris o n
b e tw e e n human and r a b b i t serum c h o l i n e s t e r a s e r , t o s tu d y
p o t o n ly t h e n e t a c t i v i t y o f t h e seru m to w a rd a c e t y l
c h o l i n e , b u t a l s o t h e f r a c t i o n a l c o n t r i b u t i o n o f t h e
a c e t y l ^ - m e t h y l c h o l i n e and b e n z o y lc h o lin e h y d r o ly z in g
co m p o n en ts to w a rd t h i s t o t a l . I n a d d i t i o n , t h e p o s s i b i l i t y
of a n enzyme c a p a b le o f h y d r o ly z in g b e n z o y l c h o l i n e o n ly
m u st be c o n s id e r e d * T h is i s made p o s s i b l e by t h e m ethod
o f G u n te r (.1 7 ), w h ic h u s e s 2 x 10 e s e r l n e t o d i f f e r e n
t i a t e b e tw e e n b e n z o y l c h o l i n e s t e r a s e and p s e u d o c h o l i n e s t e r *
a s e . H a w k in s.a n d G u n te r (1 9 ) d e m o n s tr a te d t h a t b y t h i s
c r i t e r i o n human serum c o n t a i n s no b e n z o y l c h o l i n e s t e r a s e .
B i l l s (1 2 ) s t a t e d t h a t r a b b i t serum c o n t a i n s b e n z o y l c h o l i n
e s t e r a s e *
The e x p e r im e n ts o f E l l i s ( 1 2 , 13) w i t h r a b b i t s
p o is o n e d w i t h c a rb o n t e t r a c h l o r i d e w ere o f s h o r t d u r a t i o n
an d a lt h o u g h s e v e r e l i v e r dam age was n o te d c i r r h o s i s was
h o t r e p o r t e d a s h a v in g b e e n p ro d u c e d .
The c o n c l u s i o n s draw n i n t h e s e e x p e r im e n ts a r e
l i a b l e t o c e r t a i n c r i t i c i s m s . Norm al v a l u e s f o r v a r i o u s
r a b b i t s w e re shown t o f a l l o v e r a f a i r l y w ide r a n g e . In
t h e dam aged a n im a l s , some show ed r i s e s i n serum a n d l i v e r
c h o l i n e s t e r a s e a c t i v i t y w h ile o t h e r s f e l l . The c o n c l u s i o n
t h a t t h e r e was no c h a n g e i n c h o l i n e s t e r a s e l e v e l s was b a s e d
13
a v e r a g e s o f t h e w h o le g ro u p o f a n im a ls b e f o r e and a f t e r
l i v e r dam age. I f , h o w e v e r, i n d i v i d u a l a n im a ls a r e c o n s i d e r
e d , c h a n g e s i n serum c h o l i n e s t e r a s e a r e n o t to o g r e a t , h u t
c h a n g e s i n l i v e r c h o l i n e s t e r a s e v a r y fro m 44$ t o 1 6 8 $ .
The e f f e c t s o f t h e p o i s o n i n g o f t h e com ponent
enzym es was n o t c o n s i d e r e d , so t h a t a n y c h a n g e s i n t h e
r e l a t i v e am ounts o f s p e c i f i c and n o n - s p e c i f i c enzym es
w ere n o t n o t e d . I f o n ly t h e p s e u d o c h o l i n e s t e r a s e w ere
a t t a c k e d t h i s m ig h t he t h e e x p l a n a t i o n f o r t h e d i f f e r e n c e
b e tw e e n human seru m c o n t a i n i n g a l a r g e am ount and r a b b i t
serum w i t h a low t o medium a m o u n t•
The aim o f t h e e x p e r i m e n t a l w ork r e c o r d e d on t h e
f o l l o w i n g p a g e s was t o p ro d u c e a n u n q u e s t io n e d c i r r h o s i s
o f t h e r a b b i t l i v e r s an d t o f o l l o w b o t h s p e c i f i c and n o n
s p e c i f i c enzyme — t o n o t e w h e th e r a n y c h a n g e s I n serum
enzyme l e v e l a r e f u n c t i o n s o f th e in d iv i d u a l.e n z y m e s o r
o f t h e c o m b in a tio n , a n d to d e te r m in e t h e p o s s i b l e i n v o l v e
m ent o f b e n z o y l c h o l i n e s t e r a s e .
CHAPTER III
EXPERIMENTAL WORK
R a b b it s w ere fed. c a rb o n t e t r a c h l o r i d e i n o r d e r t o
p ro d u c e l i v e r dam age. M e asu rem en ts o f c h o l i n e s t e r a s e
a c t i v i t y w ere made a t i n t e r v a l s on t h e r a b b i t se ru m .
H i s t o l o g i c a l e x a m in a tio n s w e re made on l i v e r b i o p s y
sp e c im e n s o r a t a u to p s y t o d e te r m in e t h e e x t e n t o f t h e
d am age•
I . METHODS
F e e d in g s . C arb o n t e t r a c h l o r i d e was a d m i n i s t e r e d
o r a l l y , d i l u t e d i n W esson o i l so t h a t i t s c o n c e n t r a t i o n d id
n o t e x c e e d tw e n ty p e r c e n t b y v o lu m e . W here l a r g e r am ounts
w ere g i v e n , a f o r t y p e r c e n t s o l u t i o n was a d m i n i s t e r e d b y
sto m ach tu b e •
M e a s u re m e n ts . E s t i m a t i o n s o f t h e enzyme a c t i v i t i e s
w ere made b y t h e W arburg m e th o d . The m an o m etric m ethod
d e te r m i n e s t h e am ount o f a c i d s p l i t fro m t h e c h o l i n e e s t e r s
by m e a su rin g t h e am ount o f c a rb o n d i o x id e e v o lv e d fro m £
b u f f e r e d c a r b o n a t e s o l u t i o n . The m e a su re m e n ts o f a c e t y l
c h o l i n e h y i r o l y s i s w ere made b y t h e m ethod o f Mazur and
B o dansky ( 2 2 ) . W here b e n z o y l c h o l i n e w as u s e d a s t h e s u b s
t r a t e , t h e ' f i n a l c o n c e n t r a t i o n w as 0 .0 0 6 m o la r a s recom m ended
15
"by H aw kins a n d M endel ( 2 0 ) . To d i s t i n g u i s h b e tw e e n p s e u d o -
c h o l i n e s t e r a s e and b e n z o y l c h o l i n e s t e r a s e , th e enzyme was
in c u b a te d w i t h 2 x 10” 6 m o la r e s e r i n e a s d e s c r i b e d b y
G u n te r ( 1 7 ) .
T h re e m l. o f th e f i n a l r e a c t i o n m ix tu r e was 0 .0 1 5
m o la r i n a c e t y l c h o l i n e , 0 .0 4 m o la r i n sod ium b i c a r b o n a t e ,
an d c o n t a i n e d 0 .2 5 m l. o f r a b b i t se ru m . I t was g a s s e d a t
room t e m p e r a t u r e w i t h a m ix tu r e o f f i v e p e r c e n t c a rb o n
d i o x id e i n o x ygen a t room t e m p e r a t u r e and th e f i n a l pH was
7 . 7 . A f t e r th e r m a l e q u i l i b r i u m w as a t t a i n e d , t h e a c e t y l
c h o l i n e was t i p p e d i n t o t h e m ain b o d y o f t h e f l a s k a t
z e r o t i m e . A ll r e a d i n g s w ere made a t tw e n ty m in u te s a n d
c a l c u l a t e d t o s t a n d a r d c o n d i t i o n s .
M azur and B o d an sk y (2 2 ) showed t h a t f o r t h e f i r s t
f o r t y p e r c e n t o f a c e t y l c h o l i n e h y d r o l y s i s , t h e t i m e - a c t i o n
c u rv e i s a s t r a i g h t l i n e a n d - h e n c e th e r e a c t i o n i s o f jsero
o r d e r . T im e - a e tio n c u r v e s w ere r u n w i t h s e v e r a l s e r a w i t h
b o th a c e t y l c h o l i n e a n d b e n z o y l c h o l i n e s u b s t r a t e s t o d e t e r
m ine w h e th e r t h e tw e n ty m in u te r e a d i n g s w ould f a l l upon
t h e s t r a i g h t p o r t i o n o f th e c u rv e and so b e v a l i d f o r a
c o m p a ris o n o f v a l u e s . F i g u r e 1 show s t h a t f o r b o th su b
s t r a t e s f o r p e r i o d s up t o an h o u r , th e p l o t s a r e s t r a i g h t
l i n e s , an d t h a t t h e y re m a in so a f t e r p r o d u c t i o n o f l i v e r
dam age. T h a t t h e r e i s a s m a ll e n d o g e n e o u s e v o l u t i o n o f g a s
on m ix in g a c e t y l c h o l i n e an d b u f f e r a n d s l i g h t a b s o r p t i o n o f
No. S 3 1 1, U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
^
17
c a rb o n d i o x i d e when b e n z o y l c h o l i n e i s ad d ed t o t h e b u f f e r
i s shown b y F i g u r e 2 .
S in c e th e p l o t p ro v e d to be a s t r a i g h t l i n e , i t w as .
deemed s u f f i c i e n t t o make a s i n g l e tw e n ty m in u te r e a d i n g
s e r v e a s a m easu re o f t h e r a t e o f t h e r e a c t i o n an d th e
a c t i v i t y o f th e enzym e. I n th e c a l c u l a t i o n s , no c o r r e c t i o n
was made f o r t h e s m a ll e n d o g en e o u s c h a n g e s , in asm u c h a s
t h e s e v a l u e s w e re s m a l l , c o n s t a n t , and w e l l w i t h i n th e
e x p e r i m e n t a l e r r o r . F u r th e r m o r e , a l l c o m p a ris o n s o f
r e s u l t s w ere made f o r a g iv e n a n im a l, and no a tte m p t was
made t o com pare r e s u l t s among s e v e r a l a n i m a l s . T h is h e l d
t r u e i n e v a l u a t i n g th e r e s u l t s , a f t e r p o i s o n i n g , w i t h r e g a r d
t o th e tw o enzyme s y s te m s . C o m p ariso n s a r e o n ly f o r i n d i v i d u
a l a n im a ls a t v a r i o u s t i m e s . No a t t e m p t h a s b e e n made t o
a v e r a g e v a lu e s f o r t h e w hole s e r i e s . - The two enzyme s y s te m s
w ere c o n s i d e r e d s e p a r a t e l y i n t h i s m a n n e r, and no a tte m p t
was made to c o r r e l a t e them n u m e r i c a l l y .
H i s t o l o g i c a l t e c h n i q u e s . L i v e r sp e c im e n s w ere p r e
s e r v e d i n t e n p e r c e n t f o r m a l i n . H e m a to x y lin - e o s in , K r a ja n ,
and S c h a r l a c R s t a i n s w e re made to d e m o n s tr a te g e n e r a l
m o rp h o lo g y , c o n n e c ti v e t i s s u e , an d f a t r e s p e c t i v e l y . A ll
sp e c im e n s w ere ju d g e d by c o m p a ris o n w i t h t i s s u e s fro m
n o rm a l a n im a l s .
0
N o . 6 3 1 1, U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
H i s t o l o g i c a l c r i t e r i a o f l i v e r dam age. The d e g re e
o f c i r r h o s i s was a s s i g n e d on th e b a s i s o f f o r m a t io n o f
b a n d s o f c o n n e c ti v e t i s s u e . As l i v e r damage p ro c e e d s
t h e r e i s an i n c r e a s e i n t h e am ount o f f i b r o u s t i s s u e . The
c a p s u le becom es t h i c k e n e d and f i b r o u s t i s s u e in v a d e s t h e
a r e a s ab o u t t h e c e n t r a l and p o r t a l v e i n s . As t h i s p r o c e s s
c o n t i n u e s , i s l a n d s o f parenchym a becom e i s o l a t e d by t h e
b a n d s g i v i n g t h e t y p i c a l c o n d i t i o n o f c i r r h o s i s . S l i g h t ,
m edium , and m arked i n c r e a s e s i n t h e am ount o f f i b r o u s
t i s s u e w ere c l a s s i f i e d a s e a r l y , m edium , and a d v a n c e d
c i r r h o s i s r e s p e c t i v e l y . P a t t y i n f i l t r a t i o n and a n y o t h e r
n o t a b l e m o r p h o lo g ic a l c h a n g e s w ere n o t e d .
2 . RESULTS
S t a b i l i t y o f th e enzym e. The s t a b i l i t y o f t h e r e
f r i g e r a t e d enzyme h a s b e e n n o te d b y s e v e r a l w o rk e rs ( 2 , 6 ,
3 3 ) , and was f u r t h e r t e s t e d i n t h i s w o rk . A sp e cim en o f
serum was s t o r e d i n t h e r e f r i g e r a t o r o v e r a p e r i o d o f two
w eeks and t e s t e d a t i n t e r v a l s . No s i g n i f i c a n t c h a n g e s w ere
o b s e r v e d .
Norm al r a n g e o f v a l u e s . D e t e r m in a t io n s o f n o rm a l
r a b b i t serum c h o l i n e s t e r a s e a c t i v i t i e s w e re made f o r b o t h
a c e t y l c h o l i n e and b e n z o y l c h o l i n e s u b s t r a t e s on a l a r g e
s e r i e s o f a n i m a l s . The r e s u l t s shown i n T a b le I i n d i c a t e
20
TABLE I .
VALUES FOR 20 MINUTE HYDROLYSIS OF
ACETYLCHOLINE AND BENZOYLCHOLINE
BY NORMAL RABBIT SERA
R a b b it ACh R a b b it ACh BCh.
A1 39*7 C19 3 5 .7 2 6 .1
A2 4 1 .9 C20 4 2 .5
0
A3 5 0 .7 C21
4 3 .2 0
A4 4 1 .5 C2 2 3 6 .8 0
A5 4 9 .0 C23 5 1 ,0 44 .4
A6 4 3 .0 C24
4 1 .7 1 6 .1
B7 3 8 .8 C25 3 3 .8 44 .0
B8 5 0 .0 C26
4 1 ,8
3 3 .6
B9 5 3 .2 C27 4 1 ,7
4 3 .8
BIO 3 4 .3 C28 3 4 ,0
1 9 .6
B l l 4 0 .0 C29 4 0 .8
3 0 .8
B12 2 9 .0 C30 4 1 ,0 4 6 .7
C l 5 4 .7 C31 4 1 .5 4 . 5
C2 5 1 .6 C32 4 0 .6 0
C3 4 5 .6 C35
3 2 .7 1 9 .5
C4 4 5 .1 C36 3 9 .4 2 3 .9
C5 3 7 .6 037 4 0 .3 5 7 .3
c e ; 4 1 . 2 C38 3 9 .4 4 1 .5
C7 3 8 .3 C39 4 2 .1 2 8 .6
C8 2 8 .8 C40 4 9 , 3 3 6 .9
C9 2 9 .5 C41 3 5 .7 3 5 .0
CIO 3 0 .7 0 4 2 3 4 .0 0
C l l 2 8 .8
C12 3 4 .7
C13 2 8 .5
C14 3 4 .0
Cl® 3 3 .6
C17 4 5 ,8
21
t h a t t h e r e i s a w ide r a n g e — 1 0 0 $ — o v e r w hich n o rm al
v a l u e s to w a rd a c e t y l c h o l i n e f a l l , an d t h a t t h e a c t i v i t y
to w a rd b e n z o y l c h o l i n e v a r i e s ev en m o re, and i s c o m p le te ly
a b s e n t i n s e r a o f some a n im a ls *
C o n s ta n c y o f v a l u e s o v e r _a lo n g p e r i o d . N orm al
a c e t y l c h o l i n e e s t e r a s e v a lu e s w e re m e a su re d i n a s e r i e s o f
s i x r a b b i t s o v e r a p e r i o d o f n in e w e e k s . I t w i l l be s e e n
fro m T a b le I I t h a t t h e v a l u e s a r e q u i t e c o n s t a n t o v e r t h i s
i n t e r v a l * As a r e s u l t o f t h e s e d e t e r m i n a t i o n s , a l t e r a t i o n s
o f l e s s t h a n t e n p e r c e n t I n serum c h o l i n e s t e r a s e a c t i v i t y
a f t e r l i v e r dam age c o u ld n o t be c o n s i d e r e d s i g n i f i c a n t .
E f f e c t s o f a s i n g l e d o se o f c a r b o n t e t r a c h l o r i d e .
0*4 m i l l i g r a m s o f c a rb o n t e t r a c h l o r i d e p e r k ilo g r a m o f body
w e ig h t was a d m i n i s t e r e d t o r a b b i t s . D e t e r m in a t io n s o f serum
c h o l i n e s t e r a s e a c t i v i t y w ere made b e f o r e a d m i n i s t r a t i o n o f
t h e d ru g a n d d u r i n g t h e r e c o v e r y p e r i o d . F i g u r e s 3 a and 3b ,
p a g e s 23 an d 2 4 , show t h e r e s u l t s o f t h i s w o rk . The a n im a ls
w ere s a c r i f i c e d a t i n t e r v a l s an d h i s t o l o g i c a l e x a m in a tio n s
o f t h e l i v e r s w ere made a s r e c o r d e d i n T a b le I I I , p a g e 2 5 .
E f f e c t s o f s e v e r a l sm a ll d o s e s o f c a r b o n t e t r a c h l o r i d e .
T h re e d o s e s o f c a r b o n t e t r a c h l o r i d e , 0 .1 m g /k g , w ere a d m in is
t e r e d t o a g ro u p o f a n im a ls a t two d a y i n t e r v a l s , and th e
c o u r s e o f th e ^ s e r u m c h o l i n e s t e r a s e was n o te d a s i n F i g u r e 4 .
22
TABLE I I
HYDROLYSIS OP ACETYLCHOLINE BY NORMAL
RABBIT SERA OVER A PROLONGED PERIOD
R a b b it
0 1 2
Weak
3 4 5 9
A1 3 9 .7 3 5 .4 3 1 .6 3 9 .2 3 5 .6 3 8 .6 3 8 .9
A2 4 1 .9 4 1 .0 3 3 .2 3 4 .1 4 0 .6 4 0 .0 3 3 .0
A3 5 0 .7 4 6 .4 3 4 .7 4 5 .1 4 0 .0 5 3 .4 4 4 .6
A4
4 1 .5 4 1 .6 3 7 .7 4 0 .6 3 6 .7 4 2 .6 4 0 .8
A5
4 9 .0 4 1 .7 3 9 .0 4 0 .6 3 5 .3 4 6 .1 4 5 .8
A6
4 3 .0 5 2 .9 3 8 .4 3 9 .3 3 6 .8 4 4 .1 4 3 . 2
N o . 6 3 1 1 , U n i v e r s i t y B o o k s t o r e , L a s A n g e l e s
A L COg E volved
60
6 0
40
30
20
1 0
Day
FIGURE 3b
HYDROLYSIS OF ACETYLCHOLINE BY SERA OF RABBITS
FOLLOWING A SINGLE D0SE OF 0 .4 m l/k g OF CARBON TETRACHLORIDE
TABLE III
HISTOLOGY OF RABBIT LIVERS DAMAGED BY A
SINGLE DOSE OF 0 .4 m l/k g CARBON TETRACHLORIDE
R a b b it Day A u to p s ie d
H i s t o l o g i c a l
O b s e r v a tio n s
C l 41
n o rm a l
C2 37
c o r d c e l l s s w o lle n -
C3 39
no f a t
n o rm a l
C4
39 norm al
C6 .
32 n o rm a l
C7
24
n o rm a l
C8
24 n o rm al
N o . 6 3 1 1, U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
27
H i s t o l o g i c a l e x a m in a tio n s w ere made o f t h e l i v e r s o f r a b b i t s
t h a t d i e d d u r in g t h e c o u r s e o f t h e e x p e r im e n ts . T h is d a t a
i s p r e s e n t e d i n T a b le IV .
E f f e c t o f c o n tin u o u s s m a ll d o s e s o f c a r b o n t e t r a c h l o r
i d e . R e p e a te d d o s e s o f t h e d ru g w ere a d m i n i s t e r e d t o a l a r g e
g ro u p o f r a b b i t s . The d o sa g e l e v e l , tim e s o f a d m i n i s t r a t i o n ,
and c h o l i n e s t e r a s e a c t i v i t i e s a r e r e c o r d e d i n F i g u r e s 5 a - 5 $ ,
p a g e s 2 9 -3 5 , an d th e b i o p s y o r a u to p s y f i n d i n g s a r e g i v e n
i n T ab le V, page 3<$.
28
TABLE IV
HISTOLOGY OP RABBIT LIVERS
DAMAGED BY THREE 0 .1 m g/kg DOSES
OP CARBON TETRACHLORIDE
Day
R a b b it A u to p s ie d O b s e r v a ti o n s
C l l 3 P a t t y d e g e n e r a t i o n a ro u n d c e n t r a l
and p o r t a l v e i n s . B a s o p h i l i c
hom ogeneous s w e l l i n g .
C13 5 G e n e ra l f a t t y d e g e n e r a t i o n w i t h
c o n g e s t i o n o f f a t t y a r e a s . M axi
mum f a t a ro u n d c e n t r a l v e i n s .
C14 18 Homogeneous n e c r o s i s o f p o r t a l
a r e a s . E a r l y c i r r h o s i s .
SSm i u i i i i
: : : : : :
N o . S 3 1 1, U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
, N o . 6 3 1 1, U n i v e r s i t y B o d k s t o r e , L o s A n g e l e s
^
R a b b it C24 ACh
\ BCh
R a b b it C25 ACh
; B Ch
.R abbit C26 ACh .
B C h 60
50
4G
50
Of 20
0 . 2 m l/k g CC1.
0 ,1 m l/k g CC1
1 0
Day
60
FIGURE 5c
EFFECT OF CONTXNUOtS S&ALL DOSES OF CARBON TETRACHLORIDE
ON HYDROLYSIS CF ACETYLCHOLINE AND BENZOYLCHOLINE BY RABBIT SERA
N o . 6 3 1 1, U n i v e r s i t y B o o k s t o r e , L o s a n b e l e s
■■■■■■■■I
HSKEiH
N o . 6 3 1 1, U n i v e r s i t y B o o k s t o r e , L o s A n g e l e s
^
u
%
I \
A i> J
Evolved, r
80
70
6 Q
SO
40
30
20
1 0
, Rabbit' C36 ACh
! ____ BCh
R a b b it 0 3 6 ACh.
i BCh
< R a b b i t C37 ACb
j # BCh
.. /
* A d m ira isttra tio n o f
0 . 2 ra l/k g CCI4
TT
10 20 50 40
t y * — y~~ * * '» '*! * '»■"* * ' *— * ■
. . . . . -PJOORR 5 f
EFPECT OP COHTjNXJOtlS SMALL DOSES OP CARBON
iTETRACHtORlRE O f HYDROLYSIS OP ACETYLCHOLINE
i 1 AND 3ENZ0YLCH0LINE BY RABBIT SERA
N::. <13 1 !, u k sv .ih k sty 1.1 ... a n ;
35
E v o lv e d
.7 0
60
■ SO
40
30
I
20
■ i i o
« Rabbit C39 ACh
i BCh
* . . R a b b i t C41 ACh
BCh
< R a b b i t C42 ACh
* A d m i n i s t r a t i o n o f
0 . 2 m l/k g CCi4
Day
"W y * £ ' »
L ■ . . ... ; EX O H R E 5 g
j EFFECTOR CONTINUOUS SfjiALL! DOSRS OF CARBON
TETRJACHLORIDE OR HYDROLtSIS OP ACETYLCHOLINE
AND BENZOtLCH0LIN$ BY RABBIT SERA
N > :. C tli 1 1, i.lN iv r.R ;i!T Y l i u i : ; ; '
3 6
TABLE V
HISTOLOGY OP RABBIT LIVERS DAMAGED
BY CONTINUOUS SMALL DOSES OF
CARBON TETRACHLORIDE
D a te o f
R a b b it B io p s y op O b s e r v a tio n s
A u to p sy
C15 22A A r c h i t e c t u r e n o r m a l, no f a t .
S p ia ll am ount o f f i b r o u s t i s s u e
fro m p o r t a l a r e a s .
C16 78B F i b r o u s p r o l i f e r a t i o n w i t h t h i c k
e n ed c a p s u l e . B ands o f c o n n e c tiv e
t i s s u e r u n n in g t h r o u g h p a re n ch y m a .
A dvanced c i r r h o s i s .
C17 ‘ 78B F a t t y d e g e n e r a t i o n . C a p s u le t h i c k
e n e d . C o n n e c tiv e t i s s u e p r o l i f e r
a t i o n . M o d e ra te c i r r h o s i s .
124B A r c h i t e c t u r e a l t e r e d . A dvanced
c i r r h o s i s .
C19 1 0 2B A dvanced c i r r h o s i s .
020 ISA Hom ogeneous n e c r o s i s i n p o r t a l
a r e a s . F a t t y d e g e n e r a t i o n a ro u n d
c e n t r a l v e i n s . Some f i b r o u s t i s s u e
p r o l i f e r a t i o n .
021 3A N e c r o s is a b o u t c e n t r a l v e i n s . S l i g h t
am ount o f f a t i n t h e s e a r e a s .
022 2A Hom ogeneous n e c r o s i s a ro u n d c e n t r a l
v e i n s . No f a t .
C23 102B A dvanced c i r r h o s i s .
024 93B A dvanced c i r r h o s i s .
37
TABLE V (CONTINUED)
HISTOLOGY OF RABBIT LIVERS DAMAGED
BY CONTINUOUS SMALL DOSES OF
CARBON TETRACHLORIDE
R a b b it
D ate o f
B io p s y o r
A u to p sy
O b s e r v a tio n s
C25 93B E a r l y c i r r h o s i s .
C26 • 93B A dvanced b i r r h o s i s .
C27 87B M o d e ra te c i r r h o s i s .
C28 87B A dvanced c i r r h o s i s .
C29 90B A dvanced c i r r h o s i s .
C30 90B A dvanced c i r r h o s i s .
C31 90B E a r l y c i r r h o s i s .
C32 34B M o d e rate c i r r h o s i s .
C32 90B A dvanced c i r r h o s i s .
C35 29A Hom ogeneous n e c r o s i s a b o u t
p o r t a l a r e a s a n d f a t t y d e g e n
e r a t i o n a ro u n d c e n t r a l v e i n s .
C39 48B E a r l y c i r r h o s i s .
C40 6A N e c r o s i s . H yperem ia o f n e c r o t
a r e a s . Some f a t t y d e g e n e r a t i o
a ro u n d c e n t r a l v e i n s .
C41 14 A N e c r o s is a ro u n d p o r t a l v e i n s .
E a r l y c i r r h o s i s .
C42 48B A dvanced c i r r h o s i s .
m
I n h i b i t i o n b y e s e r i n e . A c o m p a ris o n w as made b e
tw e e n r a b b i t an d human serum o f t h e e f f e c t o f t h e c h o l i n
e s t e r a s e i n h i b i t o r e s e r i n e . E s e r i n e c o m p le te ly i n h i b i t e d
th e a c t i v i t y o f human serum to b o lh a c e t y l c h o l i n e and
b e n z o y l c h o l i n e ♦ I n th e r a b b i t t h e a c t i v i t y to w a rd a c e t y l
c h o l i n e w as I n h i b i t e d c o n s i d e r a b l y m ore t h a n t h a t to w a rd
b e n z o y l c h o l i n e .
TABLE VI
A COMPARISON OP THE EFFECTS
OF 2 x 10 * " 6 M ESERINE ON
RABBIT AND HUMAN SERUM.
ACh p l u s P e r c e n t BCh p l u s P e r c e n t
ACh E s e r i n e I n h i b i t i o n BCh E s e r i n e I n h i b i t i o n
Human 55 0 100 7 8 .8 0 100
R a b b it 5 0 .9 1 0 .5 7 9 .4 6 3 .4 4 1 .5 3 4 .7
DISCUSSION OF RESULTS
3 9
A c e t y l c h o l i n e e s t e r a s e v a l u e s h a v e b e e n r e p o r t e d t o
f a l l b e lo w n o rm a l i n human l i v e r damage c a s e s . I n a n im a ls
.w ith e x p e r i m e n t a l l y c a u s e d l i v e r dam age, v a lu e s w ere .re
p o r t e d e l e v a t e d i n c e r t a i n s p e c i e s and d e p r e s s e d in o t h e r s *
I n t h e p r e s e n t w o rk , r a b b i t s w ere f e d c a r b o n t e t r a c h l o r i d e
a n d t h e e x t e n t o f l i v e r damage was a s c e r t a i n e d b y h i s t o
l o g i c a l s t u d y .
1 . SUMMARY OF EXPERIMENTAL W ORK
H y d r o l y s i s o f a c e t y l c h o l i n e , an d b e n z o y l c h o l i n e by
r a b b i t serum w as f o llo w e d d u r i n g t h e c o u rs e o f p r o d u c t i o n
o f l i v e r d am age. I n g e n e r a l , t h e r e was an i n c r e a s e i n
a c e t y l c h o l i n e h y d r o l y s i s r a t e s d u r i n g t h e f i r s t week a f t e r
p o i s o n i n g , f o llo w e d b y a s lo w , i r r e g u l a r r e t u r n t o n o r m a l.
B e n z o y lc h o lin e h y d r o l y s i s d i d n o t p a r a l l e l a c e t y l c h o l i n e
h y d r o l y s i s .
The p r o d u c t i o n o f l i v e r damage f o llo w e d t h e t y p i c a l
p a t t e r n ; s e v e r e c e n t r a l n e c r o s i s , f a t t y I n f i l t r a t i o n , f i b r o s i s
and f i n a l l y c i r r h o s i s .
S e l e c t i v e i n h i b i t i o n o f t h e serum enzym es w i t h
e s e r i n e c o n firm e d t h e f a c t t h a t b e n z o y l c h o l i n e a n d s c e t y l -
c h o l i n e h y d r o l y s e s w e re c o m p le te ly i n h i b i t e d i n . t h e hum an.
I n t h e r a b b i t , h o w e v e r, w h ile a c e t y l c h o l i n e a c t i o n was
l a r g e l y i n h i b i t e d , b e n z o y l c h o l i n e v a l u e s w ere o n ly s l i g h t l y
d e c r e a s e d .
2 . CONCLUSIONS
I n r a b b i t serum t h e r e i s no s i g n i f i c a n t d e c r e a s e i n
♦
a c e t y l c h o l i n e h y d r o l y s i s a f t e r p r o d u c t i o n o f e x p e r i m e n t a l
c i r r h o s i s .
Serum b e n z o y l c h o l i n e h y d r o l y s i s v a lu e s d i d n o t p a r -
l l e l a c e t y l c h o l i n e e s t e r a s e a c t i v i t y .
2 x 1 0 ~ 6 e s e r i n e c o m p le te ly i n h i b i t e d t h e a b i l i t y
o f human serum to., h y d r o l y s e a c e t y l c h o l i n e and b e n z o y l
c h o l i n e . T h is c o n firm s t h e r e p o r t s t h a t t h e h y d r o l y s i s o f
b e n z o y l c h o l i n e by human serum r e p r e s e n t s o n ly p s e u d o - c h o l i n
e s t e r a s e a c t i v i t y .
The r e s u l t s o f t h e a d d i t i o n o f e s e r i n e show t h a t
r a b b i t serum h a s a c o n s i d e r a b l e am ount o f b e n z o y l c h o l i n e
e s t e r a s e a c t i v i t y . The f a c t t h a t a c e r t a i n am ount o f i t s
a c t i v i t y to w a rd a c e t y l c h o l i n e p e r s i s t s i n d i c a t e s e i t h e r t h a t
a n e s e r i n e - r e s i s t a n t c h o l i n e s t e r a s e i s p r e s e n t o r t h a t t h e r e
a r e q u a l i t a t i v e d i f f e r e n c e s b e tw e e n human a n d r a b b i t s e r a
w i t h r e s p e c t t o t h i s i n h i b i t o r .
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Creator Suran, Anita Alta (author)

Core Title Cholinesterase levels in the sera of rabbits with livers experimentally poisoned by carbon tetrachloride

Degree Master of Science

Degree Program Toxicology

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

Tag Health Sciences, Toxicology,OAI-PMH Harvest

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

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