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Studies on cholesterol metabolism in the rat

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Studies on cholesterol metabolism in the rat

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Content STUDIES ON CHOLESTEROL METABOLISM IN THE RAT
A D is s e r t a tio n
P resented to
th e F a c u lty of the Graduate School
U n iv e rs ity of Southern C a lif o rn ia
In P a r t i a l F u lfillm e n t
of th e Requirements f o r the Degree
D octor of Philosophy
by
Michael C. Schotz
August 1953
ph, 0 B*o V & 37-Sf
This dissertation, written by
3 *4
fro.)
Mlchael..C,_..Schotz.....................................
under the direction of ..hi& Faculty Committee,
on Studies, and approved by all its members, has
been presented to and accepted by the Council J
on Graduate Study and Research, in partial ful
fillment of requirements for the degree of
D O C T O R OF P H IL O S O P H Y
Date...
Com m ittee on Studies
Dean
ACKNOWLEDGEMENTS
C
I would l i k e to express my deep a p p r e c ia tio n to
\
D r. Roslyn B. A l f i n - S l a t e r f o r h e r encouragement and gu id
ance throughout my work, and to Dr. Harry J . D euel, J r .
i
f o r h is sp o n so rsh ip .
I am g r a t e f u l f o r th e co o p eratio n of Ire n e L. Rice
w ith whom many of th ese experim ents were planned.
I am a l s o in d eb ted to D r. David Berman, Dr. David
Masuoka, Dr. John Ganguly, and Norman I . K rinsky f o r t h e i r
in v a lu a b le ad v ic e .
I would l i k e to express my s in c e re a p p r e c ia tio n
t o my w ife , J u d ith , f o r h e r h elp in p re p a rin g t h i s manu
s c r i p t •
TABLE OP CONTENTS
PAGE
I . INTRODUCTION AND HISTORICAL . ................................... 1
1. P recu rso rs of C h o l e s t e r o l ............................. 3
2. S i t e s of C h o le s te ro l S y n t h e s i s . . . . 6
3. Importance of th e L iv er in C h o le ste ro l
M e t a b o l i s m .......................................................... 7
4 . E ff e c t of D iet on C h o le ste ro l
S y n th esis . . . . . . . . . . . . . . 9
5 . C h o le s te ro l E s te ra s e . . . * . . . . . 10
6. Statem ent of Problem . . . . . . . . . 12
I I . EXPERIMENTAL . . . . . . . . . . . . . . . . 13
1. M a te ria l and M e t h o d s ........................ 13
(1) Animals . . . . . • . . . . • . 13
(2) D iets . .. . . ._ . .................... 1 13
(3) E x tra c tio n of C h o le ste ro l . . . 14
a . S oxhlet m e t h o d ........................ 14
b . Waring Blendor method . . 14.
c. Plasma e x t r a c tio n . . . . 15
(4) D eterm ination of C h o le s te ro l • . 15
(5) Deuterium A n aly sis . . . . . . . 18
a . I s o l a t i o n of c h o le s te r o l
d ig ito n id e . . . . . . . 18
b* Combustion and red u ctio n • 18
V
PAGE
c. D eterm ination of deuterium
j to hydrogen r a t i o • . . • 19
d. D eterm ination of deuterium
in th e body f l u i d s . . . 19
(6) P erfu sio n Technique . . . . . . . 20
(7) H om ogenization.of T issues . . . . 20
(8) P re p a ra tio n of U ltr a c e n t r i f u g a l
F ra c tio n s from 0.25 M Sucrose
L iver Homogenate . . . . . . . 20
(9) P re p a ra tio n of B u ffer . . . . . . 25
(10) P re p a ra tio n of Aqueous Suspension
of C h o le s te ro l A cetate . . . . 25
2. Plan of Experiment .......................... 25
(1) E f f e c t of D iet on Newly Formed
C h o le s te ro l 25
(2) D i s tr ib u ti o n o f C h o le ste ro l . . . 27
(3) Assay f o r C h o le s te ro l E s te ra s e . 28
I I I . RESULTS AND DISCUSSION...................................... 30
1. E ff e c t of D iet on Newly Formed
C h o le s te ro l . • ................................................ 30
2. D is tr ib u ti o n of C h o le ste ro l w ith in the
L iver C e l l ................................. 44
3 . C h o le s te ro l E s te ra s e in Rat L iver
v i
PAGE
Homogenate . . ...................................... 50
(1) D em onstration of C h o le ste ro l
E s te ra s e A c tiv ity ............................. 50
(2) D is tr ib u ti o n of C h o le s te ro l
E s te ra s e ............................. 59
IV. SUM M ARY AND CONCLUSIONS ........................................... 62
BIBLIOGRAPHY............................................................. 64
LIST OP TABLES
TABLE PAGE
I . D iets Used during P re lim in ary and
E xperim ental Periods 14
I I . E ff e c t of High-Fat and C h o le s te ro l D iets
on th e Newly Formed C h o le s te ro l P resent in
th e Livers of Rats P re v io u sly Fed Normal
and C h o le ste ro l D ie ts . . . . . .............................. 31
I I I . E ff e c t of High-Fat and C h o le s te ro l D iets
on th e Newly Formed C h o le s te ro l P resent in
th e Lungs of Rats P re v io u sly Fed Normal
and C h o le ste ro l D ie ts . . • . . . . . . . . 39
IV. E ff e c t of High-Fat and C h o le s te ro l D iets
on th e Newly Formed C h o le s te ro l P resen t in
th e Kidneys of Rats P re v io u sly Fed Normal
and C h o le s te ro l D ie ts ............................. 40
V. E f f e c t of High-Fat D iet on th e Labeled
C h o le s te ro l P resen t in th e Plasma of Rats
P re v io u sly Fed Normal and C h o le ste ro l
D ie ts ....................... 42
VI. D is tr ib u ti o n of F re e , E s t e r i f i e d and T o tal
C h o le s te ro l of U l t r a c e n t r i f u g a l F ra c tio n s
of Normal Rat L iver .................................. 45
V II. The Percent of Free C h o le s te ro l in T o ta l
v i i i
TABLE PAGE
C h o le ste ro l of F ra c tio n s from Normal
Rat L iver .......................................................... 47
V I I I . H ydrolysis of C h o le s te ro l A cetate by
Rat L iver Homogenate . . . . . . . . . . . . 52
IX. E f f e c t of B o ilin g on the A b ilit y of Rat
L iver Homogenate t o Hydrolyze C h o le s te ro l
A cetate . . . . . . . ............................................ 57
X. D is tr ib u tio n of C h o le s te ro l E s te ra s e in
Various U l t r a c e n t r i f u g a l F ra c tio n s of Rat
Liver Homogenate . . . . . . . . . . . . . . 60
LIST OP FIGURES
*
FIGURE PAGE
1* F ra c tio n a tio n Scheme f o r Rat L iver
Homogenate Prepared in 0,25 M Sucrose
S o l u t i o n ........................ . ....................... 24
2 , The E f f e c t of Changes of Hydrogen Ion
C o n cen tratio n on C h o le s te ro l E s te ra s e
A c ti v ity o f Rat L iver H om ogenate........................... 54
3, The E f f e c t of Varying Time I n te r v a ls on
C h o le s te ro l E s te ra s e A c tiv ity of Plat L iver
Homogenate ............................................... 55
I . INTRODUCTION AND HISTORICAL
I
The o r ig in and s ig n if ic a n c e of the c h o l e s te r o l which '
jis p re s e n t in th e anim al body has been a m a tte r of c o n tro - j
jv ersy sin c e i t s d isc o v ery in 1769 by P o u l l e t i e r (1) who
1 observed th a t l e a f l e t s o f a su bstance somewhat lik e b o r ic '
i :
i ,
o r benzoic a c id in appearance were d e p o sited from a concen
t r a t e d a lc o h o lic e x t r a c t o f g a l l s t o n e s . These o b se rv a tio n s
were confirmed and extended by ConradI (2) in 1775, who
i I
noted th a t th e substance in g a lls to n e s was a ls o so lu b le In I
i
e th e r and t h a t t h i s su b stan ce could be c r y s t a l l i z e d from j
!t h i s so lv e n t as w ell as from a lc o h o l. In 1816 Chevreul (3) !
j j
ic a lle d th e se c r y s t a l s c h o le s te rin e ( c h o le - b ile , s t e r e o s - i
j i
j s o l i d ) . Soon a f t e r t h i s work was p re s e n te d , c h o le s te rin e j
i was e x tra c te d from human b i l e as w e ll as from t h a t of the
b e a r and th e p ig . In 1859, B e rth e lo t (4) e s ta b lis h e d th e
|
a lc o h o lic n a tu re of c h o le s te rin e by p re p a rin g e s t e r s , and j
th e name was, t h e r e f o r e , changed to c h o l e s t e r o l . .
E a rly I n v e s tig a to r s doubted th a t c h o l e s te r o l was a |
i
normal c o n s titu e n t of th e anim al body, sin c e i t had been j
d isco v ered In only p a th o lo g ic a l and n e c r o t ic t i s s u e . This |
I
th e o ry was q u ic k ly abandoned when Couerbe (5) showed th e j
e x is te n c e of c h o le s te r o l in th e normal b r a i n , and Lecanu j
j (6) found i t in normal b lo o d . Couerbe (5) th e o riz e d th a t |
j 1
I th e la rg e amount of c h o l e s te r o l t h a t was found in th e b ra in j
i
| !
2
o r ig in a te d in the nervous system . Lehmann (7 ), however,
b e lie v e d th a t the c h o l e s te r o l was formed as a decom position
p roduct of some unknown p re c u rs o r. F l i n t (8) extended t h i s
l a t t e r view in 1862 by p o s tu la tin g th a t c h o l e s t e r o l was a
w aste product o r ig in a t in g in the b r a in . This view, however,
was h eld un ten ab le by Beneke (9 ).
In a d d itio n to the b r a in and the nervous system ,
s e v e r a l o th e r organs were suggested as the s i t e o f sy n th e
s i s , However, o th e r i n v e s tig a to r s a t the tim e (10-12)
found i t r a t h e r d i f f i c u l t to conceive of the mechanism of
s y n th e s is o f c h o le s te ro l; in t h e anim al organism . By the
study o f c h o l e s te r o l c o n c e n tra tio n in h e n ’s eggs and in
newly hatched c h ic k s , i t was found th a t th e c h o l e s te r o l
co n ten t of newly hatched chicks was n o t g r e a t e r than th a t
of h e n ’s eggs. These r e s u l t s were i n te r p r e t e d to mean t h a t
c h o le s te r o l sy n th e s is does not ta k e p lace in t h e anim ai
organism . This n e g a tiv e fin d in g in newly hatched chicks
and i n h e n ’s eggs was s u b s ta n tia te d by R itte n b e rg and
Schoenheimer (13) u sin g more modern la b e lin g methods.
However, in s p it e of th e n e g a tiv e evidence in the
in c u b atio n t e s t s w ith h e n ’s eggs, o th e r evidence has
proved th a t c h o l e s te r o l can be sy n th e siz e d in th e anim al
body. Thus, by th e use of balance experim ents i t was
dem onstrated th a t c h o l e s te r o l was ex creted even though the
3
d i e t fed was s t e r o l - d e f i c i e n t (14, 15 ). This fin d in g was
s u b s ta n tia te d in the balance experim ents of Gamble and
Blackfan (16) w ith in f a n ts on a m ilk d i e t , as w ell as in
those of Gardner and Pox (17) w ith a d u lts on a mixed d i e t .
Schoenheimer and Breusch (18) in t h e i r c l a s s i c a l balance
experim ents w ith m ice, found t h a t th e se anim als fed a d i e t
of b read , sy n th e siz e d as much c h o le s te r o l in a month as
t h e i r bodies contained a t the beginning of th e experim ent.
With th e s y n th e s is of c h o l e s te r o l in th e anim al
body e s t a b l i s h e d , in v e s tig a to r s tu rn ed t o th e problems of
the s i t e s o f t h i s s y n th e s is , of th e n a tu re of c h o le s te r o l
p re c u rs o rs and of th e e f f e c t of v a rio u s d i e t a r y and ex
p e rim e n ta l f a c t o r s on th i s r e a c t i o n . However, the g r e a t e s t
advances have occurred sin c e the advent and a p p lic a tio n
o f iso to p e s to m edical r e s e a r c h .
1. P recu rso rs of C h o le s te ro l
R itte n b e rg and Schoenheimer (13) determ ined t h e ’
deuterium co n ten t o f th e body c h o l e s te r o l of mice a f t e r
th e a d m in is tra tio n of D20 in th e d rin k in g w a te r. The i s o
la te d c h o l e s te r o l contained a p ro p o rtio n of deuterium which
in d ic a te d th a t h a l f of the s ta b le hydrogen atoms of the
m olecule were d eriv ed from th o se o f th e body f l u i d s . This
would not be p o s s ib le i f c h o l e s te r o l were formed from any
s t e r o i d in th e food, o r by c y e liz a tio n o f a long chain
i !
I I
I f a t t y a c id ; th e a u th o rs , t h e r e f o r e , concluded th a t in m ice,
! c h o l e s te r o l i s sy n th e siz e d by th e coupling of a la rg e
i
jnumber of sm all m olecules. :
i ,
i Sonderhoff and Thomas (19) recovered la rg e q u a n t i-
!
i t i e s of deuterium from th e u n sa p o n ifia b le f r a c t i o n of y e a st
i
j grown on a medium c o n ta in in g d e u te r io a c e ta te and concluded
| t h a t a c e t i c a c id was d i r e c t l y converted in to s t e r o l s . j
Bloch and R itte n b e rg (20), in o rd e r to v e r i f y t h i s l a t t e r
c o n c lu sio n , s p e c i f i c a l l y in the ease of c h o l e s te r o l , fed
d e u te riu m -la b e le d sodium a c e ta te to r a t s and m ice. The
q u a n t i t i e s of deuterium found in th e c h o l e s te r o l d e f i n i t e l y
e s ta b lis h e d the f a c t t h a t a c e t i c a c id can serve as one of
I
| th e p re c u rs o rs of c h o l e s t e r o l .
I In 1944, Bloch and R itte n b e rg (21) fed deuterium -
la b e le d compounds t o r a t s , and i s o l a t e d ca rc ass c h o l e s t e r
o l . In t h i s manner, th e y were a b le to show t h a t a c e t a t e ,
b u t y r a t e , v a l e r a t e and p a lm ita te could be converted in t o
c h o l e s t e r o l . In l a t e r experim ents w ith d o u b ly -la b e le d
a c e t i c a c id (CD»C^00H) (22), th e y i s o l a t e d from th e a n i -
m als, c h o l e s t e r o l which con tain ed b o th carbon 13 and deu-
i
i te riu m , e s ta b li s h in g p o s i t i v e l y t h a t th e carbon atom of
i
! a c e t i c a c id i s u t i l i z e d in th e s y n th e s is of c h o l e s t e r o l .
i
C a lc u la tio n s showed t h a t a t l e a s t one h a l f of th e carbon
atoms of c h o l e s te r o l were d e riv e d from a c e t a t e .
5
I t has a l s o "been e s ta b lis h e d th a t s u rv iv in g r a t
l i v e r s l i c e s are a b le to in c o rp o ra te th e carbon atoms o f
a c e ta te (23), acetone (24), pyruvate (25, 2 6 ), b u ty ra te
(26), hexanoate (26), o ctan o ate (26), iso p ro p y l fragm ents
o f is o v a le r a t e (27), e th y l a lc o h o l (28), a c e to a c e ta te (27),
acetald eh y d e (27), and is o b u ty ra te (2 9 ). Thus a number of
sm all m olecules have been shown to e n te r in to th e form ation
of c h o l e s te r o l , most of them probably through p rev io u s
breakdown t o a c e ta te (30) u n its,. However, a c e to a c e ta t e ,
b u ty r a te , is o b u ty ra te and i s o v a l e r a t e , were shown not to be
*
s p l i t to a c e ta te b efo re in c o rp o ra tio n in t^ r c h o l e s te r o l (30,
3 1 ). I t is thought t h a t an a c e to a c e ta te type u n it could
be the common in te rm e d ia te f o r th ese compounds (30).
The n a tu re o f in te rm e d ia te s o f la r g e r m olecular
w eight in th e s y n th e s is of c h o l e s t e r o l , has u n t i l r e c e n tly ,
been v ery obscure and m erely s p e c u la tiv e . In 1926, H e il- •
bron e t a l . (32) e lu c id a te d the s t r u c t u r e of sq u a le n e , and
suggested t h a t t h i s te rp e n o id might be an in te rm e d ia te in
th e b io s y n th e s is of s t e r o i d s . Balance s tu d ie s to t e s t t h i s
h y p o th e sis gave c o n f l i c t i n g r e s u l t s (33-35). More r e c e n t l y
however, Langdon and Bloch (36) dem onstrated the b io lo g i c a l
s y n th e s is of squalene from a c e t a t e and found t h a t squalene
occurred in sm all c o n c e n tra tio n s in the r a t . These workers
a ls o dem onstrated th e conversion of squalene to c h o l e s te r o l
6
in th e r a t (37).
2 . S ite s of C h o le s te ro l S y n th esis
Bloch, Borek and R itte n b e rg (23) approached t h i s
problem by in c u b a tin g s l i c e s of d i f f e r e n t organs w ith heavy
w a t e r , ' d e u t e r io a c e ta te , ~a'nd d o u b ly -la b e le d a c e t a t e . Under
t h e i r c o n d itio n s th e y were a b le to show s y n th e s is of cho-.
l e s t e r o l in l i v e r s l i c e s o n ly . However, S re re art aJL. (38)
showed t h a t b eef a d re n a ls were capable o f sy n th e s iz in g
c h o l e s te r o l in v i t r o u sin g a c e ta te as th e p re c u rs o r
m a t e r i a l . S re re e^t a_l. (39) compared th e conversion of
la b e le d C.14 a c e ta te t o ^ c h o le ste ro l by normal and e v is c e r
a te d r a t s . Since th e au th o rs found r a d i o a c t i v i t y in the
c h o l e s te r o l of bo th groups of an im als, th ey concluded
t h a t th e re was e x tra h e p a tic s y n th e s is of c h o l e s te r o l .
These a u th o rs found s y n th e s is o f c h o l e s t e r o l from a c e ta te
in r a t s which were ad ren a lec to m ized , ovariectom ized and
e v i s c e r a te d . T h erefo re, o th e r organs than those in the
v is c e r a can be re s p o n s ib le f o r c h o l e s te r o l p ro d u c tio n .
Although Bloch and a s s o c ia te s (23) were unable to
dem onstrate the in v i t r o conversion of d e u te r io a c e ta te t o
c h o l e s te r o l in anim al t i s s u e s o th e r than th e l i v e r , S re re
e t a l . (39), upon r e i n v e s t i g a t i o n of t h i s problem usin g
(A ^ -la b ele d a c e ta te ,, found t h a t th e g u t, gonadal t i s s u e ,
k id n ey , a d u lt s k in , baby skin and one day old r a t b ra in
7
i .
were a b le to sy n th e siz e c h o l e s te r o l in v i t r o ; however,
th e y could n o t dem onstrate s y n th e s is of c h o l e s te r o l in the
a d u lt b r a in .
Popjak and Beeckmans (40) have re p o rte d t h a t th e
r a b b it can sy n th e siz e c h o l e s te r o l from la b e le d a c e ta te not
only in the l i v e r , bu t a ls o in th e i n t e s t i n e and o v a r ie s .
✓
Popjak (41) has a l s o shown th a t th e r a b b it f e tu s can
s y n th e s iz e , but not degrade, c h o l e s t e r o l , w hereas, the
f e t a l l i v e r can do b o th .
More re c e n tly i t has been dem onstrated t h a t cho
l e s t e r o l can be formed in th e a r t e r i a l w all (42) and in
th e t e s t e s (43). Although th e b io s y n th e s is of c h o l e s te r o l
had been accom plished in v i t r o only by the use o f t i s s u e
s l i c e s up to 1952, s e v e r a l in v e s tig a to r s (44-46) have now
been ab le to dem onstrate t h i s s y n th e s is in homogenates.
Rabinowitz and Gurin (46) have r e p o r te d the s u c c e s s fu l
p r e p a ra tio n of a w a te r-s o lu b le enzyme system from r a t l i v e r
m itochondria capable of in c o rp o ra tin g C^-4 - la b e le d a c e ta te
in to c h o l e s t e r o l . I t has been re p o rte d t h a t , w ith the
p o s s ib le exception o f adipose (47) and a d u lt b ra in t i s s u e
(3 9 ), alm ost every organ o r t i s s u e th a t has been examined
by a s e n s i t i v e technique is a b le to s y n th e siz e c h o l e s t e r o l .
3. Importance of th e L iver in C h o le s te ro l Metabolism
D esp ite th e known p a r t i c i p a t i o n o f alm ost every
" 8
organ of the body in th e sy n th e s is of c h o l e s te r o l , i t has
been shown t h a t only th e l i v e r can exchange s i g n i f i c a n t
amounts of c h o l e s te r o l w ith the plasma o f r a t s (48, 4 9 ).
This f in d in g has been su b seq u en tly confirmed in th e dog by
Gould and co-workers (50).
Meir e t a l . (51) have shown t h a t th e c a p a c ity of
th e l i v e r f a r exceeds t h a t of any o th e r t i s s u e to o x id ize
th e te rm in a l carbons o f c h o le s te r o l to COg. S ip e r s te in
and C haikoff (52) had p re v io u sly shown t h a t the l i v e r elim i
n a te s the breakdown p ro d u cts of c h o l e s te r o l via the b i l e .
These i n v e s tig a to r s concluded ’’t h a t th e l i v e r is not only
th e c h ie f organ f o r th e sy n th e s is of c h o l e s t e r o l , but a ls o
th e t i s s u e in which i t is degraded and e lim in a te d from the
body." (51).
Since the l i v e r , th e n , had been shown to be of im
p o rtan ce in th e s y n th e s is , d e s tr u c tio n , and sto ra g e of
c h o l e s te r o l (5 3 -5 6 ), i t became of i n t e r e s t in t h i s i n v e s t i
g a tio n to approach th e problem of lo c a l i z i n g the s i t e s and
mechanisms of th e se fu n c tio n s w ith in the l i v e r c e l l .
In t h i s co n n ectio n , Kretchmer and Barnum (57)
stu d ie d th e p a r t i t i o n o f u n sa p o n ifia b le m a te r ia l in
f r a c t i o n s of th e mouse l i v e r , namely in m ito ch o n d ria, in
microsomes, and in su p e rn a ta n t f l u i d . Chauveau e t a l .
(58), re p o rte d a more comprehensive stu d y of the d i s t r i -
9
b u tio n of f r e e and e s t e r i f i e d c h o l e s t e r o l in t h e above
f r a c t i o n s as w e ll as in a d d i t i o n a l f r a c t i o n s of normal r a t
l i v e r . In th e p re s e n t work re p o rte d h e r e , l i v e r c e l l s o f
normal r a t s have been se p a ra te d in to f iv e f r a c t i o n s ("F”
la y e r , n u c l e i , m ito ch o n d ria, microsomes, and s u p e rn a ta n t) ,
by a d i f f e r e n t i a l c e n tr if u g a tio n te c h n iq u e ; f r e e and e s
t e r i f i e d c h o l e s te r o l have been determ ined in each o f th e se
f r a c t i o n s .
4. E f f e c t o f D iet on C h o le s te ro l S y n th esis
The Importance of exogenous f a t in c h o l e s te r o l me
ta b o lism and d e p o s itio n , is c o n t r o v e r s i a l. E a rly i n v e s t i
g a to rs (59, 60) have re p o rte d c o n tra d ic to r y r e s u l t s r e l a
t i v e to th e e f f e c t of in g e sted f a t on c h o l e s te r o l sy n th e sis.
Cook and Thomson (61) showed t h a t when th e anim als
were fed a d i e t h ig h in c h o l e s te r o l sim u lta n eo u sly w ith a
d i e t h ig h In f a t , th e re was s i g n i f i c a n t l y le s s c h o l e s te r o l
d e p o site d in th e l i v e r th a n when th e anim als were fed a
d i e t co n ta in in g a normal amount o f f a t and a h ig h le v e l
o f c h o le s te ro l* Cook ejt a l . (62) found t h a t when r a t s
were fed a d i e t h ig h in c h o l e s te r o l and f a t , th e re was a
lo s s of c h o l e s te r o l from the l i v e r equal to th e amount of
petroleum e t h e r - in s o lu b le ac id s which appeared in the feces.
The accum ulation o f c h o l e s te r o l In c e r t a i n t i s s u e s
o f th e anim al body may be a r e s u l t o f in c re a se d s y n th e s is ,
10
decreased d e s tr u c tio n o r breakdown, o r decreased r a t e of
t r a n s p o r t o f c h o l e s t e r o l from th e t i s s u e s t o th e blood
stream .
I t had been b e lie v e d t h a t th e r a t e of c h o le s te r o l
s y n th e s is in normal anim als was not a f f e c te d g r e a t l y by
v a r i a t i o n s in d i e t a r y com position (63). More r e c e n t l y ,
however, Gould e t a l . (64, 65) have re p o rte d t h a t dogs and
r a b b i t s fe d a h ig h - c h o le s te r o l d i e t f o r a p erio d of s ix
weeks, showed a decreased h e p a tic s y n th e s is of c h o l e s te r o l
in vivo and in v i t r o , as measured by th e in c o rp o ra tio n of
(A4 from a c e ta te in to c h o l e s t e r o l .
Larack and A l f i n - S l a t e r (66), determ ining the i n
c o rp o ra tio n of deuterium In to c h o l e s te r o l in v iv o , w ith
c h o l e s te r o l - f e d r a b b i t s , found normal s y n th e s is in th e
l i v e r , bu t d ep ressed c h o l e s te r o l sy n th e s is In th e lung and
k id n ey . More r e c e n t l y , Tomkins erfc a_l. (67) noted th a t r a t s
fed c h o l e s te r o l e x h ib ite d a marked d ecrease in th e a b i l i t y
of l i v e r s l i c e s to sy n th e s iz e c h o l e s te r o l from a c e t a t e .
In view of th e above f a c t s , i t was f e l t to be im
p o r ta n t t o determ ine th e e f f e c t of h ig h - f a t and c h o l e s te r o l
d i e t s on th e s y n th e s is of c h o l e s te r o l In th e l i v e r , lung,
and k id n ey , and on th e c o n c e n tra tio n in the plasma of r a t s .
5 . C h o le s te ro l E ste ra se
_______ Kondo, in 1910 (68), stu d ie d the e f f e c t of l i v e r
1 1
e x t r a c ts on c h o l e s te r o l e s t e r s . His r e s u l t s In d ic a te d th a t
c h o l e s te r o l e s t e r s could be s p l i t by l i v e r t i s s u e e x t r a c t s .
Heating th e e x t r a c t d estro y ed t h i s a c t i v i t y . Using im
proved methods, S ch u ltz (69) l a t e r confirmed th ese ex
p e rim e n ts. R e p e titio n of t h i s work by M ueller (70) y ie ld e d
no evidence of h y d r o ly s is , b u t seemed in f a c t , to in d ic a te
s y n th e s is of e s t e r s .
Shope in 1928 (71), t e s te d e x t r a c t s of guinea p ig
t i s s u e s , usin g serum c o n tain in g c h o l e s te r o l e s t e r s as a
s u b s t r a t e . He found t h a t a l l t i s s u e s showed some a c t i v i t y ,
alth o u g h l i v e r , kidney and muscle were th e most a c t i v e .
The use of a serum as a source of s u b s t r a te was c r i t i c i z e d
by S p erry (72) sin c e serum i t s e l f i s a p o t e n t i a l source of
enzyme a c t i v i t y . K lein (73) found t h a t s a l i n e e x t r a c t s of
l i v e r would s p l i t 70-85 p e r cent of serum c h o l e s te r o l
e s t e r s in f i f t e e n h o u rs . N ie ft and Deuel (74) in d ic a te d
th e e x iste n c e of a c h o l e s te r o l e s t e r - s p l i t t i n g enzyme in
r a t l i v e r e x t r a c t s .
In view of the c o n f lic tin g evidence f o r a c h o l e s te r
o l e s te r a s e ^ , i t seemed o f value to determ ine the v a l i d i t y
1 The term c h o l e s te r o l e s te r a s e Is not meant to
n e c e s s a r i l y imply s p e c i f i c i t y f o r c h o l e s te r o l e s t e r s , but
r a t h e r an e s te r a s e f o r which c h o le s te r o l e s t e r s a re a
s u b s t r a t e .
12
of the e x iste n c e of t h i s enzyme in th e l i v e r , to lo c a l i z e
the s i t e of t h i s enzyme in the l i v e r c e l l , and t o e s t a b l i s h
th e c o n d itio n s under which th e maximum a c t i v i t y is ob
s e rv e d .
Statem ent of Problem
I t i s proposed to i n v e s tig a te th e fo llo w in g problems
in c h o l e s te r o l m etabolism in the r a t :
1) To determ ine the e f f e c t o f c h o le s te r o l and h ig h -
f a t d i e t s on the newly formed c h o l e s t e r o l in v a rio u s organs
of the r a t .
2) To determ ine th e d i s t r i b u t i o n of c h o l e s te r o l
w ith in the l i v e r c e l l .
3) To v e r i f y th e presence of a c h o le s te r o l e s te r a s e
in th e l i v e r , to lo c a l i z e t h i s enzyme i f p r e s e n t, and to
c h a r a c te riz e th e a c t i v i t y of t h i s enzyme.
I I . EXPERIMENTAL
1. M a te ria l and Methods
In o rd e r to i n v e s t i g a t e th e e f f e c t o f v a rio u s d i e t s
on the newly formed c h o l e s te r o l in the l i v e r , k id n ey , lu n g ,
and plasma of r a t s , the fo llo w in g m a te r ia ls and methods
were used.
(1) A nim als.
Female a lb in o r § t s of th e U n iv e rs ity of Southern
C a lif o rn ia s t r a i n were used in a l l ex p erim en ts.
(2) D i e t s .
The com position of th e d ie ts used d u rin g th e p r e
lim in a ry and experim ental p e rio d s i s given in Table I .
u*
TABLE I
D iets Used during Preliminary and Experimental Periods
bietary
Component
Normal
Diet
High-Fat
Diet
Cholesterol
D iet
i
•
Cottonseed Oil
containing Vitamins A and D /I 2.2 30.0 9.0
Oats 36.6 12.3 33.6
Wheat 36.6 18.3 33.3
Skim »!ilk Powder 16. U
22.5 15.0
Teast
Anheuser-Busch, "strain G " 2.2 3.0 2.0
A lfalfa (le a f meal) U .»4
C O
•
L.O
Sodium Glycocholate /2 0.5 0.7 o.5
Sodium Chloride 0.6 0.7 0.6
Calcium Carbonate 0 .5 0.7 o.5
C holesterol /3 0.0 0.0 1.0
/ I Vitamin A, 312 U.S.P. Units per gram of Wesson O ilj
Vitamin D, 50 U.S.P. Units per gram o f Wesson O il.
/2 M allinckrodt, Los Angeles, C alifornia.
/3 Merck and Company, U.S.P.
15
(3) E x tra c tio n of C h o l e s t e r o l «
a * S o x h let method. The t i s s u e s were q u ic k ly ex
t i r p a t e d , trimmed, b l o t t e d , ground w ith a m o rtar and
p e s t l e , t r a n s f e r r e d t o S oxhlet th im b le s , weighed and r e
flu x e d f o r 8 hours w ith e th y l a l c o h o l- e th y l e th e r (3:2 by
volume) in a S oxhlet a p p a ra tu s . The e x t r a c t was then
q u a n t i t a t i v e l y t r a n s f e r r e d to a v o lu m e tric f l a s k .
b . Waring Blendor method. This e x t r a c tio n p ro
cedure was used in th e d i s t r i b u t i o n and e s te r a s e s t u d i e s .
The method i s based on a procedure of Thompson e t a l . (75).
The sample to be e x tra c te d was made up t o 10 m l. w ith d i s
t i l l e d w a te r; 20 m l. o f 95$ e th y l a lc o h o l and 125 m l. of
petroleum e t h e r ( b . p . , 63.3 - 69.3°C .) were then added,
and th e m ixture was homogenized in a Waring Blendor f o r 10
m in u tes. This homogenized m a te r ia l was t r a n s f e r r e d q u a n ti
t a t i v e l y to a s e p a ra to r y fu n n e l, and th e a lc o h o l-w a te r
la y e r was se p a ra te d fro m the petroleum e th e r l a y e r , and the
form er r e e x tr a c te d tw ice f o r 5 minute p e rio d s w ith 125 ml.
o f petroleum e t h e r in th e Waring B lendor. The petroleum
e t h e r e x t r a c t s were pooled* c o n c en trated by ev a p o ratio n
w ith a i r and h e a t, o r w ith h e a t in vacuo, f i l t e r e d in to
v o lu m e tric f l a s k s , and a d ju s te d t o a d e f i n i t e volume. The
q u a n tity of c h o l e s te r o l e x tra c te d from l i v e r by t h i s method
has been shown to be i d e n t i c a l w ith t h a t o b ta in ed by 8 hour
16
e x t r a c tio n In the S oxhlet ap p a ra tu s,, w ith a l c o h o l- e th e r or
by a l k a l i n e h y d ro ly s is (76),
c * Blasma e x t r a c t i o n . The blood o b ta in ed by h e a r t
puncture was t r e a t e d w ith sm all q u a n t i t i e s of h e p a rin to
p rev en t c l o t t i n g , and th e plasma was o b tain ed by cen
t r i f u g a t i o n of th e t r e a t e d b lo o d . F ourteen volumes of
e t h y l a lc o h o l-a c e to n e (1:1 by volume) were added to each
volume of plasma by means of a sy rin g e under s u f f i c i e n t
p re s s u re to f a c i l i t a t e m ixing, to p r e c i p i t a t e th e plasma
p r o t e i n s , and to e x t r a c t the c h o le s te ro l* The r e s u l t i n g
p r e c i p i t a t e was c e n trifu g e d f o r 10 m inutes a t 3000 RPM and
th e e x t r a c t obtained was s to re d In a sto p p ered b o t t l e .
(4) D eterm ination of C h o l e s te r o l*
The method used i s a m o d ific a tio n of th e Schoen-
heim er-S perry method developed by N ie ft and Deuel (74).
An a l iq u o t of th e t i s s u e e x t r a c t c o n ta in in g ap proxim ately
0*1 t o 0 .5 mg. of c h o l e s t e r o l was evaporated to dryness In.
a 60°C. c o n sta n t tem perature a p p a ra tu s , by means of a
stream of a i r .
One ml. of e t h y l a lc o h o l-a c e to n e s o lu tio n (1:1 by
volum e), and two drops o f 33$ potassium hydroxide were
added; th e tu b e s were corked and th e samples were kept a t
60°C. f o r 45 m inutes w ith f re q u e n t sh ak in g . The s o lu tio n
t
was then n e u t r a liz e d to th e phenophthalein end p o in t w ith
17
15$ a c e t i c acid * Then 2 m l. of e t h y l a lc o h o l- a c e to n e s o -
l u t i o n (1 :1 by volume) were added.
The c h o l e s t e r o l d ig it o n id e was p r e c i p i t a t e d by th e
a d d itio n o f one m l. o f 0 .5 $ d i g i t o n i n s o lu tio n (in 50$
e t h y l a l c o h o l ) , and allo w ed t o in c u b a te a t room te m p e ra tu re
f o r a p p ro x im a te ly 12 h o u r s . The p r e c i p i t a t e was th e n cen
t r i f u g e d a t a speed o f a p p ro x im a te ly 3000 RPM f o r about 10
m inutes* The s u p e rn a ta n t l i q u i d was then c a r e f u l l y d e c a n t
ed and th e p r e c i p i t a t e was washed w ith 3 m l. o f anhydrous
e t h e r . T his was th en c e n tr if u g e d a t a p p ro x im a te ly 3000 RPM
f o r 8 to 10 m in u te s, and the s u p e r n a ta n t e th e r was d i s
c a rd e d . The p r e c i p i t a t e was d r ie d w ith a i r a t 6 0 °C ., 0 .5
m l. o f g l a c i a l a c e t i c a c id was ad d e d , and th e m ix tu re was
h e a te d a t 60°C. u n t i l th e p r e c i p i t a t e was c o m p letely d i s
s o lv e d . E x a c tly 3 m l. o f c h lo ro fo rm were added, and th e
tu b e s w ere b ro u g h t to a te m p e ra tu re o f 35°C,
The c o lo r r e a g e n t, p r e v io u s ly p re p a re d by ad d in g one
m l. of c o n c e n tra te d s u l f u r i c a c id to each 9 m l. o f c h i l l e d
a c e t i c a n h y d rid e , was th e n ad d ed . The tu b e s were in c u b a te d
f o r e x a c tl y 10 m in u tes a t 35QC., a t w hich tim e th e tu b e s
were p la c e d in an ic e b a th f o r a t l e a s t 10 m inutes p r i o r
to r e a d in g . The c o lo r I n t e n s i t y was th e n re a d in a K l e t t -
Summerson p h o t o e l e c t r i c c o lo r im e te r a t 420 m illim ic r o n s .
A re a g e n t b la n k c o n s is tin g o f a c e t i c a cid,} ch lo ro fo rm and
18
c o lo r re a g e n t was alw ays used to s e t th e z e ro p o in t on th e
c o lo r im e te r . V alues were re a d from a s ta n d a rd curve d e
te rm in e d s im u lta n e o u s ly .
F o r th e d e te rm in a tio n of f r e e c h o l e s t e r o l , th e same
p ro ced u re used in t o t a l c h o l e s t e r o l was fo llo w e d w ith th e s e
e x c e p tio n s : 1) U s u a lly a l a r g e r a l i q u o t was ta k e n f o r
a n a l y s i s , and 2) The h y d r o ly s is s te p was o m itte d .
The e s t e r c h o l e s t e r o l was c a lc u la te d from th e
d if f e r e n c e betw een t o t a l and f r e e c h o l e s t e r o l .
(5) D euterium A n a ly s is ,
a . I s o l a t i o n o f c h o l e s t e r o l d l g l t o n l d e . The p r o
cedure f o r th e d e te rm in a tio n o f t o t a l c h o l e s t e r o l was
fo llo w ed , u sin g p r o p o r ti o n a te ly l a r g e r q u a n t i t i e s o f th e r e
a g e n ts to accommodate th e g r e a t e r q u a n t i t i e s o f c h o l e s t e r o l
p r e s e n t in th e e x t r a c t . The c h o l e s t e r o l d ig it o n id e was
p u r i f i e d by w ashing th e p r e c i p i t a t e w ith e t h y l a l c o h o l-
a c e to n e (1 :1 by volume) and t h r e e tim e s w ith e th y l e t h e r .
The p r e c i p i t a t e was th e n t r a n s f e r r e d to a sm a ll b e a k e r and
allo w ed t o d ry o v e r ca lc iu m c h lo rid e in a vacuum d e s ic c a to r
f o r a p p ro x im a te ly 12 h o u r s .
b . Combustion and r e d u c t io n . A pproxim ately 20 mg.
o f th e d r ie d c h o l e s t e r o l - d i g i t o n i d e were p la c e d in a p o rc e
l a i n b o a t, combusted w ith a stre a m o f oxygen a t 600°C. to
carbon d io x id e and w a te r, u s in g copper oxide as a c a t a l y s t .
19
The w a te r produced w as tra p p e d in a T J tu b e immersed in a
d ry i c e - s o lv e n t m ix tu re . T his fro z e n w a te r sam ple was th en
t r a n s f e r r e d t o a re d u c tio n a p p a ra tu s and th e system evacu
a t e d . The fro z e n w a te r sam ple was g r a d u a lly warmed; th e
w a te r v a p o rs formed were reduced o v er z i n c , and th e d eu -
teriu m -h y d ro g en gas m ix tu re was c o l le c te d o v er m ercury in
a sample c o n t a in e r .
c . D e te rm in a tio n o f d e u te riu m to hydrogen r a t i o .
The r a t i o o f d e u te riu m t o hydrogen was read in a C o n s o li-
d a te d -N ie r is o to p e r a t i o mass s p e c tro m e te r which had been
p r e v io u s ly c a l i b r a t e d w ith known d eu teriu m -h y d ro g en
m ix tu re s (7 7 ). S in ce th e hydrogen c o n te n t of th e d i g i t o -
n id e i s t h r e e tim e s th e hydrogen c o n te n t o f c h o l e s t e r o l ,
th e d e u te riu m v a lu e s o f c h o l e s t e r o l were c a lc u la te d on th e
b a s is o f th r e e tim es th e v a lu e o b ta in e d f o r th e c h o l e s t e r o l
d i g i t o n i d e •
d . D e te rm in a tio n of d e u te riu m in th e body f l u i d s .
To d eterm in e th e d e u te riu m c o n te n t o f th e body f l u i d s , th e
c a rc a s s e s w ere ground in a meat g r in d e r and s u b s e q u e n tly
l y o p h il lz e d . The f l u i d c o l le c te d was p u r i f i e d by d i s
t i l l a t i o n o v er a l k a l i n e perm anganate and th e d i s t i l l a t e
c o l le c te d in th e te m p e ra tu re ra n g e o f 99-101°C. was r e
duced o v er z in c a s d e s c rib e d p r e v io u s ly , and th e d eu teriu m
to hydrogen r a t i o s d eterm in ed a s ab o v e.
j In o r d e r to i n v e s t i g a t e th e d i s t r i b u t i o n o f ch o - !
i
i
l e s t e r o l w ith in th e l i v e r c e l l }th e fo llo w in g m a te r ia l s and
methods w ere u se d . ,
1(6) P e rfu s io n T ech n iq u e. |
I
A b lu n t #18 hypoderm ic n e e d le was in tro d u c e d in t o
th e a o r ta th ro u g h an i n c i s i o n in th e l e f t v e n t r i c l e . Ic e
co ld 0 .2 5 M su c ro se s o lu t io n was f o r c e d th ro u g h th e a o r ta 1
[w ith a s y r in g e . The h e p a tic v e in was se v e re d c lo s e t o th e
j l i v e r t o p ro v id e an e x i t f o r th e blood and p e r f u s io n f l u i d .
I
I P e rfu s io n w ith 60 m l. of f l u i d was found t o be s u f f i c i e n t i
1 I
ito remove most o f th e blood from th e l i v e r in th e maximum I
I 7 *
of two m in u te s . I
| i
!(7) H om ogenization o f T is s u e s .
The l i v e r o f each r a t was q u ic k ly e x t i r p a t e d ,
trim m ed, b l o t t e d , p la c e d in a b e a k e r immersed in crack ed !
l i c e , and minced w ith s c i s s o r s ; 2 .5 gm. o f th e minced l i v e r !
was homogenized w ith e x a c t l y 7 .5 m l. o f ic e co ld 0 .2 5 M ■
s u c ro se in a m o d ified P o tte r-E lv e h je m hom ogenizer immersed |
[in an ic e b a th f o r 15 s tr o k e s o f th e p e s t l e . T his p r o - \
I ,
Icedure was found t o y i e l d on h i s t o l o g i c a l e x a m in a tio n , th e I
g r e a t e s t number o f broken c e l l s w ith th e l e a s t amount o f ;
i
broken n u c l e i .
! I
•(8 ) P re p a ra tio n of U l t r a c e n t r i f u g a 1 F r a c tio n s from 0 .2 5 M !
i
!S u cro se L iv e r Homogenate. |
21
The homogenate p re p a re d a s d e s c rib e d in th e p re v io u s
s e c tio n was f i l t e r e d th ro u g h s in g le napped f l a n n e l e t t e
(7 8 ). A ll s te p s o f t h i s p ro ced u re were perform ed a t a
te m p e ra tu re o f 3-4°C . S ucrose h av in g a c o n c e n tr a tio n of
0*25 M was chosen in p re f e re n c e to th e h ig h e r concen
t r a t i o n s u s u a lly r e q u ir e d t o m a in ta in m ito c h o n d r ia l mor
ph o lo g y , b ecau se o f th e g r e a t e r ea se of f r a c t i o n a t i o n in
0 .2 5 M s u c ro s e ’ coupled w ith th e f a c t t h a t some b io c h e m ic a l
r e a c tio n s a r e i n h i b i t e d a t th e h ig h e r s u c ro s e concen
t r a t i o n s (7 9 ).
E ig h t m l. o f th e homogenate were e a r e f u l l y la y e re d
b e n e a th 4 m l. of 0 .1 2 5 M s u c ro se s o lu t io n in a 13 m l.
L u s te ro id c e n tr if u g e tu b e . The c e n tr if u g e tu b e s were
s e a le d in a p r e -c o o le d 4°G. No. 40 r o t o r o f a S p in co u l t r a -
c e n t r i f u g e , model L; and c e n tr if u g e d f o r one h o u r a t an
av e ra g e f o r c e 2 of 105,000 x £ . D uring c e n t r i f u g a t i o n , a
creamy m a te r ia l m i g r a t e d t o th e to p of th e t u b e s . This
f r a c t i o n has been d e s ig n a te d a s f l o a t i n g la y e r o r th e M FW
l a y e r . T his f r a c t i o n and a p o r tio n o f th e 0.125 ffi s u c ro se
s o lu t io n were removed w ith a s y r in g e . Any creamy m a te r ia l
a d h e rin g to th e w a ll o f th e tu b e was wiped o f f w ith a
p ie c e o f c o tto n w hich was then combined w ith th e nFM la y e r
The £ v a lu e s a r e g iv e n f o r th e c e n te r s o f th e
t u b e s . ________________________
22
f o r f u tu r e e x t r a c t i o n and a n a l y s i s . The s u p e rn a ta n t
f r a c t i o n - 1 was th e n removed a lo n g w ith th e rem aining 0.125
M s u c ro s e , le a v in g th e re s id u e w hich c o n s is te d o f c e l l
d e b r i s , n u e l e i , m ito c h o n d ria and m icrosom es. In t h i s
m anner, i t was p o s s ib le to s e p a r a te th e th r e e f r a c t i o n s
w ith minimum c o n ta m in a tio n .
The "F" l a y e r and s u p e r n a ta n t- 1 f r a c t i o n s were ex
t r a c t e d im m ed iately by th e Waring B lendor method as d e
s c r ib e d p r e v io u s ly . The r e s id u e was then rehom ogenized
w ith 4 m l. o f 0.25 M su c ro se f o r f i v e s t r o k e s , u s in g a
L u c ite p e s t l e made to f i t th e c e n tr if u g e tu b e s . Any
m a te r ia l a d h e rin g t o th e p e s t l e was r in s e d back I n to th e
tu b e . The r e s u l t i n g homogenate was f r a c t i o n a t e d by th e
fo llo w in g m o d if ic a tio n o f th e te c h n iq u e of S c h n e id e r and
Hogeboom (8 0 ).
N u c le i, whole c e l l s and c e l l d e b r i s , were sedim ented
by c e n t r i f u g a t i o n a t 600 x £ f o r 10 m in u te s . The sedim ent
was washed tw ic e by reh o m o g e n iza tio n w ith 0 .2 5 M s u c ro se
and c e n tr if u g e d a t th e same f o r c e . The f i n a l re s id u e
was la b e le d " n u c le a r f r a c t i o n " .
The s u p e rn a ta n t f r a c t i o n and w ashings from th e
n u c le a r f r a c t i o n w ere combined and c e n tr ifu g e d a t 5000 x £
f o r 10 m inutes t o sedim ent th e m ito c h o n d ria . The r e s id u e
was w ashed tw ic e w ith 0.25 M s u c ro s e ; th e f i r s t sedim en
23
t a t i o n was done a t 5000 x £ f o r 10 m in u te s , w h ile th e f i n a l
s p in was a t 24,000 x £ f o r 10 m in u te s . The r e s u l t i n g r e s i
due was c a lle d ’’m ito c h o n d ria l f r a c t i o n ” .
The s u p e rn a ta n t f r a c t i o n and w ashings o b ta in e d from
th e m ito c h o n d ria l f r a c t i o n w ere combined and c e n tr ifu g e d
a t 105,000 x £ f o r one h o u r. The s u p e rn a ta n t f r a c t i o n was
d e s ig n a te d 1 1 s u p e r n a ta n t- 2 ” and th e r e s id u e was d e s ig n a te d
’’su b m iero seo p ic p a r t i c l e s ” .
T his f r a c t i o n a t i o n scheme i s shown d ia g r a m a ti c a lly
in F ig u re 1 .
22*
HOMOGENATE
1 HOUR
1 0 5 ,0 0 0 X6
4 4
FLOATING RESIDUE
LAYER
REHOMOGENIZE
10 MIN. 6 0 0 X 6
1
NUCLEAR RESIDUE
1 IO MIN. 6 00X G
4 4
N. RESIDUE SUPER ..
’ r
SUPERNATANT-1
10 MIN 6 0 0 X 6
.SUPER.
NUCLEI
i X
SU PER .
10 MIN
5 , 0 0 0 XG
4
MITO. RESIDUE
| 10 MIN
I 5 , 0 0 0 X 6
f ................ 1.......................... 4
MITO. RESIDUE SUPER.
10 MIN.
2 4 .0 0 0 X G
I-----
MITOCHONDRIA
\
s u p e r :
SUPER.
' I HOUR
1 0 5 ,0 0 0 X 6
J *
MICROSOMES SUPERNATANT-2
F IG U R E I
FRACTIONATION SC H EM E FOR RAT LIVER HO M O G ENATE
PREPARED IN 0 -2 5 M SU C R O SE SO LU TIO N
25
In o r d e r t o d em o n strate th e p re se n c e o f a cho
l e s t e r o l e s t e r a s e In th e l i v e r and to l o c a l i z e t h i s
enzyme, th e fo llo w in g m a te r ia l s and methods were u s e d .
(9) P re p a r a tio n of B u f f e r .
The b u f f e r used was th e a c e ta te - v e r o n a l d e s c rib e d by
M ic h a e lis (8 1 ).
(10) P r e p a r a tio n o f Aqueous S u spension o f C h o le s te ro l
A c e ta te .
T his te c h n iq u e was f i r s t r e p o r te d by M eir et; a l .
(5 1 ). Tween 203 was f i r s t added to th e d ry c h o l e s t e r o l
a c e t a t e ^ a p p ro x im a te ly One dro p p e r mg. of c h o l e s te r o l
a c e t a t e , and mixed w ith j u s t s u f f i c i e n t 95$ e t h y l a l c o h o l,
t o b r in g th e c h o l e s t e r o l a c e t a t e I n to s o l u t i o n . The a l c o
h o l was th e n removed by e v a p o ra tio n . The v is c o u s s o lu tio n
o f c h o l e s t e r o l a c e t a t e in th e Tween 20 was th e n d i l u t e d t o
any d e s ir e d volume w ith fs 4 a l i n e o r b u f f e r .
2 » P lan o f E xperim ent
(1) E f f e c t of D ie t on Newly Formed C h o l e s te r o l.
Two groups of fem ale a lb in o r a t s 60 days o ld and
160 gm. in w eight were fed th e norm al o r c h o l e s t e r o l d i e t s
^ P o ly o x y eth y len e s o r b ita n m o n o la u ra te , A tla s
Powder C o ., W ilm ington, D elaw are.
^ B ios L a b o r a to r ie s , 17 W. 6 0 th S t . , New Y ork,
New Y ork.
26
l i s t e d in T able I , f o r one month a f t e r w eaning. Each
group was then d iv id e d in to th r e e groups w hich were su b se
q u e n tly p la c e d on n o rm al, h i g h - f a t o r c h o l e s t e r o l d i e t s
d u rin g th e e x p e rim e n ta l p e rio d (see T able I ) .
On th e f i r s t day o f th e e x p e rim e n ta l p e r io d , th e
an im a ls were i n j e c t e d w ith s u f f i c i e n t 25$ d e u te riu m oxide
t o b r in g th e c o n c e n tr a tio n o f d e u te riu m o x id e in th e body
f l u i d s t o a p p ro x im a te ly 1 .5 $ . T his amount was c a lc u la te d
from d e te rm in a tio n s o f th e w a te r c o n te n t o f c o n t r o l r a t s
by l y o p h i l i z a t i o n . A f te r th e i n j e c t i o n of th e 25$ deu
te riu m o x id e , th e an im als w ere allo w ed to d r in k 2 .5 $ deu
te riu m o xide ad lib itu m t o m a in ta in a c o n c e n tr a tio n of
ab o u t 1 .5 $ in th e body f l u i d s . A ll th e an im als g a in ed in
w eig h t on th e v a r io u s d i e t s d u rin g th e e x p e rim e n ta l p e r i
o d s, th e d if f e r e n c e s in w eight on th e d i f f e r e n t d i e t s b e in g
i n s i g n i f i c a n t . . >
The an im als p re fe d on th e norm al d i e t were s a c r i
f ic e d a f t e r 8 o r a f t e r 16 d a y s , and th e an im als p re fe d
c h o l e s t e r o l d i e t w ere s a c r i f i c e d a t 0 , 8 , or 16 d a y s , by
th e a d m in is tr a tio n o f an a n e s t h e t i c dose o f nem butal and
w ith d ra w a l o f blood by h e a r t p u n c tu re . The l i v e r , k id n e y s ,
and lu n g s were q u ic k ly e x t i r p a t e d , w eighed, ground w ith a
m o rta r and p e s t l e , t r a n s f e r r e d t o S o x h le t th im b le s , and
e x t r a c te d In th e S o x h le t e x t r a c t i o n a p p a r a tu s , as p r e v io u s -
l y d e s c r ib e d . j
I Each group s a c r i f i c e d c o n ta in e d e i g h t a n im a ls . The j
j
I lu n g s and k id n ey s o f f o u r an im a ls were p o o led f o r e x - j
J t r a c t i o n . The l i v e r s in g e n e r a l were e x tr a c te d individu-^ j
J a l l y . In a few c a s e s , two l i v e r s were p ooled b e fo re j
S o x h le t e x t r a c t i o n . The b lo o d was t r e a t e d w ith h e p a rin i
|
(1 drop 10% s o l u t i o n / 5 m l. whole b lo o d ) to p re v e n t c l o t - j
\
t i n g , and th e plasm a o b ta in e d by c e n t r i f u g a t i o n was e x - |
\
i
t r a c t e d a s p r e v io u s ly d e s c r ib e d . T o ta l c h o l e s t e r o l d e - j
te rm in a tio n s were perform ed on th e l i v e r and plasm a e x - J
t r a c t s . |
D euterium d e te r m in a tio n s were c a r r i e d o u t on th e j
p r e c i p i t a t e d c h o l e s t e r o l d i g it o n id e sa m p les. The r a t i o s j
of d eu teriu m t o hydrogen o b ta in e d f o r th e d ig it o n id e s were J
c a lc u l a te d t o v a lu e s f o r c h o l e s t e r o l .
(2) D i s t r i b u t i o n o f C h o l e s t e r o l .
S ix fem ale a lb in o r a t s o f th e U n iv e r s ity of S outhernJ
C a lif o r n ia s t r a i n , a p p ro x im a te ly 60 days o ld and 170 gm. j
in w eig h t were u se d . A ll an im als were r e a r e d on P u rin a
I
L a b o ra to ry Chow,
j The an im als were a n e s th e tiz e d w ith n e m b u ta l, and as j
I i
much b lood as p o s s ib le was w ithdraw n by h e a r t p u n c tu re . j
The l i v e r s o f th e r a t s were th e n p e r f u s e d , rem oved, homo- j
i
g e n iz e d and f r a c t i o n a t e d a s d e s c rib e d p r e v io u s ly . In most
28
c a s e s , d u p lic a t e sam ples o f each hom ogenate were f r a c t i o n
a te d s im u lta n e o u s ly . Each u l t r a c e n t r i f u g a l f r a c t i o n , as
w e ll a s an a l i q u o t o f th e u n f r a c tio n a te d hom ogenate, was
e x t r a c te d by th e W aring B lendor m ethod. D u p lic a te d e
te rm in a tio n s o f f r e e and t o t a l c h o l e s t e r o l were made on
each e x t r a c t .
(3) A ssay f o r C h o le s te r o l E s t e r a s e .
In g e n e ra l th e fo llo w in g p ro c e d u re was em ployed:
r a t s w ere s a c r i f i c e d by c e r v i c a l f r a c t u r e , th e p e rfu se d
l i v e r s were th en q u ic k ly rem oved, trim m ed, b l o t t e d , p la c e d
in a b e a k e r su rro u n d ed by crack ed i c e , and homogenized as
p r e v io u s ly d e s c rib e d in 0 .2 5 M s u c ro s e . Two m l. o f homo
g e n a te were added to 50 m l. .erienm eyer f l a s k s w ith 8 m l. o f
v e ro n a l b u f f e r and 2 m l. o f c h o l e s t e r o l a c e t a t e d is s o lv e d
in Tween 2 0 . The m ix tu re s were th en in c u b a te d a t 36°C. in
a i r f o r v a ry in g tim e i n t e r v a l s . The r e a c t i o n was sto p p e d
a t th e d e s ir e d tim e i n t e r v a l by th e a d d itio n o f 5 m l. o f
e t h y l a lc o h o l to th e r e a c t i o n f l a s k . C h o le s te ro l was t h e n
e x t r a c te d by th e W aring B lendor.m ethod and f r e e and t o t a l
c h o l e s t e r o l v a lu e s were d e te rm in e d .
In th e a s s a y f o r th e d i s t r i b u t i o n o f c h o l e s t e r o l
e s t e r a s e , th e homogenate was f r a c t i o n a t e d by d i f f e r e n t i a l
c e n t r i f u g a t i o n a s d e s c rib e d p r e v io u s l y . F r a c tio n s e q u iv a
l e n t to 2 m l. o f th e homogenate were used in th e a s s a y f o r
th e enzyme.
I I I . RESULTS AND DISCUSSION
1 . E f f e c t of D ie t on Newly Formed C h o le a te r o l
T ab le I I I l l u s t r a t e s th e e f f e c t o f h i g h - f a t and
c h o l e s t e r o l d i e t s on th e new ly formed c h o l e s t e r o l p r e s e n t
In th e l i v e r s o f r a t a p r e v io u s ly fe d norm al and c h o l e s t e r o l
d i e t s . x
TA B LE I I
Effect of High-Fat and Cholesterol Diets on the Newly Formed Cholesterol Present in the Livers of Rats
Previously Fed Normal and Cholesterol Diets
Diet Duration
of
experiment
Weight
of
liver
Deuterium • *
in
body fluid
Total cholesterol in liver
Previous Experimental per gm. total
Deuterium Newly
concentration | formed
days gm. atom % mg. mg. atom %
m
8 6.12 1.61 1.8 11.0 0.610 75.8 8. 3 1 *
Normal Normal
16 7.1h 1.51 1.8 12.9 0.660 8 7 .1 * 11.27
8 7.01 1.56 2.2 1 5 .1 * 0.1*10 52.6 8.10
Normal High-Fat
16 6.99 1.66 2.3 16.1 0.5Y0 68.6 11.01*
0 7.7?
-
20.2 156.6
■ _ ■ _ r — : J . . j -,
Cholesterol Normal 8 8.25 1.1*6
1U.5
119.6 0.033
1 * .6 5.50
16 7.55 1.60 9.5 71.5 0.120 i5 .o 10.73
0 7.75
-
20.2 156.6
-- ■ -r- .- - - - - -in ----
Cholesterol High-Fat 8 7.12 1.1*8 9.7 72.0 0.087 11.8 8.50
16 7.90 1.67 1 * .6 36.3
0.276 33.0 11.98
0 7.75
mmm—
20.2 156.6
...in. . _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
Cholesterol Cholesterol 8 7.73 1 . 1 * 1 * 20.2 156.3 0.037 5.2 8.13
16 8.1*5 1.51 18.1 153.0 0.061* 8 .1 * 12.85
/l Percent of newly formed cholesterol - {% Deuterium in cholesterol)/{% Deuterium in body fluid)x2xl00.
The factor of 2 is used in this calculation since experimental evidence reported by Rittenberg and
Schoenheimer (19) indicates only half of the hydrogen atoms of cholesterol are in exchange with
hydrogens of the body fluid.
[2 Mg. of newly formed cholesterol= percent of newly formed cholesterol x mg. of cholesterol per total
liver.
32
I t i s e v id e n t t h a t th e i n t e r p r e t a t i o n o f th e q u a n t i
t y o f newly formed c h o l e s t e r o l d eterm in ed by th e m easure
ment o f th e I n c o r p o ra tio n o f d e u te riu m in to th e c h o l e s t e r o l
m olecule depends upon a number o f f a c t o r s .
1) I t i s assumed in t h i s s tu d y t h a t th e p re se n c e
o f d eu teriu m in th e c h o l e s t e r o l m olecule i s n o t due to an
exchange r e a c tio n betw een th e hydrogens o f th e a lr e a d y
form ed c h o l e s t e r o l and th e d e u te riu m o xide in th e body
f l u i d s (13, 8 2 ) .
2) The amount o f new ly form ed c h o l e s t e r o l found in
a p a r t i c u l a r organ may be due e i t h e r in t o t o o r in p a r t to
th e endogenous s y n th e s is by t h a t p a r t i c u l a r o rg a n .
3) P a rt o r a l l o f th e new ly formed c h o l e s t e r o l
found in a p a r t i c u l a r organ may be formed in a n o th e r organ
and tr a n s p o r te d to th e p a r t i c u l a r organ by means o f th e
plasm a o r lymph f l u i d .
4) A p o r tio n o f th e c h o l e s t e r o l found in some o f
th e org an s may o r i g i n a t e from th e d i e t and may, t h e r e f o r e ,
d i l u t e o u t th e newly form ed c h o l e s t e r o l p r e s e n t in th e
organ a s a r e s u l t o f endogenous s y n th e s is and t r a n s p o r
t a t i o n .
T h e re fo re , th e d e u te riu m -la b e le d c h o l e s t e r o l p r e s e n t
in an organ o f an an im al a t t h e end o f 8 and 16 days
fo llo w in g a d m in is tr a tio n o f d e u te riu m o x id e i s n o t a
33
q u a n t i t a t i v e m easure o f endogenous s y n th e s is o f a p a r t i c u
l a r o rg a n , b u t i s m e re ly th e amount o f newly form ed cho
l e s t e r o l p r e s e n t in th e organ a t t h a t tim e , and i s d e
p en d en t on th e c o n d itio n s l i s t e d abo v e.
I t can be seen from T able I I t h a t a h i g h - f a t d i e t
h as no e f f e c t on th e c h o l e s t e r o l c o n c e n tr a tio n in th e
l i v e r s o f an im als w hich have been fed a h i g h - f a t d i e t
d u rin g th e e x p e rim e n ta l p e rio d and t h a t th e amount o f newly
formed c h o l e s t e r o l p r e s e n t a t th e end o f 8 and 16 days i s
th e same as i n th e c o n t r o l a n im a ls .
The an im als w hich have been p re f e d a c h o l e s t e r o l
d i e t f o r 30 days show a la r g e in c re a s e in th e t o t a l cho
l e s t e r o l in th e l i v e r . A f te r th e c h o l e s t e r o l d i e t i s
d is c o n tin u e d , and th e an im als a r e p la c e d on norm al o r h ig h -
f a t d i e t s , th e r e i s a marked d e c re a se in th e s to r e d cho
l e s t e r o l in th e l i v e r , p a r t i c u l a r l y in th e a n im als fe d a
h i g h - f a t d i e t . However, th e an im a ls t h a t a r e c o n tin u e d on
#
th e c h o l e s t e r o l d i e t showed no f u r t h e r in c re a s e in th e
amount o f c h o l e s t e r o l in th e l i v e r , i n d i c a t i n g t h a t p erh ap s
a p la te a u in th e d e p o s itio n o f c h o l e s t e r o l in th e l i v e r
h as been re a c h e d .
I t would a p p e a r from th e d a ta o f th e p e rc e n ta g e o f
new ly form ed c h o l e s t e r o l In th e an im als p re f e d c h o l e s t e r o l ,
a s g iv en In T able I I , t h a t th e r e i s a d e c re a s e in th e
34
new ly form ed c h o l e s t e r o l in th e l i v e r s o f th e s e an im als on
th e d i f f e r e n t d i e t a r y regim ens a t 8 o r 16 days when com
p ared t o t h a t in t h e l i v e r s o f th e a n im als on th e norm al
d i e t . I t sh o u ld be n o te d , t h a t c o n c lu s io n s drawn from th e
c a l c u l a t i o n o f th e p e rc e n t o f new ly form ed c h o l e s t e r o l a r e
m is le a d in g , s in c e th e y do n o t ta k e in t o c o n s id e r a tio n th e
f a c t t h a t th e r e i s an in c r e a s e due to d i e t a r y c h o l e s t e r o l
in t h e t o t a l c h o l e s t e r o l o f th e l i v e r s o f th e s e an im als
w hich d i l u t e s th e new ly formed c h o l e s t e r o l p r e s e n t and
t h e r e f o r e , le a d s to lo w er p e rc e n ta g e v a l u e s .
However, when th e t o t a l amount o f new ly formed
c h o l e s t e r o l in th e l i v e r i s c a l c u l a t e d , i t can be seen to
be th e same, r e g a r d le s s o f th e d i e t a r y reg im en , a t th e end
o f th e 16 day e x p e rim e n ta l p e r io d . However, a t th e end o f
8 d ay s, th e amount o f new ly formed c h o l e s t e r o l in th e
l i v e r s o f a n im als p re fe d c h o l e s t e r o l and th e n fe d a norm al
d i e t , i s le s s th a n t h a t found in th e c o n tr o l a n im a ls and
in th e o th e r e x p e rim e n ta l a n im a ls .
I t had been found by A l f i n - S l a t e r (83) in s i m i l a r
in v iv o ex p e rim en ts t h a t t h e r a t e o f in c o r p o r a tio n o f
d e u te riu m i n t o th e l i v e r c h o l e s t e r o l approached a p la te a u
a t 8 d a y s . A lthough th e amount o f new ly formed c h o l e s t e r o l
in th e l i v e r i s th e same In th e groups o f an im als fe d
c h o l e s t e r o l and c o n t r o l d i e t s , th e r a t e a t which th e ch o -
35
l e s t e r o l i s form ed and d e s tro y e d could be d i f f e r e n t in th e
two c a s e s . One can c a l c u l a t e th e r a t e o f fo rm a tio n and
d e s t r u c t i o n from th e d a ta p r e s e n te d in T able I I , u s in g th e
f o llo w in g fo rm u la 5 :
K. = chol*. In - cho:Lt _______
ch o It - c h o ld
where ch o l^ e q u a l s t h e amount o f d e u te riu m c h o l e s t e r o l
p r e s e n t a t a tim e , t ; chol^. e q u a ls th e t o t a l mg. c h o l e s t e r
o l , and K e q u a ls th e r a t e o f fo rm a tio n and d e s t r u c t i o n .
T his e q u a tio n i s t r u e i f one assum es t h a t th e r e i s a com
p l e t e m ixing o f th e c h o l e s t e r o l a lr e a d y p r e s e n t and w ith
t h a t w hich i s d e p o s ite d from t h e d i e t and o th e r o rg a n s .
One must a l s o assume t h a t th e c h o l e s t e r o l in th e l i v e r i s
in th e " s te a d y s t a t e " i . e . , th e t o t a l amount o f c h o l e s t e r o l
« ... T
i s c o n s ta n t d u rin g t h e e x p e rim e n ta l p e rio d s o f 8 and 16
d a y s .
S u b s t i t u t i o n o f a v e ra g e v a l u e s f o r th e t o t a l cho
l e s t e r o l c o n te n t a t 8 and 16 days in th e c a s e o f th e norm al
and c h o l e s t e r o l - f e d an im als y ie ld s K v a lu e s f o r th e norm al
an im al o f 1.76 a t 8 d a y s , and 2 .1 3 a t 16 d a y s , w hereas th e
v a lu e s f o r th e c h o l e s t e r o l - f e d an im a ls a t 8 and 16 days a re
5 The a u th o r i s in d e b te d to D r. J . Mehl f o r th e
d e r iv a tio n o f th e fo rm u la .
j l . 1 3 and 0*83 r e s p e c tiv e ly *
I
i Prom th e s e d a t a , i t a p p e ars t h a t th e r a t e o f f o r -
i
jm ation and d e s tr u c tio n a r e d e c re a se d by c h o l e s t e r o l f e e d -
i
jin g t o a p p ro x im a te ly 50 p e r c e n t. Yet the amount o f new ly
form ed c h o l e s t e r o l a t th e end of 8 and 16 days i s th e same
I
in b o th g ro u p s • 1
These r e s u l t s a r e in good agreem ent w ith th e j
f in d in g s o b ta in e d by L arack and A l f i n - S l a t e r (66) in
s i m i l a r ex p erim en ts w ith r a b b i t s given d e u te riu m o x id e .
However, th e s e r e s u l t s a r e o n ly in p a r t i a l agreem ent w ith 1
th o se o b ta in e d by Gould and h i s a s s o c i a t e s (65) in in v i t r o
ex p e rim en ts w ith d o g s. These l a t t e r w o rk ers used th e amountj
o f in c o r p o r a tio n of C^-4 from la b e le d a c e t a t e in to c h o l e s te r -
i
i
o l o f l i v e r as an in d i c a t i o n of th e s y n th e s is o f c h o l e s t e r - !
o l . T h e ir r e s u l t s showed t h a t le s s th a n one p e r c e n t of
th e amount o f s y n th e s is o c c u rre d in th e l i v e r s l i c e s ob
ta in e d from a n im als fe d c h o l e s t e r o l a s compared w ith th e
l i v e r 3 l i c e s of th e c o n t r o l a n im a ls . In s i m i l a r experim ents
w ith r a t s , in v i t r o . Tomkins el; a l . (67) o b ta in e d r e s u l t s
;com parable w ith th o s e of Gould and a s s o c i a t e s (6 5 ). '
i i
j S in ce th e r a t e o f fo rm a tio n and d e s tr u c tio n in th e
ex p erim en t on c h o l e s t e r o l fe e d in g u sin g d e u te riu m o x id e ,
1 i s n o t d e c re a se d as m arkedly as o b ta in e d by o th e r i n v e s t i -
i j
g a to r s u sin g C14 a c e t a t e (65, 67) p o s s ib le e x p la n a tio n s
37
f o r th e d if f e r e n c e in a c tio n betw een a c e t a t e and d e u te riu m
a r e g iv e n below .
I t could be p o s tu la te d t h a t a c e t a t e i s an end
p ro d u c t of c h o l e s t e r o l m etab o lism as w e ll as a p r e c u r s o r
f o r c h o l e s t e r o l s y n t h e s i s . G ra n tin g t h i s p o s t u l a t e , i t i s
v e ry p o s s ib le t h a t th e c h o l e s t e r o l and a c e t a t e a r e in r e
v e r s i b l e e q u ilib r iu m and t h a t when th e l i v e r c h o l e s t e r o l
i s in c re a s e d th e amount o f a c e t a t e p r e s e n t would be i n
c re a se d a s w e ll . T his in c r e a s e in th e c o n c e n tr a tio n o f
a c e t a t e m ight th en ac co u n t f o r th e la r g e d e c re a se in cho
l e s t e r o l s y n t h e s i s , a s m easured by th e G ^-4 c h o l e s t e r o l
i s o l a t e d , r e p o r te d by Gould e t aJL. (65) and Tomkins e t a l .
(6 7 ), s in c e th e r e w ould be a d i l u t i o n o f th e la b e le d acetete
added by w hich th e s y n th e s is o f c h o l e s t e r o l p ro c e e d s . How
e v e r , t h i s th e o ry seems u n te n a b le s in c e a c e t a t e i s a l s o a
p r e c u r s o r of o th e r l i p i d s ’ such a s f a t t y a c i d s , and in th e s e
e x p e rim en ts o f Gould e t a l . (65) and Tomkins e t a l . (6 7 ),
th e r a t e o f f a t t y a c id s y n th e s is from a c e t a t e was n o t
a f f e c t e d by c h o l e s t e r o l fe e d in g a s would be ex p e cte d i f
th e a c e t a t e p o o l w ere .d ilu t e d .
An a l t e r n a t e e x p la n a tio n could be t h a t th e in c o rp o
r a t i o n o f d e u te riu m from d e u te riu m oxide p ro cee d s by a
mechanism d i f f e r e n t from t h a t o f th e a c e t a t e r e a c t i o n . The
mechanism by w hich d e u te riu m oxide i s in c o rp o ra te d does n o t
38
seem t o be a p p r e c ia b ly a f f e c t e d by th e ac cu m u latio n of
e x c e ss c h o l e s t e r o l in th e l i v e r , w hereas th e a c e t a t e
mechanism i s a p p r e c ia b ly d e c re a s e d .
The e f f e c t of th e s e v a rio u s d i e t a r y regim ens on th e
amount o f new ly formed c h o l e s t e r o l in th e lung and k id n e y
o f r a t s p r e v io u s ly fe d c h o l e s t e r o l o r th e norm al d i e t can
be se e n in T ab le s I I I and IV r e s p e c tiv e ly *
T A B L E III
Effect of H igh -F at a n d Cholesterol Diets o n the N e w ly F o r m e d Cholesterol P resent in the L u n g s of R a ts
Previously F e d N o r m a l a n d C holesterol Diets
Diet l)uration
of
experiment
Weight
of
lungs
Deuterium
in
body fluid
Total cholesterol in lungs
Previous Experimental per gm.
r
total
Deuterium
concentration
Newly
formed
clays
ffi*
atom % m g. mg. atom %
« 1 1 1
m g.^2
8
0.77 1.61 5.3 U.l 0.357 lili.li 1.82
Normal Normal
16 0.81
1.51 5.1i h.k 0.166 60.1 - 2.66
8
0.73 1.56 ii.8
3.5 0.312 llO .O l.llO
Normal High-Fat
16 0.68 1.66
lull
3.0 0.180 57.8 1.73
0 0.77
.
5.2 3.7
urn - II ---- -
Cholesterol Normal 8 0.79 I.I1 6 5.3 lu?
0.132 18.0 0.76
16 O.6L 1 1.60 k .l 3.0 0.282 35.2 1.06
0 0.71
- -
5.2 3.7
, m rr-i. ---
Cholesterol High-Fat 8 0.67 l.i» 8
5.1i
3.6 0.150 20.2
0.73
16 0.71 1.67 5.5 3.9 0.29k 35.2 1.37
0 0.71 5.2 3.7
,------ — -
Cholesterol Cholesterol 8 0.71 l.UU 5.2 3.7 o.oho 5.6 0.21
16 0.80
l.5 i
5.6
li.5
0.110 1 1 1 . 6 0.82
/ l See foot-note for corresponding column, Table II.
/2 See foot-note for corresponding column, Table II.
T A B L E I V
Effect of H igh-F at a n d Cholesterol Diets o n the N e w ly F o r m e d Cholesterol Present in the K id n ey s of
R ats Previously F e d N o r m a l a n d Cholesterol Diets
Diet duration Weight Deuterium
T
total cholesterol in kidneys
Previous
Experimental
of
experiment
of
kidneys
in
body fluid per gm . total
Deuterium I Newly
concentration formed
days
£ “•
atom %
mg. m g. atom % % A m g.^2
Normal Normal
8
16
1.10
1.22
1.61
1.51
i*.2
l*.l
ii.6
5.0
0.222
0.285
27.6
37.8
1.27
1.89
Normal High-Fat
8
16
1.08
1.06
1.56
1.66
3.6
3.6
3.9
3.8
0.237
0.360
30.lt
1 * 3 .1 *
1.19
1.65
Cholesterol Normal
0
8
16
1.10
1.18
1. 2U
1.16
1.60
l*.l
1*.2
1*.9
1.5
5.0
6.1
0.099
0.201
13.6
25.2
0.68
1.51*
Cholesterol High-Fat
0
8
1A
A W
1.10
1. 1 1 *
1.30
1.18
1.67
l*.l
1 1 .2
1 1 .3
1*.5
li.8
5.6
0.088
0.156
11.8
18.6
0.56
l.ou
Cholesterol Cholesterol
0
8
16
1.10
1.10
1.12
1. 1 * 1 *
1.51
l*.l
Ji.l
1 » .3
1 *.5
li.5
U .8
0.063
0.137
8.8
18.2
0.1 * 0
0.87
/ l See foot-note for corresponding column, Table II.
/2 See foot-note for corresponding column, Table II.
41
There ap p e a rs to be no s i g n i f i c a n t change in th e
t o t a l c h o l e s t e r o l c o n c e n tr a tio n in th e lung and k id n e y
o f r a t s . There does a p p e a r to b e , how ever, a n o tic e a b le
d e c re a s e in th e amount o f new ly form ed c h o l e s t e r o l in a l l
c a s e s in w hich th e an im als a r e f e d c h o l e s t e r o l p r e v io u s ly ,
and t h i s d e c re a s e i s more marked in th o s e anim als w hich
a r e c o n tin u e d on th e c h o l e s t e r o l d i e t f o r 8 and 16 d a y s.
The h i g h - f a t d i e t h a s no e f f e c t on t h e amount o f new ly
form ed c h o l e s t e r o l in th e lu n g and k id n e y of th e an im als
p r e f e d a norm al d i e t .
E f f e c t of h i g h - f a t d i e t on the amount o f new ly
form ed c h o l e s t e r o l i n th e plasm a o f an im als p r e v io u s ly
fe d c h o l e s t e r o l and norm al d i e t s i s shown in T able V.
T A B L E V
Effect of H igh-F at Diet o n the L ab eled Cholesterol Present in the P la sm a of R ats Previously F e d N o r m a l
a n d Cholesterol Diets
Diet Duration
of
experiment
Deuterium
m
body fluid
Total cholesterol in plasma
Previous Experimental per 100 ml.
Deuterium
concentration labeled 100 ini.
days atom %
El*
atom %
1 1 1 2£‘ 11
Normal Normal
8
16
1.61
1.51
69
5 1 *
0.1 * 1
0.1*7
50.9
62.3
35.1
33.6
Normal High-Fat
8
16
1.56
1.66
62
5 1 *
0.1 * 1 *
0.60
56.1*
72.3
35.0
39.5
Cholesterol Normal
8
16
1. 1 * 6
1.60
68
51
0.10
0.26
13.7
32.5
9.3
16.6
Cholesterol High-Fat
8
16
1.1 * 8
1.67
70
1 * 2
0.11
0.1,5
1 1 *.?
53.9
10.1 *
22.6
/ I See foot-note for corresponding column, Table I I .
/2 See foot-note for corresponding column, Table II .
ro
- 43
As can be seen in T able V, th e amount o f new ly
form ed c h o l e s t e r o l a p p e a rin g in th e plasm a o f norm al a n i
mals fe d a h i g h - f a t d i e t i s t h e same as in th e plasm a o f
th e c o n t r o l a n im a ls . However, th e q u a n t i t y o f new ly formed
c h o l e s t e r o l in th e plasm a o f r a t s p r e v io u s ly fe d a cho
l e s t e r o l d i e t and th e n p la c e d on a norm al o r h i g h - f a t d i e t
i s m arkedly l e s s th a n th e c o n t r o l s .
A p o s s ib le e x p la n a tio n f o r th e d e c re a s e in th e t o t a l
amount o f new ly formed c h o l e s t e r o l found in th e lung and
k id n e y o f th e a n im a ls .p r e fe d a c h o l e s t e r o l d i e t may be
g iv e n a s f o llo w s : A lthough th e amount o f new ly formed
c h o l e s t e r o l in th e l i v e r s o f r a t s u n d er th e s e d i e t a r y
regim ens i s com parable to th e c o n tr o l v a lu e s , th e d e u te riu m
c o n te n t o f t h i s c h o l e s t e r o l i s low (see T able I I ) * As
suming t h a t th e main so u rc e o f th e plasma c h o l e s t e r o l i s
th e l i v e r , i t may be su g g e ste d t h a t th e low er d e u te riu m
c o n te n t o f th e plasm a c h o l e s t e r o l (see T able V) o f r a t s
p r e fe d c h o l e s t e r o l , i s due t o th e f a c t t h a t th e c h o l e s t e r o l
coming from th e l i v e r i n t o th e plasma c o n ta in s a h ig h e r
r a t i o o f u n la b e le d c h o l e s t e r o l to la b e le d c h o l e s t e r o l due
to th e la r g e ex cess of d i e t a r y c h o l e s t e r o l s to r e d in th e
l i v e r d u rin g c h o l e s t e r o l f e e d in g . As th e s to r e d cho
l e s t e r o l in th e l i v e r i s d e c re a s e d by th e norm al o r h ig h - .
f a t d i e t , th e r a t i o o f la b e le d to u n la b e le d c h o l e s t e r o l
I
44 ]
in c r e a s e s (see T able I I ) . T his c h o l e s t e r o l i s th e n d i s
charged in t o th e plasm a and i s su b s e q u e n tly m easured t h e r e .
I f th e d i l u t e d la b e le d c h o l e s t e r o l a s compared t o th e
c o n t r o l (see T able V ), i s tr a n s p o r te d to th e k id n e y and
lung* th e d e c re a se in th e new ly formed c h o l e s t e r o l can th u s
1
be e x p la in e d .
2 . D i s t r i b u t i o n of C h o le s te r o l w ith in th e L iv e r C e ll
Wihen th e l i v e r s of norm al r a t s a r e s u b je c te d to
d i f f e r e n t i a l c e n t r i f u g a t i o n , 65 p e rc e n t o f th e f r e e cho
l e s t e r o l o f th e l i v e r i s fo u n d in th e su b m ic ro sc o p ic
p a r t i c l e s (see T able V I). The rem ain d er of th e f r e e cho
l e s t e r o l i s f a i r l y e v e n ly d i s t r i b u t e d among th e o th e r
f r a c tio n s *
T A B L E VI
D istribution of Free, E sterified and Total Cholesterol of
U ltracentrifugal Fractions of Normal Rat Liver
Description of fraction
Average % of to ta l recovered / l
Free E sterified Total
cholesterol cholesterol cholesterol
Floating 3.6
( 2.U - h.7)
62.2
(51.6 - 79.7)
13.3
( 9.2 - 15.7)
Nuclei 6.9
( h»U - 11.5)
1.5
( 0.0 - h.9)
6.0
( 3.7 - 9.3)
Mitochondria
13.5
( 6.9 - 15.8)
3.1
( 0.0 - 12.8)
13.7
( 5.6 - 15.3)
Sutmicroscopic
p articles
65.8
(58.6 - 75.2)
19.6
( 8.6 - 30.3)
58.2
(50.1* - bk.9)
Supernatant - 1 ii.6
( 1.7 - 7.2)
6.5
( 1.1 - 9.6)
U.9
( 3.0 - 6.1)
Supernatant - 2 6.6
( 3.0 - 9.5)
8.6
( h .l - 11.9)
7.1
( h.3 - 9.3)
/ l Values are averages of analyses on liv ers of 6 ra ts . Figures in
parenthesis represent range of values.
4 6
The d i s t r i b u t i o n o f e s t e r i f i e d c h o l e s t e r o l , how ever,
i s q u i t e d i f f e r e n t ; 60 p e r c e n t i s lo c a te d in th e uFn
l a y e r , 2 0 p e r c e n t in th e su b m icro sco p ic p a r t i c l e s , 15 p e r
c e n t in th e s u p e r n a ta n t, and o n ly m inute amounts a r e found
in th e n u c le a r and m ito c h o n d ria l f r a c t i o n s (se e T able V I).
The r a t i o o f f r e e to t o t a l c h o l e s t e r o l , e x p re sse d
as p e r c e n t f r e e c h o l e s t e r o l , in each o f th e u l t r a c e n
t r i f u g a l f r a c t i o n s o f norm al r a t l i v e r i s shown in T able
V II.
Ii7
TABLE V n
The Percent of Free Cholesterol in Total Cholesterol of Fractions
from Normal Rat Liver
Description of fraction % of to ta l cholesterol in each fraction
in free form
Floating 22.8 (17.7 - 26. 0)
Nuclei 95.7 (85.3 - 100 )
Mitochondria 96.6 (85.3 - 100 )
Subnicroscopic p articles 914.2 (91.1 - 97.6)
Supernatant - 1 75.2 ; U5.1 - 96.9)
Supernatant - 2 76.2 (55.6 - 89.9)
Unfractionated homogenate
•»
81.9 (78.7 - 83.5)
/ l Values are averages of analyses on livers of 6 rats* Figures in
parenthesis represent range of*values*
48
I t can be seen from T able V II t h a t th e p e rc e n ta g e of
f r e e c h o l e s te r o l in th e u n f r a c tio n a te d hom ogenates of
norm al l i v e r s averaged 82 p e r c e n t and shows l i t t l e v a r i
a t io n among th e s ix a n im a ls . At l e a s t 95 p e r c e n t o f cho
l e s t e r o l in th e n u c le a r , m ito c h o n d ria l and su bm icroscopic
p a r t i c u l a t e f r a c t i o n s , and 75 p e r c e n t o f th e c h o l e s t e r o l
in th e s u p e rn a ta n t f r a c t i o n s i s in th e f r e e s t a t e . The
f l o a t i n g la y e r i s th e o n ly component in which th e cho
l e s t e r o l i s found to e x i s t p re d o m in a n tly in th e e s t e r i f i e d
form .
The sum o f th e f r e e and t o t a l c h o l e s t e r o l v a lu e s in
th e s e s ix f r a c t i o n s amounts to 87 - 99 p e r c e n t o f th e f r e e
and t o t a l c h o l e s t e r o l o f th e u n f r a c tio n a te d hom ogenate.
However, th e re c o v e ry o f e s t e r i f i e d c h o l e s te r o l which is
c a lc u la te d from th e d if f e r e n c e between f r e e and t o t a l cho
l e s t e r o l v a r ie s m ore, due to th e s l i g h t i n e q u a l i t i e s in th e
re c o v e ry of f r e e and t o t a l c h o l e s t e r o l .
When em ploying th e te c h n iq u e o f d i f f e r e n t i a l cen
t r i f u g a t i o n to d eterm in e th e i n t r a c e l l u l a r d i s t r i b u t i o n of
a s u b s ta n c e , th e p o s s i b i l i t y o f a d s o rp tio n o f t h i s substance
on th e c e l l u l a r p a r t i c u l a t e s must be c o n s id e re d . S c h n e id e r
and Hogeboom (84) re p o r te d " . . . i f a la r g e p e rc e n ta g e o f th e
su b sta n c e p r e s e n t in th e whole t i s s u e can be shown to be
lo c a l i z e d in a s in g le f r a c t i o n , th e n th e p r o b a b i l i t y o f
49
a d s o rp tio n would a p p e a r to be m inim al. . . . . . . On th e
o th e r h a n d , th e f in d in g o f a sm a ll p e r c e n ta g e o f a
su b sta n c e in a f r a c t i o n must be s e r i o u s l y c o n s id e re d as an
a d s o rp tio n phenomenon." A pplying th e s e c o n s id e r a tio n s to
th e r e s u l t s r e p o r te d h e r e , i t i s e v id e n t t h a t most o f th e
f r e e c h o l e s t e r o l i s c o n c e n tra te d in th e su b m icro sco p ic
p a r t i c l e s , w h ile th e m a jo r ity o f th e e s t e r i f i e d c h o l e s t e r o l
i s lo c a te d in th e f l o a t i n g l a y e r . However, in view o f th e
d e m o n stra tio n o f c h o l e s t e r o l in more h ig h ly p u r i f i e d p re p a
r a t i o n s o f m ito c h o n d ria (57, 85, 86) and n u c l e i (5 8 ), i t
a p p e a rs u n lik e ly t h a t -the s m a lle r amounts o f c h o l e s t e r o l
found in th e s e f r a c t i o n s in - t h e p r e s e n t work a r e e n t i r e l y
due to a d s o r p tio n .
I t i s o f i n t e r e s t to compare th e v a lu e s f o r th e
d i s t r i b u t i o n of f r e e and e s t e r i f i e d c h o l e s t e r o l in norm al
r a t l i v e r r e p o r te d h e re w ith th o s e o b ta in e d by Chauveau
and co-w orkers (5 8 ). These w orkers a l s o employed a p r e
lim in a ry c e n t r i f u g a t i o n to remove " f r e e f a t " '("F" la y e r )
p r i o r to f r a c t i o n a t i o n o f th e c e l l u l a r p a r t i c u l a t e s . How
e v e r , th e y re c o g n iz e d t h a t t h e i r i n v e s t i g a t i o n was lim ite d
by th e use o f a c e n tr if u g e which p ro v id e d a maximal cen
t r i f u g a l f o r c e o f 5 0 ,0 0 0 x g . The f a c t t h a t t h i s f o r c e was
i n s u f f i c i e n t to b r in g ab o u t se d im e n ta tio n o f a l l th e cho
l e s t e r o l - c o n t a i n i n g p a r t i c l e s in a 0 .8 8 M su c ro se medium i s
50
b orne o u t by a com parison o f Chauveau e t a l . (58) in t h e i r
f in d in g t h a t 55 p e r c e n t of th e f r e e c h o l e s t e r o l i s found
in th e s u p e rn a ta n t f r a c t i o n , and 25 p e r c e n t i s p r e s e n t in
th e m icrosom al f r a c t i o n w ith th e p r e s e n t r e p o r t of 12 p e r
c e n t in th e s u p e rn a ta n t f r a c t i o n and alm o st 60 p e r c e n t
in th e m icrosom al f r a c t i o n . Chauveau e t a l . (58) a l s o
found a h ig h p e rc e n ta g e (80 p e r c e n t) o f th e e s t e r i f i e d
c h o l e s t e r o l in th e " f r e e f a t " f r a c t i o n . T hus, th e r e s u l t s
o f th e two i n v e s t i g a t i o n s a r e in good a g re e m e n t, i f th e
assu m p tio n i s made t h a t Chauveau and a s s o c i a t e s (58) would
have found a g r e a t e r p e rc e n ta g e o f f r e e c h o l e s t e r o l in th e
m icrosom al f r a c t i o n had th e y a c h ie v e d a more com plete s e p a
r a t i o n o f th e m lcrosom es from th e s u p e rn a ta n t f r a c t i o n .
3 . C h o le s te r o l E s te r a s e in Rat L iv e r Homogenate '
(1) D em o n stratio n o f C h o le s te r o l E s te r a s e A c t i v i t y .
The f a c t t h a t th e r a t i o o f f r e e t o t o t a l c h o l e s te r o l
in r a t l i v e r i s rem ark ab ly c o n s ta n t (see T able V II) le d
t o an i n v e s t i g a t i o n of th e p o s s i b i l i t y o f th e p re se n c e o f
an enzyme o r enzyme system s in th e l i v e r ca p ab le of m ain
t a i n i n g t h i s c o n s ta n t r a t i o .
In an a tte m p t to d e m o n stra te th e p re se n c e In th e
l i v e r o f a c h o l e s t e r o l e s t e r a s e , th e a s s a y c o n d itio n s d e
s c r ib e d p r e v io u s ly were em ployed. These c o n d itio n s a r e
a n a la g ous to th o s e used t o d e m o n stra te a v ita m in A e s te r a s e
(8 7 ). The r e s u l t s o f t h i s ex p erim en t a r e seen in Table
V I I I .
52
TA BLE VIII
Hydrolysis o f C holesterol A cetate by Rat Liver Homogenate
Exp.
#
f ix tu re / I C holesterol values %
Hydrolysis /2
C holesterol
Homogenate a c e ta te /3
Tween
20
Control
Free 'to ta l
Incubate
Free t o ta l
mg. mg. mg. rag. mg.
+ 0 .9 6 4 1.22 2.30 1.87 2.23 67.7
1 +
— - - 1.21
1.3b 1.17 1.35
—— .
4 4 1.21 1.31* 1.26
1.35 5.2
4 1.30 4 1.16 2.50 2.31 2.55 88.5
2 4
------
4A
1.19 1.27 1.16 1.25
— —
1.30 4 0 .0 0 1.30 0 .0 0 1.30 0 .0
/ l Each fla s k contained 2 ml* homogenate, 2 m l. c h o le stero l ac eta te so lu tio n
and 6 ml. vero n al-acetate b u ffe r, pH 8 .5 . Incubation time 1 * hours a t 37°C.
/2 % hydrolysi3= ...P R .t.., S °p tro l (free, c h o l j 100
L— mg* ch o le stero l a c eta te
C alculation based on assumption th a t the increase in mg. o f fre e ch o lester
o l was due to hydrolysis of only the added c h o lestero l a c e ta te , since no
hydrolysis o f the endogenous e s te rs occurs where the homogenate i s incubated
alone*
1 1 C holesterol a c eta te was dissolved in 0.855? sodium chloride w ith the a id of
Tween 20*
/It S aline so lu tio n containing same amount of Tween 20 as c h o le stero l a c eta te
so lu tio n was added to incubation mixture*
53
I t can be seen t h a t when th e homogenate i s in c u b a te d
a lo n e th e r e i s no change in f r e e o r t o t a l c h o l e s t e r o l
c o n c e n tr a tio n ; i t i s e v id e n t t h a t th e homogenate i s c a p a b le
o f h y d ro ly z in g 80 p e r c e n t o f th e added c h o l e s t e r o l
a c e t a t e . In o r d e r to i n v e s t i g a t e t h i s a c t i v i t y f u r t h e r and
t o s e t up more o p tim a l c o n d itio n s f o r th e h y d r o ly tic a c
t i v i t y , th e fo llo w in g ex p e rim en ts w ere p erfo rm e d .
The e f f e c t o f v a ry in g hydrogen io n c o n c e n tr a tio n on
th e c h o l e s t e r o l e s te r a s e a c t i v i t y o f r a t l i v e r homogenate
i s shown in F ig u re 2 . In F ig u re 3 th e e f f e c t of v a ry in g
th e In c u b a tio n tim e on th e c h o l e s t e r o l e s t e r a s e a c t i v i t y
o f r a t l i v e r homogenate I s p l o t t e d .
o 30-
20 -
z
pH
FIGURE 2
THE EFFECT OF CHANGES OF HYDROGEN ION CONCENTRATION
ON CHOLESTEROL ESTERASE ACTIVITY OF RAT LIVER
HOMOGENATE
PERCENT HYDROLYSIS
55
801
70-
6 0 -
THE EFFECT OF VARYING TIME
INTERVALS ON CHOLESTEROL
ESTERASE ACTIVITY OF RAT
L i v e r h o m o g e n a t e
50-
4 0 -
30-
20 -
10 -
30 60 90 120 150 180
TIME(MIN)
FIGURE 3
56
The pH v a lu e s t h a t a r e g iv e n in F ig u re 2 were d e
te rm in e d on th e t o t a l c o n te n ts o f th e in c u b a tio n f l a s k a t
th e end o f th e in c u b a tio n p e r io d . Under th e s e c o n d itio n s
th e maximum a c t i v i t y o f t h i s r e a c tio n i s found to be a t
a b o u t pH o f 8 , However, th e r e i s a c o n s id e ra b le amount o f
a c t i v i t y on e i t h e r s id e of th e pH optimum.
I t i s e v id e n t from F ig u re 3 t h a t th e r e i s a v e ry
r a p id h y d r o ly s is o f th e added c h o l e s t e r o l a c e t a t e w ith in
th e f i r s t 30 m in u te s , w hich becomes more g r a d u a l t h e r e
a f t e r .
The e f f e c t of b o i l i n g th e homogenate p r i o r to in c u
b a tio n i s shown in T able IX.
57
TABLE IX
E ffect of Boiling on the A b ility of Rat Liver Homogenate
to Hydrolyze C holesterol Acetate
C holesterol values H
%
Hydrolysis /2
Treatment Control Incubate
Free| (Total Free ] (Kotal
mg. mg. mg. mg.
1.16 2.50
2.31 2.55 93.5
Boiled /3 1.12 2.1*5 1.11 2.50
--------
l l Incubation mixtures contained 2 ml. homogenate, 3 ml*
cholesterol acetate (0.143 mg./ml.) and 7 ml. veronal-
acetate b u ffer, pH 5 .5. Samples incubated 3 hours a t
37°C.
/2 See foot-note fo r corresponding column, Table V III.
U ?»e homogenate was heated in a b o iling water bath fo r
1 minute.
58
These d ata in T able IX i n d ic a t e t h a t th e c h o l e s t e r o l
e s t e r a s e a c t i v i t y o f th e r a t l i v e r homogenate i s c o m p le te ly
d e s tro y e d upon b o i l i n g f o r one m in u te .
The p o s s i b i l i t y t h a t th e Tween 20 used to d is s o lv e
th e c h o l e s t e r o l a c e t a t e , b e fo re i t s a d d i tio n to th e homo-
g e n a te , m ight have a h y d r o ly tic e f f e c t on th e e s t e r , was
r u le d out by th e fo llo w in g e x p e rim e n t: Tween 20 was added
to a homogenate w ith no added c h o l e s t e r o l a c e ta te and in c u
b a te d . The Tween 20 d id n o t have an e f f e c t on th e hy
d r o l y s i s o f th e endogenous e s t e r s o f c h o l e s t e r o l as shown
in T able V I I I . I t i s a l s o shown in T able V III t h a t when
c h o l e s t e r o l a c e t a t e a lo n e I s in c u b a te d in b u f f e r th e r e I s
no h y d r o ly tic a c t i v i t y .
These r e s u l t s in d ic a t e t h a t th e r e i s an enzyme
p r e s e n t in r a t l i v e r homogenate which can h y d ro ly ze cho
l e s t e r o l a c e ta te to f r e e c h o l e s t e r o l and presum ably t o
a c e t i c a c i d . I t i s p o s s ib le t h a t t h i s enzyme i s concerned
w ith th e fo rm a tio n o f f r e e c h o l e s t e r o l from th e c h o l e s t e r o l
e s t e r s p r e s e n t in th e l i v e r .
As th e t o t a l c o n c e n tra tio n o f c h o l e s t e r o l rem ains
c o n s ta n t d u rin g th e in c u b a tio n p e r io d , i t may be assumed
t h a t th e c h o l e s t e r o l o b ta in e d from th e h y d r o ly s is o f th e
added c h o l e s t e r o l a c e t a t e i s n o t f u r t h e r m e ta b o liz e d .
At t h i s p o in t i t i s r a t h e r i n t e r e s t i n g t o s p e c u la te
59
on th e re a s o n s why t h e homogenate w ith no added s u b s t r a t e
can n o t h y d ro ly z e th e c h o l e s t e r o l e s t e r s a lr e a d y p r e s e n t in
th e hom ogenate. The fo llo w in g e x p la n a tio n s a r e s u g g e s te d .
1) The s u b s t r a t e and enzyme a r e in e q u ilib r iu m , and
by m easuring t o t a l changes one can d e t e c t no h y d r o ly s is .
2) The enzyme i s n o t s p e c i f i c f o r th e s e e s t e r s .
3) The e s t e r s a r e in a p h y s ic a l s t a t e su ch a s t h a t
th e enzyme i s p re v e n te d from r e a c t i n g w ith i t .
(2) D i s t r i b u t i o n o f C h o le s te r o l E s t e r a s e .
I t now seemed d e s i r a b l e to a tte m p t to c o r r e l a t e th e
enzyme a c t i v i t y . w i t h a p a r t i c u l a r u l t r a c e n t r i f u g a l f r a c t i o n
o r f r a c t i o n s o f r a t l i v e r hom ogenate. T h e re f o re , a s e p a
r a t i o n o f th e p a r t i c u l a t e m a tte r o f l i v e r was ach iev ed
a c c o rd in g t o th e scheme p re s e n te d in F ig u re 1 . Each s e p a
r a t e f r a c t i o n , e x c lu d in g th e "F" l a y e r , was th e n in c u b a te d
w ith added c h o l e s t e r o l a c e t a t e s u b s t r a t e a t th e o p tim a l
c o n d itio n s d e te rm in e d .
T able X shows th e d i s t r i b u t i o n o f th e c h o l e s t e r o l
e s t e r a s e in th e d i f f e r e n t u l t r a c e n t r i f u g a l f r a c t i o n s o f
r a t l i v e r hom ogenate.
TABLE X
D istrib u tio n of C holesterol E sterase in Various U ltra c e n trifu g a l
Fractions of Rat Liver Homogenate
C holesterol values
1 1
Fraction Control Incubate Hydrolysis /2 D istrib u tio n
of
a c tiv ity /3
Free Total Free Total
mg. mg.
mg.
mg.
I t
U nfractionated
homogenate 0.45 1.75 1.19 1.74 66.1 100.0
Nuclei 0.00 1.23 0.11 1.24 9.8 14.8
Mitochondria 0.15 1.17 0.13 1.24 0.0 0.0
Submicroscopic
p a rtic le s 0.32 1.45 1.15 1.51 74.1 112.2
Supernatant 0.03 1.23 0.06 1.16 2.7 4.1
/ l Each fla s k contained 2 ml. homogenate or the equivalent amount o f each
fr a c tio n , 2 ml. ch o lestero l acetate solution (1.12 mg*) and 8 ml. of
veronal b u ffe r, pH 8 .5 . Incubation time 2 hours a t 36°C.
/2 See foot-note for corresponding column, Table V III.
l l Based on the assumption th a t the unfractionated homogenate has 100#
a c tiv ity .
61
I t is e v id e n t from th e s e d a ta t h a t th e a c t i v i t y o f
t h i s enzyme i s lo c a te d a lm o st e x c lu s iv e ly In th e m icrosom al
f r a c t i o n . The sm a ll amount of a c t i v i t y t h a t I s found in
th e n u c l e i i s p ro b a b ly due to th e f a c t t h a t a few whole
c e l l s were p r e s e n t in t h i s f r a c t i o n , w hereas th e sm a ll
amount o f enzyme a c t i v i t y found in th e m ito c h o n d r ia l
f r a c t i o n was q u i t e p o s s ib ly due to a d s o r p ti o n , w hich has
p r e v io u s ly been d is c u s s e d .
IV. SUM M ARY AND CONCLUSIONS
1* The amount o f new ly form ed c h o l e s t e r o l in th e
l i v e r , k id n e y , lu n g , and plasm a u n d er v a rio u s e x p e rim e n ta l
d i e t a r y c o n d itio n s h as been d e te rm in e d by th e m easurem ent
o f th e u p tak e o f d e u te riu m i n t o c h o l e s t e r o l a t t h e end of
d e f i n i t e tim e i n t e r v a l s .
2 . I t h as been found t h a t th e amount o f new ly
form ed c h o l e s t e r o l p r e s e n t in t h e above t i s s u e s o f r a t s
p r e f e d a norm al d i e t i s unchanged when th e an im als a re
p la c e d on a h i g h - f a t d ie t*
3 . The amount o f new ly form ed c h o l e s t e r o l in th e
l i v e r s o f an im a ls p re fe d c h o l e s t e r o l d i e t i s unchanged when
th e r a t s a re p la c e d on th e h i g h - f a t d i e t and is- com parable
t o th e a n im a ls p re fe d a norm al d i e t .
4 . A h i g h - f a t d i e t ap p e a re d t o be more e f f i c i e n t
th a n a norm al d i e t in rem oving th e c h o l e s t e r o l w hich h a s
accu m u lated in th e l i v e r d u rin g th e p re e x p e rira e n ta l cho
l e s t e r o l f e e d in g .
5 . The amount o f new ly formed c h o l e s t e r o l in th e
k id n e y and lung of r a t s p re fe d c h o l e s t e r o l i s d ec re ase d '.
6 . The new ly form ed c h o l e s t e r o l a p p e a rin g in th e
plasm a of r a t s p re f e d a c h o l e s t e r o l d i e t i s much low er th a n
t h a t found i n th e c o n tr o l a n im a ls .
7 . I t i s p o s tu la te d t h a t d e u te riu m oxide and
a c e t a t e a r e I n c o rp o ra te d in th e b io s y n t h e s is o f th e cho-
63
l e s t e r o l m olecule by d i f f e r e n t m echanism s.
8 . A method h as been d e s c rib e d f o r th e s e p a r a tio n
of l i v e r f r a c t i o n s by u l t r a c e n t r i f u g a l m eans.
9 . A q u a n t i t a t i v e d i s t r i b u t i o n o f f r e e and e s t e r i
f i e d c h o l e s t e r o l h a s been d e te rm in e d in v a r io u s u l t r a -
c e n t r i f u g a l f r a c t i o n s o f l i v e r horaogenates o f norm al r a t s .
I t h as been found t h a t over 60 p e r c e n t o f th e f r e e cho
l e s t e r o l was lo c a te d in th e m icrosom al f r a c t i o n s , w h ile a
m a jo r ity o f th e e s t e r i f i e d c h o l e s t e r o l was a s s o c ia te d w ith
a c e n t r l p e t a l l y - m i g r a t i n g f r a c t i o n .
10. C h o le s te r o l e s t e r a s e a c t i v i t y h as been demon-
s t r a t e d In r a t l i v e r horaogenates. j
1 1. The e f f e c t o f pH, b o i l i n g and tim e of In c u b a tio n j
i n d i c a t e s t h a t t h i s a c t i v i t y i s en zym atic in n a t u r e .
12. A q u a n t i t a t i v e d i s t r i b u t i o n o f th e c h o l e s t e r o l
e s t e r a s e has been d eterm in ed in v a r io u s u l t r a c e n t r i f u g a l
f r a c t i o n s o f r a t l i v e r hom ogenates. I t h as been found t h a t
th e en zy m atic a c t i v i t y i s e x c lu s iv e ly a s s o c ia te d w ith th e
m icrosom al f r a c t i o n .
BIBLIOGRAPHY
j 1. P o u l l e t i e r , de la S a l l e , c ir c a 1769. C ite d by B i l l s ,
j C .E ., P h y s io l. R e v s., Iff. 2l~ (1 9 3 5 ).
|- 2 . C o n ra d i, B .G .F ., 1775. C ite d by B i l l s . C .E ., P h y s io l.
R e v s ., 15, 21 (1 9 3 5 ).
3 . C h e v re u l, M .B., Ann, chim . e t p h y s . . 1., 95, 5 (1 8 1 5 ).
4 . B e r t h e l o t , M., Ann. chim . e t phy3. . 3 , 56, 51 (1 8 5 9 ).
5 . C ouerbe, J . P . , Ann, chim . e t p h y s . . 2 , 5 6 , 160 (1 8 3 4 ).
6 . Lecanu, L .R ., Ann, chim . e t phy3. . 2 , 67, 54 (1 8 3 8 ).
7 . Lehmann, C .G ., Lehrb-uch d e r p h y s io lo g is c h e n Chemie.
L e ip z ig , 2nd e d i t i o n , 1 , 282 (1 8 5 0 ;. C ite d by
B i l l s , C .E ., P h y s io l. R e v s ., 1 5 . 21 (1935).
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Asset Metadata

Creator Schotz, Michael C. (author)

Core Title Studies on cholesterol metabolism in the rat

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1953-08

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

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-602162

Unique identifier UC11354884

Identifier DP21556.pdf (filename),usctheses-c17-602162 (legacy record id)

Legacy Identifier DP21556.pdf

Dmrecord 602162

Document Type Dissertation

Rights Schotz, Michael C.

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...

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