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

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

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Content A STUDY OF THE ALTERATIONS
IN THE AM OUNT OF CHOLESTEROL PRESENT
IN RAT LIVER HOMOGENATES AFTER INCUBATION
A T h e s is
P r e se n te d t o
th e F a c u lty o f th e Departm ent o f B io c h e m is tr y
The U n iv e r s i t y o f Sou thern C a lif o r n ia
In P a r t i a l F u l f il l m e n t
o f th e R equirem ents f o r th e Degree
M aster o f S c ie n c e
by
W illa r d P. Adams
June 1949
UMI Number: EP41301
All rights reserved
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This thesis, written by
WILLARD ) P. ADAMS
under the guidance of h..is.. Faculty Committee,
and approved by all its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fulfill
ment of the requirements for the degree of
MASTER OF SCIENCE
Emory S» Bogardus
Dean
Date............ Augnst 194$
Faculty Committee
Chairman
TABLE OP CONTENTS
CHAPTER. PAGE
I . INTRODUCTION..................................... 1
I I . HISTORICAL REVIEW OP PROBLEM........................................... 3
I I I . M ETHODS.............................................................................................. 21
T reatm ent o f Anim als ......................... 21
P r e p a r a tio n o f L iv e r Homogenates ........................... 21
Treatm ent o f In cu b a ted Samples . . . . . . . 31
E x t r a c t io n o f C h o le s t e r o l .......................................... 32
C h o le s t e r o l D e te r m in a tio n .......................................... 41
IV. STABILITY OP PURE CHOLESTEROL UNDER CERTAIN
CONDITIONS.................................................................................. 45
V. EFFECT OP THE LENGTH OP CHOLESTEROL FEEDING
AND THE SEX OP THE ANIMAL UPON THE DEGREE
OF RECOVERY OP CHOLESTEROL PROM LIVER
HOMOGENATE AFTER INCUBATION..................................... 4.8
VI. RESULTS OP ATTEMPTS TO DEMONSTRATE CHANGES IN
THE AM OUNT OP CHOLESTEROL IN HOMOGENATES
AFTER INCUBATION......................................................... . . 51
V II. DISCUSSION............................................... 61
V I I I . SUMMARY.............................................................................................. 69
IX. BIBLIOGRAPHY .................................................................... 71
LIST OF TABLES
HUM BER PAGE
I . C om p osition o f D ie t . . . . . . . . . . . . . 22
I I . E f f e c t o f V a r ia tio n s in th e Time o f
C e n t r if u g a t io n upon th e H om ogeneity and
C h o le s t e r o l C on ten t o f L iv e r Homogenate . . 26
I I I . E f f e c t o f V a r ia tio n s in th e Time o f
C e n t r if u g a t io n upon the H om ogeneity and
C h o le s t e r o l C on ten t o f L iv e r Homogenate . . 28
IV. E f f e c t o f R eh o m ogen ization a f t e r
C e n t r if u g a t io n upon th e H om ogeneity and
C h o le s t e r o l C on ten t o f L iv e r Homogenate . . 30
V. E f f e c t o f H y d r o ly s is o f Homogenate b e fo r e
E x t r a c t io n o f L ip id s upon th e Amount o f
C h o le s t e r o l R ecovered .................................................... 36
VI. A ttem p ts t o I n c r e a s e th e E x t r a c t a b i l i t y o f
C h o le s t e r o l from L iv e r Homogenates . . . . 38
V II. E f f e c t s o f P o ta ssiu m H y d ro x id e, H y d r o c h lo r ic
A c id , and G la c ia l A c e t ic A cid upon th e
R eco v ery o f Pare C h o l e s t e r o l .................................. 47
V I I I . A ttem pt t o C o r r e la te th e L ength o f
C h o le s t e r o l F e e d in g , th e Sex o f th e Animal
and th e W eight o f th e L iv e r w ith th e Degree
o f R eco v ery o f C h o le s t e r o l a f t e r In c u b a tio n 49
i i
NUMBER PAGE
IX. E f f e c t o f S to r a g e o f L iv e r Homogenate un d er
D i f f e r e n t C o n d itio n s upon th e D egree o f
R eco v ery o f C h o le s t e r o l a f t e r In c u b a tio n . • 52
X. E f f e c t o f A g it a t io n and th e P resen ce o f
Oxygen d u rin g In c u b a tio n o f L iv e r
Homogenate upon th e R eco very o f C h o le s t e r o l 54
X I. E f f e c t o f L ength o f C h o le s t e r o l F eed in g upon
th e D egree o f R ecovery o f C h o le s t e r o l
from L iv e r Homogenate a f t e r I n c u b a tio n . . . 56
X I I. E f f e c t s o f Added C o fa c to r s upon th e R ecovery
o f C h o le s t e r o l from L iv e r Homogenate a f t e r
I n c u b a t i o n ................................................................................... 58
CHAPTER I
INTRODUCTION
The im portance o f c h o l e s t e r o l in th e p h y s io lo g y o f
th e anim al organism i s a t t e s t e d to by th e f a c t t h a t i t i s
fo u n d in a l l c e l l s an d , in d e e d , i s r e g a r d e d as an e s s e n t i a l
s t r u c t u r a l component o f p r o to p la sm . The c l o s e ch em ica l
r e l a t i o n s h i p betw een c h o l e s t e r o l and su ch im p ortan t m etab
o l i t e s as th e s t e r o i d horm ones, v ita m in D, and th e b i l e
a c id s ren d ers an u n d e r sta n d in g o f c h o l e s t e r o l m eta b o lism
r e q u i s i t e to an a p p r e c ia t io n o f c e r t a in norm al body
p r o c e s s e s . F u rth erm ore, In s e v e r a l im p o rta n t d i s e a s e s t a t e s ,
n o t a b ly a t h e r o s c l e r o s i s , th y r o id d i s o r d e r s , d i a b e t e s , and
th e x a n th o m a to se s, p rofou n d a l t e r a t i o n s In th e norm al
m eta b o lism o f c h o l e s t e r o l o c c u r , and i t i s p o s s i b l e th a t
w it h e l u c i d a t i o n o f th e p a t h o l o g ic a l p h y s io lo g y in v o lv e d ,
means o f c u r in g or p r e v e n tin g su ch d i s e a s e s may be p r o v id e d .
A r t e r i a l d i s e a s e , s p e c i f i c a l l y a t h e r o s c l e r o s i s , w it h i t 3
r e s u l t a n t c o m p lic a t io n s , c o n s t i t u t e s th e m o s t . im p ortan t
. . . r .
s i n g l e problem c o n fr o n tin g m e d ic a l i n v e s t i g a t o r s a t th e
p r e s e n t tim e . However, an tin d ersta n d in g o f norm al m e ta b o lic
p r o c e s s e s i s p r e lim in a r y t o an a p p r e c ia t io n o f th e a l t e r
a t i o n s im posed upon su ch p r o c e s s e s In d i s e a s e s t a t e s .
An im p ortan t a s p e c t o f c h o l e s t e r o l m eta b o lism th a t
has lo n g p u z z le d i n v e s t i g a t o r s Is th e problem o f how th e
anim al organism d is p o s e s o f e x c e s s c h o l e s t e r o l . The answ er
t o t h i s q u e s t io n m ig h t , in d e e d , have im p ortan t a p p l i c a t i o n s
t o c l i n i c a l m e d ic in e .. The v a r i a t i o n s in th e r e sp o n s e o f
a n im a ls o f d i f f e r e n t s p e c i e s t o e x c e s s c h o l e s t e r o l in ta k e
seem s t o i n d ic a t e th a t some a n im a ls a re b e t t e r e n a b le d to
cope w it h and m e ta b o liz e c h o l e s t e r o l th an o t h e r s . I s th e
e x c e s s s t e r o l e x c r e t e d or i s i t degraded by th e organism
i n t o s im p le r compounds? I f su ch d e g r a d a tio n d oes o ccu r in
th e anim al b o d y , w h ich organs or t i s s u e s a r e in v o lv e d , and
what a re th e breakdown p r o d u c ts? F u r th e r , are t h e r e sy n
t h e t i c and d e s t r u c t i v e p r o c e s s e s o c c u r r in g c o n c o m it a n tly ,
a n d , i f s o , what f a c t o r s e f f e c t a prep on d eran ce o f one
p r o c e s s over th e o th e r?
Some o f t h e s e q u e s t io n s have b een p a r t i a l l y answ ered
I t was in an a ttem p t to throw a d d i t io n a l l i g h t upon t h i s
a s p e c t o f c h o l e s t e r o l m eta b o lism t h a t the p r e s e n t stu d y was
u n d e r ta k e n .
CHAPTER I I
HISTORICAL REVIEW OP PROBLEM
I t has lo n g been known on th e b a s i s o f jLn v iv o
b a la n c e s t u d ie s t h a t c e r t a i n anim al s p e c i e s a r e a b le to
d e s t r o y c h o l e s t e r o l . Dam (2 4 ) in 1931 o b se r v e d a l o s s o f
th e s t e r o l in young c h ic k e n s , w h ich c o u ld n o t be a c co u n ted
f o r on th e b a s i s o f e x c r e t i o n . Page and M enschick (5 5 ) in
1932 co n d u cted b a la n c e s t u d i e s on c h o l e s t e r o l f e d r a b b it s
and co n clu d ed t h a t th e an im als were a b le t o d e s t r o y 0 .8 t o
1 .8 g . c h o l e s t e r o l p e r w eek. Schoenheim er and B reu sch (6 3 )
in 1933 foun d t h a t m ic e , when on d i e t s r i c h in c h o l e s t e r o l ,
can in a month d e s t r o y f i v e tim es t h e i r body c o n te n t o f th e
s t e r o l .
S e v e r a l I n v e s t ig a t o r s have a tte m p te d t o d e ter m in e
w here in th e an im al body su ch d e s t r u c t io n ta k e s p l a c e .
Artom (4 ) in 1923 r e p o r te d t h a t th e c h o l e s t e r o l c o n te n t o f
i s o l a t e d dog l i v e r I n c r e a s e d or d e c r e a se d d u r in g a u t o l y s i s
o f th e t i s s u e , d ep en d in g upon th e tim e o f in c u b a tio n and th e
c o n d it io n s t o w h ich th e anim al w a s - s u b j e c t e d j u s t p r io r t o
i t s d e a th . T akagi (7 3 ) in 1936 r e p o r te d t h a t serum from
c h o l e s t e r o l i n j e c t e d r a b b it s when In cu b a te d a t 3 7 ° G. f o r
tw e n ty -fo u r h o u rs w it h a c h o l e s t e r o l h y d r o s o l p r e p a r a tio n
b rou gh t about a d e c r e a s e In th e c h o l e s t e r o l c o n te n t o f th e
m ix tu r e . He r e p o r t e d f u r t h e r t h a t th e serum c h o l e s t e r o l ,
w h ich rem ained low a f t e r r e p e a te d c h o l e s t e r o l i n j e c t i o n s in
th e norm al r a b b i t , became h ig h i f th e r e t i c u l o - e n d o t h e l i a l
sy stem were b lo c k e d or i f th e s p le e n w ere removed or s u b j e c
t e d t o x - r a y i r r a d i a t i o n .
N i e f t ( 8 9 ) , w h ile s tu d y in g e s t e r i f i c a t i o n o f f r e e
c h o l e s t e r o l and s p l i t t i n g o f c h o l e s t e r o l e s t e r s b y s a l i n e .
e x t r a c t s o f v a r io u s t i s s u e s o f th e r a t , foun d t h a t th e
r e c o v e r y o f c h o l e s t e r o l was som etim es n o t com p lete a f t e r
in c u b a tio n w it h l i v e r e x t r a c t ; in c u b a tio n w ith o th e r r a t
t i s s u e e x t r a c t s , h o w ev er, y i e l d e d co m p lete r e c o v e r y . I t was
c o n c lu d e d t h a t r a t l i v e r p r e p a r a tio n s are somehow c a p a b le o f
d e g r a d in g c h o l e s t e r o l .
Marx and L i p s e t t ( 4 9 ) , f o l l o w i n g up N i e f t ’ s o b se r v a
t i o n s , r e p o r te d r e c e n t l y t h a t l i v e r e x t r a c t s from c h o l e s t e r o l
f e d r a t s showed a d e c r e a s e in c h o l e s t e r o l c o n te n t a f t e r in c u
b a t io n f o r from tw e lv e to s i x t e e n h ou rs a t 3 7 ° C.
More r e c e n t l y i t has been r e p o r te d by S w e ll and
T r ea d w ell (7 0 ) th a t th e c h o l e s t e r o l c o n c e n tr a t io n o f r a t
l i v e r hom ogenates in c u b a te d a t 3 8 ° C. w ith added sodium
g l y c o c h o la t e d e c r e a s e d d u rin g th e f i r s t s i x hours o f .in cu
b a t i o n . W ith c o n tin u e d in c u b a tio n th e t o t a l c h o l e s t e r o l
in c r e a s e d u n t i l a f t e r abou t t h i r t y - s i x h ou rs th e c h o l e s t e r o l
c o n te n t was u s u a l l y g r e a t e r th a n b e fo r e in c u b a t io n . The
i n i t i a l d e c r e a s e in c h o l e s t e r o l was o b ser v ed to ta k e p la c e
o v er a pH range o f 5 .8 t o 8 . 0 . The in c r e a s e in c h o l e s t e r o l
was fo u n d t o o c cu r o n ly In th e pH range 6 .8 t o 7.2;. The
ch an ges r e p o r te d were a s s e r t e d l y n o t due t o th e p r e se n c e o f
m ic r o o r g a n ism s.
Of some im p ortan ce t o th e p r e s e n t c o n s id e r a t io n i s
th e f a c t t h a t c e r t a i n m icroorgan ism s a r e known t o be ca p a b le
o f d e g r a d in g th e s t e r o i d m o le c u le . C e r ta in F la v o b a c te r ia
and p s e u d o d ip h th e r ia organism s in g e n e r a l o x i d i z e th e n u c le a r
h y d r o x y l grou p s o f s t e r o i d compounds d e v o id o f a carbon
s e v e n te e n s id e c h a in . C h o le s t e r o l decom posing organism s
f a l l e s s e n t i a l l y w it h in th e genus P r o a c tin o m y c e s. The p o in t s
o f a t t a c k on th e c h o l e s t e r o l m o le c u le are th e h y d r o x y l grou p ,
th e dou ble bond, and a l s o th e s id e c h a in . R ing A. i s b ro k en ,
and is o c a p r o ic a c i d may be form ed from th e s id e c h a in .
E s t e r i f i e d c h o l e s t e r o l i s n o t a t ta c k e d ( 7 8 ) .
R egard ing th e p r o d u cts o f d e g r a d a tio n o f th e c h o l e s
t e r o l m o le c u le in th e an im al organ ism , n o t much i s known w ith
c e r t a i n t y . Cook (22:) r e p o r te d o n ly about s e v e n t y t o e i g h t y
p e r c e n t r e c o v e r y o f c h o l e s t e r o l f e d t o r a t s on a h ig h f a t
d i e t . H is l i p o i d b a la n c e s h e e t o f f e c a l e x c r e t i o n .showed
t h a t th e amount o f u n a c c o u n te d -fo r c h o l e s t e r o l was about
e q u a l t o th e amount o f h ig h m o le c u la r w e ig h t f a t t y a c id s
fou n d t o be in c r e a s e d in th e s t o o l s . B ergstrom and
W in t e r s t e in e r (5 ) (6 ) (7 ) (8 ) (8 3 ) have r e p o r te d a s e r i e s o f
i n t e r e s t i n g r e a c t i o n s w hereby c h o l e s t e r o l in v i t r o in c o l
l o i d a l aqueous s o l u t i o n s i s f a i r l y r e a d i l y o x id iz e d to
s e v e r a l d i f f e r e n t s t e r o i d compounds, th e m ost im p ortan t o f
w h ich perhaps b e in g 7*C- and 7 0 - h y d r o x y c h o le s t e r o l and
7 - k e t o c h o l e s t e r o l . V a ld ig u ie and S e g u e la s (8 0 ) r e c e n t l y
i
r e p o r t e d t h a t , upon in c u b a tio n o f c h o l e s t e r o l a t 3 7 ° C.
w it h an aqueous l i v e r hom ogen ate, a c e to n e was o b ta in e d in
y i e l d s up t o n in e t y - o n e p e r c e n t , p resu m ab ly d e r iv e d from th e
is o p r o p y l group in th e s i d e ch a in o f th e s t e r o l .
I t m ust be r e c o g n iz e d t h a t much, i f n o t a l l , o f th e
‘'breakdown1 1 o f c h o l e s t e r o l r e p o r te d by v a r io u s i n v e s t i g a t o r s
m ig h t be a c c o u n te d f o r on t h e . b a s i s o f c o n v e r s io n t o a n o th e r
r e l a t e d s t e r o i d compound. S e v e r a l 3uch c o n v e r s io n s a re
known t o ta k e p la c e i n th e anim al. S ch oen h eim er, H it t e n b e r g ,
and G raff (6 4 ) in 1935 d em o n stra ted t h a t , in th e d o g , c h o l e s
t e r o l a p p a r e n tly i s c o n v e r te d t o c h o le s te n o n e in th e
i n t e s t i n a l w a l l . The c h o le s t e n o n e i s e x c r e t e d i n t o th e
i n t e s t i n a l lum en, and p u t r e f a c t i v e b a c t e r i a o f th e c o lo n
th e n e f f e c t a c o n v e r s io n t o c o p r o s t e r o l v ia c o p r o s ta n o n e .
B lo c h , B e r g , and R it t e n b e r g (1 2 ) in 1943 r e p o r te d t h a t dogs
g iv e n in tr a v e n o u s deuateroncholesterol were shown t o e x c r e t e
d eu teriu m l a b e l e d c h o l i c a c i d - i n t h e i r b i l e , i n d i c a t i n g a
b i o l o g i c a l c o n v e r s io n o f th e s t e r o l t o c h o l i c a c i d . The
s i m i l a r i t y in s t r u c t u r e b etw een th e s e x hormones and c h o l e s
t e r o l l e d B lo c h (1 1 ) t o f e e d d e u t e r o - c h o l e s t e r o l t o a woman
in th e e ig h t h month o f p reg n a n cy . U r in e c o l l e c t e d
7
s u b s e q u e n tly c o n ta in e d d eu teriu m l a b e le d p r e g n a n d io l, w h ich
i s a r e d u c tio n p ro d u ct o f p r o g e s te r o n e .
The s t r u c t u r a l s i m i l a r i t i e s b etw een th e v a r io u s D
v ita m in s and c h o l e s t e r o l have lo n g b een known. One o f th e
a n t i r a c h i t i c s u b s t a n c e s , v ita m in Dg, was shown t o h ave the
same s id e ch a in as c h o l e s t e r o l and h as b een proven t o be
d e r iv e d from 7 - d e h y d r o c h o le s t e r o l, w h ich i t i s presum ed, may
be form ed from c h o l e s t e r o l in some a n im a ls b u t n o t t o any
c o n s id e r a b le e x t e n t in mammals. G eig er and L assen (3 1 )
r e p o r te d in 1943 th a t f e e d in g o f 7 - h y d r o x y c h o le s t e r o l and
7 - k e t o - c h o l e s t e r o l t o r a t s on a r a c h it o g e n ic d i e t p r e v e n te d
r i c k e t s i f th e a n im a ls were g iv e n u l t r a - v i o l e t i r r a d i a t i o n .
I t has b een shown th a t 7 - h y d r o x y c h o le s t e r o l can in v i t r o be
c o n v e r te d i n t o v ita m in Lg v ia 7 - d e h y d r o c h o le s t e r o l.
A nother group o f i n t e r e s t i n g and h i g h l y im p ortan t
compounds c h e m ic a lly r e l a t e d t o c h o l e s t e r o l i s t h a t o f th e
a d r e n a l c o r t i c a l horm ones. The e x tr e m e ly h ig h c h o l e s t e r o l
c o n c e n tr a t io n o f th e a d r e n a l c o r t e x h as l e d t o th e p o s t u l a
t i o n th a t th e s t e r o l i s a p r e c u r so r f o r th e c o r t i c a l
horm ones. The e v i d e n c e ' t o d a te on t h i s problem i s o n ly
i n f e r e n t i a l . I t h a s been shown t h a t f o l l o w i n g i n j e c t i o n s o f
a d r e n a l c o r t i c o t r o p h ic hormone or f o l lo w in g p e r io d s o f s t r e s s
th e c h o l e s t e r o l c o n te n t o f th e a d r e n a l c o r t e x f a l l s m ark ed ly,
t h e r e b e in g no su ch e f f e c t s upon th e c h o l e s t e r o l c o n te n t o f
o th e r t i s s u e s . I t i s te m p tin g t o co n clu d e t h a t c h o l e s t e r o l
i s u t i l i z e d f o r th e hormone fo r m a tio n .
The q u e s t io n r a i s e d a t th e b e g in n in g o f t h i s t r e a t i s e
as t o w h eth er s t e r o l s y n t h e s i s and d e s t r u c t io n o ccu r s im u l
t a n e o u s ly in th e anim al h a s been p a r t i a l l y an sw ered .
Channon (1 9 ) in 1 9 2 5 , b y means o f b a la n c e e x p e r im e n ts ,
showed s y n t h e s i s o f c h o l e s t e r o l by th e r a t . Schoenheim er
and B reu sch (6 3 ) in th e work above c i t e d , d e m o n stra ted t h a t
m ice on low c h o l e s t e r o l d i e t s can s y n t h e s i z e a s much s t e r o l
in a m onth a s th e y had in t h e i r b o d ie s a t th e b e g in n in g o f
th e e x p e r im e n t. The a u th o r s c o n c lu d e d ttt h a t in th e t i s s u e s
c h o l e s t e r o l i s c o n t i n u a ll y b e in g form ed and d e s tr o y e d .
E it h e r a p o s i t i v e or n e g a t iv e b a la n c e may be fo u n d , d ep en d in g
upon e x p e r im e n ta l c o n d i t i o n s , i . e . s y n t h e s i s may be in e x c e s s
o f d e s t r u c t io n or v ic e v e r s a . 1 1
As R it te n b e r g and Schoenheim er (5 9 ) p o in t o u t , i t i s
im p o s s ib le by means o f b a la n c e e x p e r im e n ts a lo n e t o d e c id e
w h eth er b i o l o g i c a l s t e r o l s y n t h e s i s in v o lv e s new fo r m a tio n
o f th e r in g s k e le t o n or m e r e ly m in or changes by w h ich a non -
p r e c i p i t a b l e s t e r o i d may be r e n d e r e d p r e c i p i t a b l e in th e •
d i g i t o n i n m ethod o f d e te r m in in g c h o l e s t e r o l . I f a c o n s ta n t
c o n c e n tr a t io n o f h ea v y w a ter i s m a in ta in e d in th e body f l u i d s
o f an an im al b y a d m in is t r a t io n o f d eu teriu m o x id e in th e
d r in k in g w a te r , a l l s y n t h e t i c r e a c t i o n s ta k e p la c e in a medium
o f I> 2 0 j and a l l compounds s y n t h e s iz e d w i l l c o n ta in d e u te riu m .
Prom a stu d y o f th e u p ta k e o f h ea v y hydrogen i t i s p o s s i b l e
t o m easure th e r a t e o f s y n t h e s i s o f th e compound even i f
n o th in g i s known o f th e a c t u a l ch em ica l m echanism .
R itt e n b e r g and Schoenheim er have made su ch s t u d i e s on m ice
and fou n d c h o l e s t e r o l c o n t a in in g d eu teriu m in s u f f i c i e n t
q u a n t it y t o i n d ic a t e s y n t h e s i s in v o lv in g c o u p lin g o f a la r g e
number o f sm a ll m o le c u le s . I t was c a lc u l a t e d t h a t h a l f o f
th e body c h o l e s t e r o l was d e s tr o y e d and r e s y n t h e s i z e d in about
t h i r t y d a y s , ab o u t two p e r c e n t b e in g r e p la c e d e v e r y d ay.
A ttem p ts t o f i n d th e p r e c u r so r s' in v o lv e d have l e d t o th e
f e e d in g o f l a b e l e d in te r m e d ia t e s o f f a t and c a rb o h y d ra te
m eta b o lism w ith s e a r c h f o r th e i s o t o p e in c h o l e s t e r o l .
B lo c h and R itt e n b e r g (1 4 ) b y means o f su ch e x p e r im e n ts r e
p o r t th a t r a t s and m ice f e d d e u t e r o - a c e t a t e show s i g n i f i c a n t
amounts o f d eu teriu m in t h e i r c h o l e s t e r o l in b o th th e n u c le u s
and th e s id e c h a in . L a b eled p r o p io n ic , b u t y r i c , and s u c c i n i c
a c id s a l l f a i l e d t o g iv e d e u t e r o - c h o l e s t e r o l , as d id a l s o
l a b e l e d a c e t o a c e t a t e and p y r u v a te . More r e c e n t l y i t has
b een d em o n stra ted t h a t s u r v iv in g r a t l i v e r s l i c e s in c u b a te d
w ith ' l a b e le d a c e t a t e s y n t h e s i z e l a b e le d c h o l e s t e r o l , i n d i
c a t in g a g a in t h a t a c e t i c a c i d i s a p r e c u r so r o f th e s t e r o l (13).
S r e r e , C h a ik o ff, and Dauben (6 9 ) o b ser v ed t h a t th e c o n v e r s io n
o f a c e t a t e to c h o l e s t e r o l ta k e s p la c e in th e h e p a te c to m iz e d
r a t , i n d ic a t in g t h a t s y n t h e s i s i s n o t c o n fin e d t o th e l i v e r .
10
They s u b s e q u e n tly have d e m o n stra ted th e c o n v e r s io n o f C14
l a b e l e d a c e t a t e t o c h o l e s t e r o l by s u r v iv in g s l i c e s o f b e e f
a d r e n a l c o r t e x .
One f u r t h e r q u e s t io n r a i s e d in th e in t r o d u c t io n w h ich
s h o u ld be c o n s id e r e d h e r e i s what are th e f a c t o r s w h ich
c a u se a p rep on d eran ce o f e i t h e r th e s y n t h e s i s or d e s t r u c t io n
o f c h o l e s t e r o l by t h e o rg a n ism . The problem has b een s t u d ie d
la r g e ly - in v iv o by i n v e s t i g a t i o n o f th e f a c t o r s w h ich
i n f lu e n c e th e a n im a l!s a b i l i t y t o d is p o s e o f c h o l e s t e r o l .
I t m ust f i r s t be r e c o g n is e d t h a t th e a b i l i t y t o d i s
p ose o f f e d c h o l e s t e r o l v a r ie s c o n s id e r a b ly in d i f f e r e n t
s p e c i e s . H erb ivorou s a n im a ls d i f f e r m ark ed ly from c a r n iv
orous a n im a ls in t h i s r e g a r d . H erb ivorou s a n im a ls absorb
f a t t y s u b s ta n c e s and c h o l e s t e r o l s l o w l y , but when t h e s e
s u b s ta n c e s g e t i n t o th e c i r c u l a t i o n and t i s s u e s , t h e y a re
e x c r e t e d o n ly w ith d i f f i c u l t y . C arn ivorou s and om nivorous
a n im a ls r e a d i l y show lip e m ia a f t e r f a t i n g e s t i o n b u t do n o t
r e a d i l y form c h o l e s t e r o l d e p o s i t s in th e t i s s u e s . Thus
Wacker and Hueck (q u o ted b y H ueper) (8 2 ) produced i n c r e a s e s
in b o th th e f r e e and bound c h o l e s t e r o l in th e b lo o d o f
r a b b it s b y c h o l e s t e r o l f e e d in g b u t c o u ld n o t produce an
in c r e a s e in c a t s and d o g s. The e a s e w ith w h ich f a t t y l i v e r s
and ath erom ata a r e produ ced in r a b b it s b y c h o l e s t e r o l f e e d i n g
i s w e l l known and i s in sharp c o n t r a s t t o th e r e s i s t a n c e
shown b y th e dog and c a t . H owever, ev en in th e c a r n iv o r o u s
11
an im al and more so in an om nivorous a n im a l, su ch a s th e r a t
or man, p r o lo n g e d f e e d i n g or h ig h c o n c e n tr a t io n o f c h o l e s
t e r o l in th e d i e t r e s u l t s in e x c e s s d e p o s i t i o n o f c h o l e s t e r o l
in th e t i s s u e s , p a r t i c u l a r l y in th e l i v e r .
O b v io u s ly , t h e n , th e d i e t a l s o p la y s an im p orta n t
p a r t in i n f l u e n c in g th e d i s p o s a l o f c h o l e s t e r o l by th e
a n im a ls . A's Schoenheim er and B r eu sc h f s work (6 3 ) i n d i c a t e s ,
a t l e a s t in m ic e , c h o l e s t e r o l s y n t h e s i s i s s t im u la t e d by a
low c h o l e s t e r o l d i e t , and c h o l e s t e r o l d e s t r u c t io n i s
s t im u la t e d by a h ig h c h o l e s t e r o l d i e t . The f a t c o n te n t o f
th e d i e t a l s o ap p ears t o be im p o r ta n t. C h o le s t e r o l d e p o s i
t i o n in th e t i s s u e s o f an anim al i s a c c o m p lish e d o n ly w ith
d i f f i c u l t y , i f a t a l l , in th e a b sen ce o f f a t in th e d i e t .
T h is ap p ears t o be lia r g e ly a m a tte r o f a b s o r p t io n , f o r l i t t l e
/
c h o l e s t e r o l i s a b so rb ed in th e a b sen ce o f f a t in th e d i e t .
S in c e a c e t a t e has b een shown to be a p r e c u r s o r o f c h o l e s t e r o l ,
h o w ev e r, i t i s c o n c e iv a b le t h a t a h ig h f a t d i e t m igh t le a d
t o endogenous s y n t h e s i s . In d e ed , as a l l th r e e m ajor c l a s s e s
o f f o o d s t u f f s h a v e been shown t o g iv e r i s e t o 2 -c a r b o n f r a g
ment's i t i s t h e o r e t i c a l l y p o s s i b l e t h a t f a t s , c a r b o h y d r a te s ,
and p r o t e in s m igh t g iv e r i s e to c h o l e s t e r o l . However, B loch
and R itt e n b e r g (14.) w ere u n a b le to d em o n stra te p y ru v a te con
v e r s io n t o c h o l e s t e r o l .- The p r o t e in c o n te n t o f th e d i e t i s
im p ortan t n o t o n ly from th e s ta n d p o in t o f su p p ly o f p o s s i b l e
2 -c a r b o n p r e c u r s o r s , but a l s o b e c a u se c e r t a i n amino a c i d s ,
p a r t i c u l a r l y m e th io n in e , g iv e r i s e t o l i p o t r o p i c s u b s ta n c e s
w h ic h , as w i l l be d is c u s s e d b e lo w , i n f lu e n c e f a t and c h o l e s
t e r o l m e ta b o lis m . I t m igh t be m e n tio n e d t h a t b i l e s a l t s
added t o th e d i e t g r e a t l y f a c i l i t a t e a b s o r p tio n o f c h o l e s t e r o l.
B i l e f i s t u l a a n im a ls have b een shown to be r e s i s t a n t t o
c h o l e s t e r o l f e e d i n g .
I t h a s b een found t h a t c e r t a i n hormones a f f e c t c h o l e s
t e r o l m eta b o lism ; T h yroid horaone a d m in is te r e d c o n c u r r e n t ly
w it h c h o l e s t e r o l t o e x p e r im e n ta l a n im a ls p o t e n t i a t e s th e
a c t i o n o f c h o lin e in p r o t e c t i n g a g a i n s t th e d evelop m en t o f
f a t t y l i v e r s ( 2 9 ) , T h io u r a c il or th y r o id e c to m y g r e a t l y
a c c e l e r a t e s b lo o d c h o l e s t e r o l e l e v a t i o n and f a t a c cu m u la tio n
in th e t i s s u e s . I t has r e c e n t l y been p o s t u la t e d by
F leisch m an (2 8 ) t h a t t h y r o id hormone p erh ap s d oes n o t a f f e c t
th e t o t a l amount o f bod y c h o l e s t e r o l so much a s i t te n d s t o
ca u se a d e p o s i t i o n o f b lo o d c h o l e s t e r o l in th e t i s s u e s .
Turner and DeLamater (7 9 ) fo u n d t h a t t h y r o t r o p ic hormone o f
th e a n t e r i o r p i t u i t a r y c a u se s a f a l l in th e b lo o d c h o l e s t e r o l
o f h y p e r c h o le s t e r o le m ic r a b b it s r e g a r d le s s o f th e p r e se n c e
o r a b sen ce o f th e t h y r o id g la n d s .
A lth o u g h th e b lo o d c h o l e s t e r o l i s a lm o st i n v a r ia b l y
e l e v a t e d in human d i a b e t e s , K ap lan , Entenm an, and
C h a ik o ff (4 0 ) w ere u n a b le t o a l t e r th e b lo o d l i p i d s s i g n i f i
c a n t l y b y i n j e c t i o n o f m a ss iv e d o se s o f i n s u l i n i n t o human
s u b j e c t s . The c o n c e n tr a t io n o f f r e e and e s t e r i f i e d
13
c h o l e s t e r o l , t o t a l f a t t y a c i d s , and p h o s p h o lip id s in th e
b lo o d were u n a f f e c t e d b y a lm o st co m p lete rem oval o f g lu c o s e
from th e b lo o d sbream .
The e f f e c t o f th e a d r e n a l c o r t i c a l hormones upon
c h o l e s t e r o l m eta b o lism i s f a r from c l e a r . J o e ls o n and
Shorr (3 8 ) r e p o r t t h a t th e b lo o d c h o l e s t e r o l o f dogs i s '
in c r e a s e d f o l l o w i n g a d r e n a le c to m y , th e r i s e b e in g more
marked in th e f r e e c h o l e s t e r o l f r a c t i o n . R e is s (5 8 ) r e p o r ts
t h a t c o r t i c a l e x t r a c t g iv e n to normal a n im a ls c a u s e s a
lo w e r in g o f b lo o d c h o l e s t e r o l , b u t t h a t a f t e r lo n g c o n tin u e d
a d m in is t r a t io n th e r e i s an in c r e a s e in th e t o t a l c h o l e s t e r o l
c o n te n t o f th e bod y. I f h e f i r s t ’’b lo c k e d ” th e r e t i c u l o
e n d o t h e l i a l system b y i n j e c t i o n s o f I n d ia in k , R e is s fou n d
no lo w e r in g o f b lo o d c h o l e s t e r o l on su b seq u e n t a d m in is t r a t io n
o f c o r t i c a l e x t r a c t . Thaddea and P a ssh a u e r (7 4 ) r e p o r t a
f a l l o f t o t a l serum c h o l e s t e r o l one t o t h r e e h ou rs a f t e r
i n j e c t i o n o f a d r e n a l c o r t i c a l e x t r a c t i n t o norm al d o g s , th e
f a l l b e in g l a r g e l y due t o a d e c r e a s e in th e e s t e r i f i e d
f r a c t i o n . B i l a t e r a l a d r e n a lec to m y in c a t s c a u s e s a r i s e in
th e serum c h o l e s t e r o l and a d e c r e a se in th e t o t a l c h o l e s t e r o l
o f l i v e r and s t r i a t e d m u s c le . T hese a u th o rs co n clu d e th a t
a d r e n a l c o r t i c a l hormones e x e r t a f i x i n g a c t i o n upon c h o l e s
t e r o l , c a u s in g a d e p o s i t i o n in th e t i s s u e s from th e b lo o d .
V i l l e l a (8 1 ) more r e c e n t l y r e p o r ts t h a t d e s o x y c o r t ic o s t e r o n e
14
e x e r t s a s i m i l a r e f f e c t , i n j e c t i o n s o f one m illig r a m p e r day
f o r s i x days i n t o g u in e a p ig s , c a u s in g a lo w e r in g o f plasm a
c h o l e s t e r o l .
Many i n v e s t i g a t o r s have been u n a b le t o c o n firm th e
above w ork, h o w ev er, and th e r e a re seme r e p o r t s t h a t b lo o d
, c h o l e s t e r o l i s * a c t u a l l y in c r e a s e d b y th e a d m in is t r a t io n o f
a d r e n a l c o r t i c a l e x t r a c t s . Thus C o lla z o , M aranon, Roda, and
T o rres (2 1 ) r e p o r te d t h a t f r e s h l y p rep a red e x t r a c t o f a d r e n a l
c o r t e x produ ced an in c r e a s e in b lo o d c h o l e s t e r o l in d o g s ,
r a b b i t s , norm al p a t i e n t s , and p a t i e n t s w ith A ddison’s d i s e a s e .
Thompson e t a l . (7 5 ) r e p o r te d t h a t a d r e n a lec to m y a b o lis h e d
th e h y p e r c h o le s te r o le m ia produced in th r e e dogs b y t h y r o id e c
tom y. Yasue (8 4 ) r e p o r te d t h a t a f t e r a d r e n a lec to m y th e
c h o l e s t e r o l e s t e r c o n c e n tr a t io n o f c a t b r a in and l i v e r f e l l ,
w hereas th e c h o l e s t e r o l e s t e r c o n c e n tr a t io n o f k id n e y was
in c r e a s e d abou t t h r e e f o l d . F i n a l l y , i t h as been r e p o r te d b y
S p o s it o (6 8 ) and in d e p e n d e n tly b y L ev in (4 7 ) t h a t th e serum
c h o l e s t e r o l i s in c r e a s e d by d e s o x y c o r t ic o s t e r o n e i n j e c t i o n s
in man and th e r a t r e s p e c t i v e l y .
The e f f e c t o f a d r e n o c o r t ic o t r o p h ic hormone upon th e
c h o l e s t e r o l c o n te n t o f th e a d r e n a ls h as a lr e a d y been a llu d e d
t o . C a r r e y e t t , G o lla , and R e iss, (1 6 ) r e p o r te d t h a t c o r t i c o -
t r o p h ic hormone c a u se s an i n i t i a l d e c r e a s e and th e n a marked
in c r e a s e a f t e r th r e e days in th e c h o l e s t e r o l c o n te n t o f th e
a d r e n a ls o f th e r a t . On th e o th e r h a n d , th e y foun d th a t
15
g o n a d o tro p h ic hormone a d m in is t r a t io n c a u se s an i n i t i a l i n
c r e a s e in th e a d r e n a l c h o l e s t e r o l c o n t e n t , f o llo w e d by a
marked d e p l e t io n i f th e i n j e c t i o n s were c a r r ie d on f o u r days
or lo n g e r .
I t h a s b een known f o r y e a rs t h a t d e p a n c r e a tiz e d dogs
on i n s u l i n d e v e lo p f a t t y l i v e r s . A l l a n , B ow ie, MacLeod, and
R obinson ( 3 ) in 1924 f i r s t r e p o r te d t h i s phenomenon and fo u n d
t h a t i t c o u ld be p r e v e n te d b y th e f e e d i n g o f raw p a n c r e a s.
H ersh ey (3 4 ) in 1930 and H ersh ey and S o sk in (3 5 ) in 1931
fou n d t h a t crude l e c i t h i n c o u ld s u b s t i t u t e f o r raw p an creas
in p r e v e n tin g th e f a t t y l i v e r s . B e s t , H er sh ey , and
Huntsman (9 ) in 1932 produ ced f a t t y l i v e r s in r a t s and p r e
v e n te d them by f e e d in g p u r i f i e d l e c i t h i n or c h o l i n e . Large
amounts o f c a s e in were a l s o fou n d to i n h i b i t f a t t y l i v e r
p r o d u c tio n ; a f t e r Tucker and E c k s t e in ’ s work (7 7 ) showed
t h a t m e th io n in e had a marked l i p o t r o p i c e f f e c t , i t was con
c lu d e d t h a t c a s e in a c te d by v ir t u e o f i t s m e th io n in e c o n te n t .
Subsequent work by du V igneaud ( 2 7 ) , w h ich d em o n stra ted th a t
m e th io n in e s u p p lie s l a b i l e m eth y l groups f o r fo r m a tio n o f
c h o l i n e , c o r r e l a t e d th e l i p o t r o p i c a c t i o n s o f th e two
compounds•
C h o lin e h as been found' t o e x e r t -a,m ore pronounced
e f f e c t upon p r e v e n tio n o f th e a c cu m u la tio n o f g l y c e r i d e s in
th e l i v e r than upon c h o l e s t e r o l e s t e r a c c u m u la tio n . In .
r-
c u r a t iv e e x p e rim en ts i t c a u se s a r a p id d im in u tio n o f
16
g l y c e r i d e s and a slo w e r d e c r e a s e in th e c h o l e s t e r o l e s t e r
c o n t e n t . Mawson and W elch (5 0 ) c o n c lu d e d t h a t th e h y d r o x y l
group i s e s s e n t i a l b u t th e m eth y l groups a r e n o t , s in c e t r i
e t h y l c h o lin e i s l i p o t r o p i c . T r o w ell (7 6 ) foun d th a t c h o lin e
added t o l i v e r s l i c e s i n h i b i t e d a c e t o a c e t i c a c id fo r m a tio n
and th e r e b y c o n c lu d e d t h a t f a t m eta b o lism was i n h i b i t e d , a
c o n c lu s io n t h a t i s n o t w arran ted on th e b a s i s o f th e e v id e n c e
s u p p lie d by B lo ch and R it te n b e r g (1 5 ) t h a t a c e t o a c e t i c a c id
i s n o t an o b li g a t o r y in te r m e d ia te in m eta b o lism o f f a t s .
I n v e s t i g a t i n g f u r t h e r th e m echanism o f a c t i o n o f c h o l i n e ,
Perlman and C h a ik o ff ( 5 6 ) , u s i n g F ^ , d em o n stra ted t h a t
c h o lin e s t im u la t e s p h o s p h o lip id tu r n o v e r in th e l i v e r o f
c h o l e s t e r o l f e d rats;: c h o l e s t e r o l d e c r e a s e d th e p h o s p h o lip id
tu r n o v e r as soon a s t h i r t y h o u rs a f t e r f e e d in g was b eg u n .
C h a ik o ff and co -w o rk ers (8 5 ) s u b s e q u e n tly showed t h a t
c h o lin e d o es n o t in c r e a s e l e c i t h i n tu r n o v e r in th e plasm a
and r e a so n e d th a t i f th e l i p o t r o p i c a c t i o n o f c h o lin e
o p e r a te s th ro u g h p h o s p h o lip id m eta b o lism th en th e rem oval o f
l i v e r f a t b y c h o lin e d o es n o t in v o lv e an in c r e a s e d tr a n s p o r t
o f f a t from th e l i v e r t o th e t i s s u e s v ia th e plasm a p h o s
p h o l i p i d s . They c o n c lu d e d t h a t th e e v id e n c e s u g g e s t s r a t h e r
t h a t c h o lin e a c t s on th e u t i l i z a t i o n o f f a t w it h in th e
l i v e r i t s e l f .
D r a g ste d t and co -w o rk ers (2 6 ) r e p o r te d in 1936 t h a t
th e amount o f raw p an creas n e c e s s a r y d a i l y t o p r e v e n t f a t t y
17
l i v e r s in d e p a n c r e a tis e d , in s u lin - m a in t a in e d dogs c o n ta in e d
o n ly s;ix ty m illig ra m s; c h o l i n e , w hich amount was f a r l e s s
than th e one gram o f c h o lin e th e y found was n e c e s s a r y as a
s u b s t i t u t e . They c o n c lu d e d t h a t th e p a n cr e a s s u p p lie d a
hormone o f i n t e r n a l s e c r e t i o n o th e r than i n s u l i n w h ich t h e y
d e s ig n a t e d l i p o c a i c and w hich th e y b e l i e v e d t o be r e s p o n s ib l e
f o r th e l i p o t r o p i c a c t i o n o f raw p a n c r e a s . Subsequent work
from t h i s la b o r a t o r y h as b rou gh t f o r t h r e p o r t s o f f r a c t i o n s
i s o l a t e d from p a n crea s w hich e x e r t a l i p o t r o p i c e f f e c t when
i n j e c t e d s u b c u ta n e o u s ly ( 2 5 ) , K aplan and C h a ik o ff (3 9 ) r e
p o r te d th e i s o l a t i o n o f a f r a c t i o n o f p a n cr e a s w h ich th e y
fo u n d c o n ta in e d two f a c t o r s , a h e a t - l a b i l e b lo o d f a c t o r ,
w hich on i n g e s t i o n c a u se s an in c r e a s e above norm al o f a l l
b lo o d l i p i d s e s p e c i a l l y c h o l e s t e r o l , and a h e a t - s t a b l e l i v e r
f a c t o r w h ich on i n g e s t i o n p r e v e n ts f a t d e p o s i t i o n in th e
l i v e r . C h a ik o ff erb a l . ( 1 7 , 1 8 ) s u b s e q u e n tly c o n clu d ed t h a t
th e l i v e r f a c t o r may be a p r o t e o l y t i c enzyme w h ich i s s e c r e t e d
b y th e p a n crea s i n t o th e i n t e s t i n e , b e in g n e c e s s a r y f o r th e
l i b e r a t i o n o f c h o lin e and m e th io n in e from th e p r o t e in s o f
th e d i e t . P ree c h o lin e or m e th io n in e w i l l o b v ia te th e need
f o r t h i s a n ti£ H f t't’ y, l i v e r f a c t o r , b u t p r o t e i n c o n t a in in g
s u b s t a n t i a l amounts o f bound c h o lin e and m e th io n in e e x e r t s
no l i p o t r o p i c e f f e c t in th e a b sen ce o f t h i s p a n c r e a tic f a c t o r .
McHenry a n d .c o -w o r k e r s (5 1 ) have fo u n d t h a t f a t t y
l i v e r s cah be produ ced in an im als in a few days by.
18
c a rb o h y d r a te and th ia m in f e e d i n g ; th e l i v e r f a t can be m ain
t a in e d a t a normal l e v e l i f c h o lin e i s a l s o g i v e n . H owever,
i f in a d d it io n l i v e r i s f e d , or an a l c o h o l i c e x t r a c t o f
l i v e r i s g i v e n , o r i f o th e r B v ita m in s a re added to th e d i e t ,
f a t t y l i v e r s a r e produ ced w hich do n o t resp o n d c o m p le t e ly t o
c h o l in e b u t w h ich are c o m p le te ly am enable t o c h o l in e p lu s
i n o s i t o l # McHenry a s s e r t s th a t in a l l th e work In h i s la b o
r a t o r y i n o s i t o l h a s b een fou n d to e x e r t a g r e a t e r e f f e c t
upon l i v e r c h o l e s t e r o l than has c h o l in e . I t i s r e p o r te d
t h a t in s t a n c e s have been o b serv ed in w h ich i n o s i t o l m ark ed ly
red u ced l i v e r c h o l e s t e r o l w ith o u t any d e f i n i t e e f f e c t upon
f a t t y a c i d s .
I t has been s u g g e s te d t h a t b i o t i n may be e s s e n t i a l
f o r d e p o s i t i o n o f e x c e s s d i e t a r y c h o l e s t e r o l in th e l i v e r ,
w h ich e f f e c t i s r e p u t e d ly s p e c i f i c a l l y com bated by i n o s i t o l .
Okey (5 3 ) r e p o r t s t h a t eg g w h ite f e e d in g p r e v e n ts c h o l e s t e r o l
a c cu m u la tio n and im p lie s f u r t h e r t h a t a v id in may even remove
c h o l e s t e r o l a lr e a d y d e p o s it e d in th e t i s s u e s .
A. group o f e n t i r e l y d i f f e r e n t su b s ta n c e s has been
r e c e n t l y i n v e s t i g a t e d f o r e f f e c t s upon c h o l e s t e r o l d i s p o s a l
b y th e an im al body— th e d e t e r g e n t s .
K e l l n e r , C o r r e l l , and Ladd (4 1 ) (4 3 ) r e p o r t t h a t s u s
t a in e d e l e v a t i o n s in b lo o d c h o l e s t e r o l and p h o s p h o lip id s
were produ ced In r a b b it s on a norm al d i e t b y means o f i n t r a
venous i n j e c t i o n s o f th e d e t e r g e n t s Tween 80 and T r it o n A -2 0 .
19
R ep eated i n j e c t i o n s o f Tween 80 ca u sed a s u s t a in e d h y p e r
c h o le s t e r o le m ia w it h l e v e l s f i v e to f i f t e e n tim e s th e normal
l e v e l and a s i m i l a r h u t somewhat g r e a t e r i n c r e a s e in th e
b lo o d p h o s p h o lip id l e v e l s . A s i n g l e i n j e c t i o n o f T r ito n A -20
r e s u l t e d in an in c r e a s e in b lo o d c h o l e s t e r o l and phospho
l i p i d s t o more than f i v e tim es normal w it h th e peak a t two
days a f t e r th e i n j e c t i o n and a r e tu r n t o norm al l e v e l s in
f i v e t o tw e lv e d a y s . T hese w orkers p la c e d a group o f r a b
b i t s on a norm al d i e t and in tr a v e n o u s Tween 80 i n j e c t i o n s
tw ic e w e e k ly f o r e i g h t t o fo u r t e e n w e e k s. C o n tr o ls were f e d
c h o l e s t e r o l bu t g iv e n no d e t e r g e n t . D e s p ite th e f a c t t h a t
th e an im a ls g iv e n d e te r g e n t had much h ig h e r mean c h o l e s t e r o l
b lo o d l e v e l s than th e c o n t r o l s , t h e y had s i g n i f i c a n t l y l e s s
a t h e r o s c l e r o s i s . T h is f i n d in g th e a u th o r s a t t r i b u t e t o th e
f a c t t h a t th e b lo o d p h o s p h o lip id s in th e d e t e r g e n t - t r e a t e d
a n im a ls were a t a l e v e l about tw ic e th a t o f th e c o n t r o l s .
These d e te r g e n ts" w e r e found, to be u n a b le t o e f f e c t a r e s o r p
t i o n o f a t h e r o s c l e r o s i s p r e v io u s l y produ ced b y c h o l e s t e r o l
f e e d i n g .
F i n a l l y some m en tion m igh t be made r e g a r d in g f a c t o r s
t h a t have been fou n d t o a f f e c t in v i t r o m e ta b o lism o f s t e r o id s .
L i p s e t t (8 7 ) fou n d t h a t r a t l i v e r hom ogenates a p p eared to
c o n t a in an in c r e a s e d amount o f c h o l e s t e r o l a f t e r in c u b a tio n
in th e p r e s e n c e o f added in o r g a n ic p h o sp h a te . Sweat and
Sam uels ( 7 1 ) ( 7 2 ) , w ork in g w ith r a t l i v e r e x t r a c t s , have
2 0
shown t h a t d ip h o sp h o p y r id in e n u c le o t id e and c i t r a t e
a c c e l e r a t e th e breakdown o f t e s t o s t e r o n e . D ir e c t a d d it io n
o f s e v e r a l o th e r im p o rta n t compounds o f Krebs c y c le d id n o t
in c r e a s e th e r a t e o f m eta b o lism o f th e horm one, h o w ev er.
I t may be se e n from th e above r e v ie w th a t a lth o u g h
many f a c t o r s a re known t o be c a p a b le o f i n f l u e n c in g th e
c h o l e s t e r o l c o n te n t and tu r n o v e r in th e anim al b o d y , n o t
much i s known o f th e a c t u a l m echanism o f c h o l e s t e r o l d e s t r u c
t i o n by th e anim al o rg a n ism . In an a ttem p t t o d i s c l o s e th e
s i t e o f su c h d e s t r u c t io n and th e mechanism in v o lv e d , t h i s
i n v e s t i g a t i o n was u n d e r ta k e n .
CHAPTER I I I
METHODS
A. T reatm ent o f A n im a ls.
The a n im a ls u s e d w ere a lb in o r a t s , m ale and fe m a le ,
o f th e U n i v e r s i t y o f Southern C a lif o r n ia s t r a i n . They were
o b ta in e d a t an age o f two t o th r e e m o n th s, h a v in g been on a
s t o c k d i e t from th e tim e o f w ea n in g , and p la c e d upon a d i e t
c o n t a in in g one p er c e n t added pure c h o l e s t e r o l and o n e - h a lf
p e r c e n t sodium g l y c o c h o la t e ( s e e T ab le I ) . A nim als were
s a c r i f i c e d a f t e r v a r y in g p e r io d s o f tim e on th e h ig h c h o l e s
t e r o l d i e t ; th e l i v e r s were e x c i s e d and s u b j e c t e d t o th e
p ro ced u res s u b s e q u e n tly t o be d i s c u s s e d .
B. P r e p a r a tio n o f L iv e r H om ogenates.
The p r e p a r a tio n o f a l l hom ogenates e x c e p t th o s e o f
e x p e rim en ts f i v e and s i x was done in th e c o ld room a t a
tem p era tu re o f abou t 29 C. t o 4 ° C. The h o m o g en izin g ap
p a r a tu s c o n s i s t e d o f a p e s t l e a t t a c h e d to a s t i r r i n g m o to r,
t o be u s e d w ith a t w e n t y - f i v e by two hundred m i ll i m e t e r
p y rex t e s t tu b e . A s t e e l p e s t l e was u s e d in e x p e rim en ts one
th ro u g h s i x and was o f su ch a s i z e t h a t th e c le a r a n c e b e
tw een th e p e s t l e and th e tub e was c o n s id e r a b ly in e x c e s s o f
th e o n e - t e n t h m i ll i m e t e r recommended by P o t t e r ( 5 7 ) . I t was
f e l t th a t th e hom ogenates o b ta in e d in t h e s e e a r l y ex p erim en ts
TABLE I
COMPOSITION OP DIET
I n g r e d ie n ta Per c e n t
O ata, Ground 3 3 .7 5
Whole W heat, Ground 3 3 .7 5
Skim M ilk , Powdered 1 5 .0 0
A l f a l f a M eal 4 .0 0
Weaaen O il 7 .0 0
F o r t i f i e d Oil'*' 2 .0 0
Y e a 3 t, S t r a in n'G tt2 2;. oo
Sodium C h lo r id e 0 .5 0
Calcium C arbonate 0 .5 0
B i l e S a l t a 3
0 .5 0
C h o le s te r o l^
1 .0 0
1 O il c o n t a in in g 1600 I.TJ. o f V itam in A and
160 I.TJ, o f V itam in D p er gram o f o i l .
2 Bre we r s ye a a t , a t ra in ,f G'*, Anheu s e r-B u a c h , I n c . ,
S t . L o u is , Mo.
3 Sodium g ly c o c h o la t e ( p u r e ) , C ity C hem ical
C o r p o r a tio n , New Y ork, N. Y.
4 C h o le a te r in C .P ., Merck and C o ., I n c . , Rahway,
New J e r a e y .
were n o t so u n ifo rm and f i n e l y d iv id e d a s d e s i r e d . C onse
q u e n t ly , a p e s t l e o f s l i g h t l y la r g e r d ia m e ter was p r e p a r ed
from l u c i t e , and a s t a i n l e s s s t e e l d u p lic a t e was a l s o m ade,
h a v in g i d e n t i c a l m easu rem en ts. P yrex tu b es were th en
s e l e c t e d , the i n s i d e d ia m e te r s o f w hich were s l i g h t l y l e s s
than th e b ore o f th e p e s t l e s . The tu b e s were ground to g iv e
a u n ifo rm in n e r d ia m e ter b y means o f th e s t a i n l e s s s t e e l
p e s t l e and carborundum in m in e r a l o i l . Thus tu b es were ob
t a i n e d w h ich s n u g ly f i t th e l u c i t e p e s t l e u s e d in e x p e r im e n ts
sev en th ro u g h t h i r t e e n .
The a n im a ls were s a c r i f i c e d in th e c o ld room e i t h e r
by 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 nem butal ( f i v e m illig r a m s
p e r one hundred grams body w e ig h t) or by a b lo w on th e h ea d .
The l i v e r was q u ic k ly e x c i s e d and w e ig h e d , and th en an
a p p r o x im a te ly f i v e gram p o r t io n o f known w e ig h t was m inced
and w ashed i n t o th e h o m o g e n iz in g tu b e w it h tw o m i l l i l i t e r s
o f h o m o g en izin g medium p er gram o f l i v e r u s e d . A f t e r th e
t i s s u e was hom ogenized and c e n t r if u g e d as d i s c u s s e d b e lo w ,
th e su p e r n a ta n t was d i l u t e d w ith an e q u a l p o r t io n o f p o t a s
sium c h l o r i d e - s u c r o s e s o l u t i o n ( s e e b elo w ) b e fo r e a l i q u o t s
w ere ta k e n , g i v in g a f i n a l c o n c e n tr a t io n o f a b ou t one p a r t
l i v e r t o s i x p a r ts hom ogen ate. (In e x p e rim en ts f i v e th rou g h
e i g h t th e hom ogenate was p rep a red w it h fo u r m i l l i l i t e r s o f
p o ta ssiu m c h l o r i d e - s u c r o s e s o l u t i o n p e r gram o f l i v e r , and no
f u r t h e r s o l u t i o n was added t o th e su p e r n a ta n t a f t e r
c e n t r i f u g a t i o n o f th e hom ogenate. T h is p roced u re g i v e s a
c o n c e n tr a t io n o f ab ou t one p a r t l i v e r t o f i v e p a r ts homoge
n a t e . ) The rem ain d er o f th e l i v e r , i f i t was t o be u s e d in
p r e p a r a tio n o f h om ogen ate, was k e p t c o ld u n t i l u s e d , e i t h e r
in a p e t r i d i s h su rroun ded by c h ip p e d i c e or b y im m ersion in
p o ta ssiu m c h l o r i d e - s u c r o s e s o l u t i o n a t about 0 ° C.
I n i t i a l l y , d i s t i l l e d w a ter was u s e d a s th e homoge
n i z i n g medium. P r e lim in a r y work by S ch o tz (9 1 ) on c h o l e s
t e r o l d e s t r u c t io n by l i v e r hom ogenates seem ed t o i n d ic a t e
t h a t hom ogen ates p rep a red w ith 0 .8 8 m olar s u c r o s e t o w h ich
was added s u f f i c i e n t p o ta ssiu m c h lo r id e t o g iv e a 0 .9 p er
c e n t s o l u t i o n showed more a c t i v i t y than th o s e p rep a red w ith
w a te r . Hogeboom, S c h n e id e r , and P a lla d e (3 7 ) r e p o r t th a t
from r a t l i v e r hom ogenates p rep a red w it h 0 .8 8 m olar s u c r o s e ,
m o r p h o lo g ic a lly i n t a c t m ito c h o n d r ia were o b ta in e d by d i f
f e r e n t i a l h ig h sp e e d c e n t r i f u g a t i o n . In w a te r o r i s o t o n i c
s a l i n e h om ogenates m ic r o s c o p ic g r a n u le s , presum ed t o be
m ito c h o n d r ia , were fou n d to be d i s t o r t e d in shapej In s a l i n e
t h e r e was c o n s id e r a b le a g g l u t i n a t i o n o f t h e s e g r a n u le s .
T hese w orkers r e p o r te d t h a t s i x t y - f i v e t o e ig h t y - tw o p e r c e n t
o f th e s u c c in o x id a s e a c t i v i t y o f t h e i r o r i g i n a l hom ogenates
was foun d in th e m ito c h o n d r ia i s o l a t e d by means o f th e 0 .8 8
m olar s u c r o s e . B ecau se o f th e s e f i n d in g s i t was c o n je c tu r e d
t h a t hom ogenates p rep a red w ith s u c r o s e m ig h t g iv e more
25
c h o l e s t e r o l d e s t r u c t i o n , assu m in g t h a t th e r e e x i s t s in
h e p a t ic c e l l m ito c h o n d r ia an enzyme system f o r th e d eg ra d a
t i o n o f c h o l e s t e r o l .
The le n g t h o f tim e o f h o m o g e n iz a tio n was a r b i t r a r i l y
s e t a t f i v e m in u te s in m ost o f th e e x p e rim en ts in w h ich th e
s t e e l p e s t l e was u s e d and a t fo u r m in u te s in m ost o f th e
e x p e r im e n ts in w h ich th e l u c i t e p e s t l e was u s e d . D uring
h o m o g e n iz a tio n th e p r e p a r a tio n was k e p t a t 0 ° C. b y im m ersion
o f th e h o m o g e n iz in g tub e in an i c e b a th .
F o llo w in g h o m o g e n iz a tio n th e t i s s u e s u s p e n s io n was
d e c a n te d i n t o f i f t y m i l l i l i t e r c e n t r i f u g e tu b es and sp u n , in
m ost i n s t a n c e s , a t 5 ,0 0 0 RPM f o r f i v e m in u te s . I t was a t
tem p ted t o show th e e f f e c t s o f v a r ia t i o n s in th e le n g t h o f
tim e o f c e n t r i f u g a t i o n upon th e amount o f c h o l e s t e r o l fou n d
in th e hom ogenate and a l s o upon th e h o m o g en eity o f th e
p r e p a r a tio n . T ab le I I , E xp erim ent F iv e , shows th e r e s u l t s
o f one su ch a tte m p t. The hom ogenate was p rep a red a s p r e
v i o u s l y d e s c r ib e d . Four volum es o f p o ta ssiu m c h lo r id e - s u c r o s e
s o l u t i o n w ere u s e d p e r u n i t w e ig h t o f l i v e r , and th e
hom ogenate was n o t d i l u t e d a f t e r c e n t r i f u g a t i o n . A f t e r fo u r
m in u te s c e n t r i f u g a t i o n a t 3 ,0 0 0 RPM a p o r tio n o f th e su p e r
n a ta n t was withdraw n by means o f a s y r in g e and lo n g n e e d le
d ip p ed un der th e l i p i d l a y e r f l o a t i n g on t o p . T h is p o r t io n
o f th e hom ogenate was th o r o u g h ly adm ixed, and a l i q u o t s were
ta k en f o r d e te r m in a tio n o f t o t a l c h o l e s t e r o l . The o r i g i n a l
TABLE I I
Experim ent 5
EFFECT OF VARIATIONS IN THE TIME OF CENTRIFUGATION UPON THE
HOMOGENEITY AND CHOLESTEROL CONTENT OF LIVER HOM OGENATE
Time ' Tube K le t t Mean K le tt Reading S . E. Error o f Mean x -j_qq Mg. C h o le ste r o l
C e n t r if . No. Readings t S. E. .of-Mean# Mean Reading" _________Per Gram L iv er
4 m in. la 47 44 t 2 .4 5 .5i 2 .5 5
b 39
2a 39
b 50
5a 50
b 38 .. . -
8 m in. l a 36 38 1 0 .7 1 .8 $ 2 .2 5
b 36
2a 39
b 39
3a 39
b 41
4a 36
b 36
12 m in. la 73 70 t 1 .0 1 .4$ 2 .1 0
b 69
2a 66
b 69
3a 69 «
b 72
1 m l. a liq u o t s were taken f o r t o t a l c h o le s t e r o l d eterm in a tio n from th e homoge
n ate c e n tr ifu g e d 4 and 8 m in u te s.
2 m l. a liq u o t s were taken from th e homogenate c e n tr ifu g e d 12 m in u te s.
*For c a lc u la t io n o f th e stan dard e rr o r o f th e mean th e fo llo w in g form ula was u sed :
S .E .« ( x ^ ) , where S .E . i s stan dard e r r o r o f the mean, x i s th e d e v ia t io n o f each i n d i-
N(N-1) v id u a l rea d in g from th e mean r e a d in g , and N i s the number o f rea d in g s in
a d e te r m in a tio n . For th e s ig n if ic a n c e o f th e stan dard e rr o r o f the mean, see any t e x t
on s t a t i s t i c a l m ethods. m
2 7
hom ogenate was r e c e n t r if u g e d in th e same c o n t a in e r f o u r
a d d i t i o n a l m in u te s a t 3 ,0 0 0 RPM, and a l i q u o t s w ere a g a in
ta k en in a s i m i l a r m anner. The rem a in in g o r i g i n a l hom ogenate
was f i n a l l y c e n t r if u g e d a n o th e r fo u r m in u te s , m aking a t o t a l
o f tw e lv e m in u tes c e n t r i f u g a t i o n , and a l iq u o t s were once more
rem oved f o r t o t a l c h o l e s t e r o l d e t e r m in a t io n . The d ata o f
T ab le I I i n d ic a t e t h a t more c o n s i s t e n t r e s u l t s w ere o b ta in e d
in th e d e te r m in a tio n o f th e c h o l e s t e r o l c o n te n t o f th e
hom ogenate c e n t r if u g e d e i g h t and tw e lv e m in u tes than in th e
c a se o f th e sam ples spun f o r fo u r m in u t e s , s u g g e s t in g t h a t a
more u n ifo rm hom ogenate m ig h t be o b ta in e d by lo n g e r c e n t r i f u
g a t i o n . The r e s u l t s f u r t h e r i n d ic a t e th a t th e lo n g e r a
hom ogenate i s c e n t r i f u g e d , th e l e s s i s i t s c h o l e s t e r o l con
t e n t , a f i n d i n g th a t one m igh t a n t i c i p a t e s in c e th e l i g h t e r
l i p i d s move t o th e to p .
The d a ta o f e x p e rim en t s i x , T able I I I , do n o t le n d to
th e same i n t e r p r e t a t i o n , h ow ever. Here th e p roced u re was
somewhat a l t e r e d from t h a t o f ex p erim en t f i v e . The homoge
n a te was p rep a red as above and th en c e n t r if u g e d f o r th r e e
m in u te s a t 3 ,0 0 0 RPM, f o l lo w in g w h ich m ost o f th e su p e r n a ta n t
was w ithdraw n w it h a s y r in g e and lo n g n e e d le and th o r o u g h ly
adm ixed. A liq u o t s w ere ta k en f o r im m ediate d e te r m in a tio n o f
t o t a l c h o l e s t e r o l and f o r d e te r m in a tio n a f t e r in c u b a tio n a t
3 7 ° C. The r em a in in g hom ogenate was t r a n s f e r r e d t o a n o th e r
c o n t a in e r and resp u n f o r th r e e a d d i t io n a l m in u te s; th e
28
TABLE I I I
E xperim ent 6
EFFECT OF VARIATIONS IN THE TIME OF CENTRIFUGATION UPON TEE
HOMOGENEITY AND CHOLESTEROL CONTENT OF LIVER HOMOGENATE
Time C e n tr ifu g e d 3 m in . 6 m in . 9 m in. 12 m in .
Mean K l e t t R eading
jf S . E . o f Mean
U n in cu b a ted 251 t 5 .8 201 ± 5 .8 320 t* 9 .4 2522 ± - 8 .7
S .E . o f Mean y in n
Mean 2.3% 2.8% 2.9% 3.5%
Mg. C h o le s t e r o l
Per Gram L iv e r
Un in c u b a te d 1 9 .6 1 3 .6
-----it
9 .9
Mean K l e t t R eading
± S. E . o f Mean
In cu b a te d 235 t 6 .9 162 t 2 2 .1 329 ± - 1 1 .7 249 ± -'6.5
S .E . o f Mean _ in n
------- = n s ------ ------- X 1UO
Mean 2:. 9% 13.7% 3 . 6 % 2.6%
Mg. C h o le s t e r o l
Per Gram L iv e r
In cu b a te d 1 7 .1 1 0 .6
m m
9 .6
1 m l. a l lq u o t a were tak en f o r t o t a l c h o l e s t e r o l d e te r m in a tio n
from th e hom ogenates c e n t r if u g e d 3 and 6 m in u t e s .
2 m l. a l i q u o t s w ere ta k e n from th e hom ogenates c e n t r if u g e d
9 and 12 m in u te s .
•KThe r e a d in g s o b ta in e d f o r th e 9 m in u te a l i q u o t s w ere so h ig h t h a t
th e y f e l l o f f th e l i n e a r p o r t io n o f th e sta n d a r d c h o l e s t e r o l cu rve;
th e c h o l e s t e r o l c o n t e n ts o f t h e s e sa m p le s, t h e r e f o r e , c o u ld n o t be
o b ta in e d a c c u r a t e l y .
29
s u p e r n a ta n t was s i m i l a r l y rem oved and a l i q u o t s tak en as
a b o v e. The p r o c e s s was r e p e a te d t w ic e m ore, so t h a t th e
f i n a l hom ogenate had been c e n t r if u g e d a t o t a l o f tw e lv e
m in u te s . The d a ta do n o t su p p o rt th e c o n t e n t io n t h a t more
c o n s i s t e n t r e s u l t s are o b ta in e d w it h lo n g e r c e n t r i f u g a t i o n ,
as fo u n d in ex p erim en t f i v e . H owever, i t was fo u n d , a s in
th e p r e v io u s e x p e r im e n t, t h a t th e c h o l e s t e r o l c o n c e n tr a t io n
in th e hom ogenate v a r ie s i n v e r s e l y a s th e le n g t h o f tim e th e
p r e p a r a tio n i s c e n t r if u g e d .
In a f u r t h e r a ttem p t t o p rep a re a more u n ifo rm homoge
n a t e , th e more t i g h t l y f i t t i n g l u c i t e p e s t l e and s p e c i a l l y
ground -hom ogenizing tu b e were u s e d . The hom ogenate p rep a red
was a l s o reh om ogen ized a f t e r c e n t r i f u g a t i o n in an a ttem p t t o
o b ta in more c o n s i s t e n t r e a d in g s in d u p lic a t e sa m p le s. In
ex p erim en t s e v e n , T ab le IV, th e hom ogenate was p rep a red w it h
f o u r m i l l i l i t e r s o f p o ta ssiu m c h l o r i d e - s u c r o s e s o l u t i o n p e r
gram o f l i v e r u s e d , h o m o g e n iz a tio n b e in g c a r r ie d ou t f o r
f i v e m in u te s . The p r e p a r a tio n was th e n c e n t r if u g e d e i g h t
m in u te s a t 3 ,0 0 0 RPM and th e su p e r n a ta n t w ithdraw n w it h a
s y r in g e and n e e d le and reh om ogen ized f o r f i v e m in u te s .
Q u a d r u p lic a te a l i q u o t s w ere ta k e n f o r c o n t r o l s and in c u b a te s
before- and a f t e r r e h o m o g e n iz a tio n . The d ata i n d ic a t e th a t
more c o n s i s t e n t ch eck s were o b ta in e d u s in g th e reh o m o g en ized
p r e p a r a t io n , p resu m ab ly b e c a u se o f th e f a c t th a t a more u n i
form hom ogenate was o b ta in e d . Ho s i g n i f i c a n t a l t e r a t i o n s in
30
TABLE IV
E xp erim en ts 7 , 8
EFFECT OF REHOMOGENIZATION AFTER CENTRIFUGATION UPON THE
HOMOGENEITY AND CHOLESTEROL CONTENT OF LIVER HOMOGENATE
E xperim ent Number 7 7 8 8
C o n d itio n s hom ogenized hom ogenized h om ogen ized hom ogenized
once tw ic e once tw ic e
Mean K l e t t R eading
+ S . E . o f Mean
U n in cu b a te d 178 ± 2.Q 173 t 3 .2 92 t 3 . 0 82 + 0 .5
S .E . o f Mean 100 ' '
Mian X X 1 .6 $ 1 .9 $ 3 .3 $ 0 .6 $
Mg. C h o le s t e r o l
P er Gram L iv e r
U n in cu b a ted 2 1 .8 2 1 .0 1 0 .2 9 .1
Mean K l e t t R eading
+ S . E . o f Mean
In c u b a te d 152 t 9 .8 183 + 2 .4 85 f 3 .8 8 0 + - 1 .6
S .E . o f Mean v i n n
Mean ; • 6 .4 $ 1 .3 $ 4 .5 $ 2 .0 $
Mg. C h o le s t e r o l
Per Gram L iv e r "'
In cu b a te d 1 8 .2 2 2 .4 9 .4 8 . 8
31
th e amount o f c h o l e s t e r o l w ere o b se r v e d in th e in c u b a te d a s
compared w ith th e u n in c u b a te d sa m p le s. E xperim ent e i g h t ,
T ab le IV , was done in e x a c t l y th e same manner a s ex p erim en t
s e v e n , and s i m i l a r r e s u l t s were o b ta in e d . The reh om ogen ized
p r e p a r a tio n s gave more c o n s i s t e n t r e a d in g s than th e s i n g l y
hom ogenized sa m p le s. A g a in , no s i g n i f i c a n t d i f f e r e n c e s in
th e c h o l e s t e r o l c o n te n t o f th e c o n t r o l s and in c u b a te s were
o b s e r v e d . I t was; fo u n d , h o w ev er, t h a t th e c o n s i s t e n c y o f
th e r e a d in g s was b e t t e r in th e c o n t r o l than in th e in c u b a te d
sa m p le s, i n d i c a t i n g perhaps t h a t i n c o n s i s t e n c i e s m ig h t be due
t o v a r ia t i o n s in th e amount o f c h o l e s t e r o l e x t r a c t e d from
d u p lic a t e a l i q u o t s o f hom ogen ate. T h is p o s s i b i l i t y was i n v e s
t i g a t e d , as i s r e p o r te d e ls e w h e r e in t h i s p a p er.
■ C* T reatm ent o f In cu b a te d S am p les.
In a l l th e e x p e rim en ts r e p o r te d i n w h ich hom ogenate '
was in c u b a te d , th e sam ples were k e p t a t 3 7 ° C. f o r from one
t o tw e n ty -o n e h o u r s , in m ost i n s t a n c e s ab ou t s i x t e e n h o u r s .
S c h o tz ’ (9 1 ) fo u n d t h a t th e r e was s i g n i f i c a n t change in
th e c h o l e s t e r o l c o n te n t o f r a t l i v e r hom ogenate d u r in g th e
f i r s t f i v e h ou rs o f in c u b a t io n . At tw e n ty h o u r s , h o w ev er, a
s e e m in g ly s i g n i f i c a n t d e c r e a s e was d e t e c t e d . No d e ter m in a
t i o n s were made b etw een f i v e and tw e n ty h o u r s .
In ex p erim en t t h r e e , t o be r e p o r t e d b e lo w , i t was
o b ser v ed t h a t a g r e a t e r d e c r e a s e in th e c h o l e s t e r o l c o n te n t
32
o c c u r r e d when th e hom ogenate was in c u b a te d u n d er oxygen than
when in c u b a te d u n d er n it r o g e n . The d i f f e r e n c e was s m a ll and
p r o b a b ly n o t s i g n i f i c a n t . H owever, in a l l su b seq u e n t e x p e r i
m ents th e in c u b a te d sam p les were p la c e d u n sto p p e r e d in a
c o n s ta n t h u m id ity chamber f i l l e d w ith oxygen and m a in ta in e d
a t 370 C.
D. E x t r a c t io n o f C h o l e s t e r o l .
In e x p e r im e n ts one th ro u g h e i g h t , th e u n in c u b a te d and
th e in c u b a te d sam ples w ere e x t r a c t e d w ith te n m i l l i l i t e r s o f
a t h r e e t o two m ix tu r e o f e t h a n o l - d i e t h y l e t h e r . In e x p e r i
m ents two t o fo u r one m i l l i l i t e r o f d i l u t e d h o m o gen ate,
r e p r e s e n t in g abou t o n e - s i x t h gram o f l i v e r (w et w e i g h t ) , was
e x t r a c t e d w it h te n m i l l i l i t e r s o f a l c o h o l - e t h e r , g i v in g a
r a t i o o f t i s s u e t o s o l v e n t o f a p p r o x im a te ly one to s i x t y .
In e x p e rim en ts f i v e and s i x , two m i l l i l i t e r s o f hom ogenate
r e p r e s e n t in g about t w o - f i f t h s gram o f l i v e r was e x t r a c t e d
w it h te n m i l l i l i t e r s o f a l c o h o l - e t h e r , a t i s s u e - s o l v e n t
r a t i o o f one to t w e n t y - f i v e . In e x p e rim en ts se v e n and
e i g h t , one m i l l i l i t e r o f hom ogen ate, r e p r e s e n t in g a p p r o x i
m a te ly o n e - f i f t h gram o f l i v e r , w as e x t r a c t e d w it h te n m i l l i
l i t e r s o f s o l v e n t , a t i s s u e - s o l v e n t r a t i o o f one t o f i f t y .
For e x t r a c t i o n o f c h o l e s t e r o l in th e above e x p e r im e n ts
i t was c o n s id e r e d th a t a g i t a t i o n o f th e so lv e n t-h o m o g e n a te
m ix tu r e f o r te n to f i f t e e n m in u tes a t room tem p era tu re
33
was a d e q u a te . N i e f t fou n d t h a t c h o l e s t e r o l e x t r a c t i o n from
t i s s u e hom ogenates was v i r t u a l l y co m p lete when t h e y w ere
a g i t a t e d w it h .a l c o h o l- e t h e r a t room tem p eratu re f o r a few
m in u t e s .
H ow ever, in th e p r o c e s s o f a tte m p tin g to e lim in a t e
th e i n c o n s i s t e n t r e s u l t s o b ta in e d in d e te r m in in g th e c h o l e s
t e r o l c o n te n t o f d i f f e r e n t a l i q u o t s o f th e same h om ogen ate,
th e q u e s t io n a r o s e as t o w h eth er th e e x t r a c t i o n p roced u re was
a d e q u a te . C ould n o t some o f th e d is c r e p a n c ie s be due t o
v a r ia t i o n s in th e amount o f c h o l e s t e r o l e x tr a c t e d ? R ic e (9 0 ),
a tte m p tin g to answ er t h i s q u e s t io n , fo u n d t h a t when l i v e r
hom ogenate was e x t r a c t e d w it h th e a l c o h o l - e t h e r s o l u t i o n a t
room tem p eratu re w ith a t i s s u e - s o l v e n t r a t i o o f ab ou t one to
s i x t y th e c h o l e s t e r o l e x t r a c t i o n was e s s e n t i a l l y c o m p le te ,
as shown b y th e sm a ll amount o f c h o l e s t e r o l o b ta in e d from
th e t i s s u e r e s id u e on r e e x t r a c t i o n w it h a h o t a l c o h o l - e t h e r
s o l u t i o n . H ow ever, i f th e hom ogenate had b een h e a te d t o
100° C. f o r a few m in u te s p r io r t o e x t r a c t i o n , th en th e p r o
ced u re was in a d e q u a te , f o r r e e x t r a c t i o n o f th e r e s id u e s
y i e l d e d s i g n i f i c a n t amounts o f c h o l e s t e r o l . E x t r a c t io n o f
su ch a p r e p a r a tio n was v i r t u a l l y c o m p lete i f th e t i s s u e -
s o l v e n t r a t i o was a l t e r e d to about one t o one hundred f i f t y ,
and th e p r e p a r a tio n was r e f lu x e d a t 6 0 ° C. f o r o n e - h a lf h o u r .
I t was assum ed t h a t d u rin g b o i l i n g th e c o a g u la t io n o f th e
p r o t e in s p r e v e n te d th e e x t r a c t i o n o f a s i g n i f i c a n t p o r t io n
34
o f th e s t e r o l u n l e s s more d r a s t i c c o n d it io n s were u s e d .
S in c e d u r in g in c u b a tio n some sam p les o f hom ogenate l o s e t h e i r
f l u i d c o n s i s t e n c y and become c l o t t e d , i t was d e c id e d t o a l t e r
th e e x t r a c t i o n p roced u re ev en in sam p les w hich w ere n o t su b
j e c t e d t o th e h ig h tem p eratu re w h ich R ice fo u n d i n t e r f e r e d
w ith co m p lete e x t r a c t i o n b y th e o r i g i n a l p r o c e d u r e . H en ce,
in e x p e rim en ts n in e and f o l l o w i n g , th e t i s s u e - s o l v e n t r a t i o
u s e d was one t o one hundred f i f t y and th e p r e p a r a tio n s were
r e f l u x e d f o r o n e - h a lf hour a t 5 5 ° t o 60° c. S p e c i f i c a l l y ,
one m i l l i l i t e r a l i q u o t s o f hom ogenate r e p r e s e n t in g o n e - s i x t h
gram o f l i v e r were p la c e d in t w e n t y - f i v e m i l l i l i t e r g l a s s -
s to p p e r e d v o lu m e tr ic f l a s k s , w h ich were f i l l e d abou t tw o -
t h i r d s f u l l w it h a l c o h o l - e t h e r and r e f lu x e d a t 55° t o 60° c.
f o r o n e - h a l f h o u r . A f t e r r e f l u x i n g , th e f l a s k s were f i l l e d
t o th e mark w ith more s o l v e n t , and th o r o u g h ly m ixed b e f o r e
a l i q u o t s were ta k e n .
I t has b een d e m o n stra ted b y e l e c t r o p h o r e s i s s t u d i e s
t h a t some n a t u r a l l y o c c u r r in g c h o l e s t e r o l e x i s t s in th e
form o f a p r o t e in com plex (3 0 ) . Schoenheiraer and
B reu sch (63) in 1933 n o te d th a t some o f th e s t e r o l o f w ool
i s so i n t i m a t e l y bound t o p r o t e in t h a t e th e r d o es n o t e x t r a c t
i t . F orbes and h i s co-w ork ers (30) d i s t i n g u i s h b etw een a
r e a d i l y e x t r a c t a b l e c h o l e s t e r o l f r a c t i o n in plasm a w h ich i s
p resum ab ly f r e e or o n ly l o o s e l y bound t o p r o t e in and a more
d i f f i c u l t l y e x t r a c t a b l e p o r t io n w h ic h , i t i s p o s t u l a t e d ,
35
i s bound t o p r o t e i n . In view o f t h e s e f a c t s , i t was a ttem p ted
t o d is c o v e r w h eth er c h o l e s t e r o l i s r e n d e r e d more r e a d i l y
e x t r a c t a b l e by h y d r o ly s is : o f th e t i s s u e u n d e r 'm ild c o n d it io n s
p r io r t o e x t r a c t i o n . The hom ogenate o f e x p e rim en t n in e ,
T ab le V, was p rep a red as d e s c r ib e d a b o v e , and one m i l l i l i t e r
a l i q u o t s w ere t r a n s f e r r e d t o t w e n t y - f iv e m i l l i l i t e r v o lu m et
r i c f l a s k s . O n e -h a lf o f th e c o n t r o l (u n in c u b a te d ) and o n e-
h a l f o f th e in c u b a te d f l a s k s were h y d r o ly z e d a t 6 0 ° C. f o r
t h i r t y m in u tes a f t e r th e a d d it io n o f f i v e d rops o f c o n cen
t r a t e d h y d r o c h lo r ic a c id t o e a ch f l a s k . The f l a s k s were
th en f i l l e d about t w o -t h ir d s f u l l w it h a l c o h o l - e t h e r s o l u
t i o n and r e f l u x e d f o r t h i r t y m in u tes a t 55° t o 6 0 ° C. The
f l a s k s t o w h ich h y d r o c h lo r ic a c id had n o t b een added were
r e f l u x e d s i m i l a r l y , and a f t e r a l l f l a s k s were f i l l e d t o th e
m ark, a l i q u o t s were taken f o r d e te r m in a tio n o f t o t a l c h o l e s
t e r o l . A's th e d a ta o f T ab le V i n d i c a t e , th e r e a d in g s were
s i g n i f i c a n t l y h ig h e r in th e h y d r o ly z e d sa m p le s. H owever, in
a l l th e sam ples t r e a t e d w ith h y d r o c h lo r ic a c i d , a brown
pigm ent d e v e lo p e d , w h ich was n o t c o m p le t e ly rem oved by th e
d i g i t o n i n p r e c i p i t a t i o n and w h ich u n d o u b te d ly i n f lu e n c e d th e
c o lo r im e t r i c d e te r m in a tio n o f c h o l e s t e r o l . T h is pigm ent was
p r o b a b ly hum in, lo n g known t o be form ed d u rin g th e h y d r o l y s is
o f p r o t e i n s w it h m in e r a l a c i d s . I t i s b e l i e v e d t h a t th e
pigm ent i s form ed by th e c o n d e n s a tio n o f an a ld e h y d e w ith
th e im ino group o f th e in d o le r in g o f try p to p h a n ( 3 2 ) ( 4 8 ) .
TABLE V
Experim ent 9
EFPECT OP HYDROLYSIS OP HOM OGENATE BEFORE EXTRACTION O P
. . LIPIDS UPON THE AM O UNT OP CHOLESTEROL RECOVERED
C on d ition s
. Mg. C h o le ste r o l Per
Gram L iv er t S .E . o f Mean
U nincubated
Mg. C h o le ste r o l Per
Gram L iv er ± S . E . o f Mean
Incubated
Per Cent
Change
Not h y d ro ly z ed 2 8 .4 f 0 .8 2 9 .3 ± 1 . 1
+ 3
H ydrolyzed w ith hydro
c h lo r ic a c id p r io r
to e x t r a c t io n 3 4 .4 t 1 .3 * 3 7 .0 ± 2 . 3 * + 8
In cr ea se due to
h y d r o ly s is 6 .0 (21$ in c r e a s e ) 7 .7 (26$ in c r e a s e )
------
*The sam ples h y d ro ly zed w ith h y d ro c h lo r ic a c id d evelop ed a brown pigm ent which un d ou b ted ly
a f f e c t e d th e c o lo r im e tr ic d eterm in a tio n o f c h o l e s t e r o l.
C /l
a
37
A ld eh yd es may "be fou n d b y th e a c t i o n o f th e m in e r a l a c i d on
any ca rb o h y d ra te p r e s e n t t o g iv e f u r f u r a l ( 3 2 ) . O lc o tt and
F ra e n k el-C o n ra t (5 4 ) r e p o r t th a t p y r u v ic a c id w i l l a l s o
r e a c t w it h try p to p h a n t o g iv e th e brown t o b la c k humin.
They s t a t e t h a t upon a d d it io n o f c y s t i n e t o th e h y d r o ly z in g
m ix tu r e , p a r t i c u l a r l y a t 1 0 0 ° C ., try p to p h a n i s d e s tr o y e d
w ith o u t humin f o r m a t io n , and th e s o l u t i o n rem ains w ater
c l e a r .
In ex p erim en t t e n , T ab le V I, i t was a tte m p te d t o
h y d r o ly z e th e hom ogenate w ith o u t fo r m a tio n o f th e brown p i g
m ent o b ta in e d a b o v e . Sam ples7 number f o u r , c o n t r o l and
in c u b a te d , w ere m e r e ly r e f lu x e d w ith o u t h y d r o l y s i s . Sam ples
number o n e , c o n t r o l and in c u b a te d , were h e a te d a t 60° C. f o r
o n e - h a lf hour w ith f i v e drops o f c o n c e n tr a te d h y d r o c h lo r ic
a c i d . F la s k s number two w ere h y d r o ly z e d w it h f i v e drops o f
t h i r t y p er c e n t aqueous p o ta ssiu m h y d ro x id e a t 60° C. f o r
o n e - h a lf h o u r. To f l a s k s number th r e e were added 0 .4 gram
o f u r e a , g i v i n g a f o r t y p er c e n t u r e a so lu tio n ;* t h e s e were
s i m i l a r l y h e a te d a t 6 0 ° C .- f o r o n e - h a l f h o u r . Then a l l
f l a s k s one t o fo u r were f i l l e d t o ab ou t th e t w o - t h ir d s mark
w ith a l c o h o l - e t h e r and r e f l u x e d a t 5 5 ° t o 6 0 ° C. f o r o n e - h a lf
h o u r . They were th en f i l l e d t o th e m ark, and a l i q u o t s were
taken f o r t o t a l c h o l e s t e r o l d e te r m in a tio n . Brown pigm ent
a g a in was form ed in th e sam ples t r e a t e d w it h h y d r o c h lo r ic
a c id and p r o b a b ly e x p la in s the u n u s u a lly h ig h r e a d in g s , as
TABLE VI
Experim ents 1 0 , 11
ATTEMPTS TO INCREASE THE EXTRACTABILITY OP CHOLESTEROL PRO M LIVER HOM OGENATES
Exp.
No.
S p l.
No.
Treatment
B efore
E x tr a c tio n
Mean K le tt
Reading
■ fr S .E .o f Mean
U nincubated
Mg. C h oles
t e r o l Per
Gram L iv er
U nincubated
Mean K le t t
Reading
+ S .E .o f Mean
Incubated
Mg. C h oles
t e r o l Per
Gram L iv er
Incubated
Per
Cent
Change
10 1 H ydrolyzed
w ith HC1
94 t 2 .9 * 1 5 .0 94 + 1 .4 * 1 5 .0 . 0
2: H ydrolyzed
w ith KO H
46 t 0 .9 7 .1 51 i 3 .0 7 .9
+ 1 1
5 H eated
w ith urea
54 t 0 .9 8 .2 61 ± 1 .3 9 .4 + 15
4 None 55 * 1 . 2 8 .6 76 ± 1 . 4 1 2 .0 + 4 0
11
Sample 4 above r e - 23 ± 0
e x tr a c t e d a f t e r
t i s s u e r e s id u e was
h y d ro ly zed w ith HC1
T o ta l e x tr a c te d
from sample 4
(un incub ated)
0 .5
9 a
4 5 iT 1 .0 1 .1 '
T o ta l e x tr a c te d 1 3 .1
from sample 4
( in cu b ated )
+ 44
*A "brown pigment d evelop ed in th e sam ples h y d ro ly zed w ith h y d r o c h lo r ic a c id prob ab ly
a c co u n tin g f o r th e h ig h rea d in g s o b ta in e d .
2 m l. a liq u o t s were taken fo r t o t a l c h o le s t e r o l d e ter m in a tio n from a l l f l a s k s in Exp. 10
10 m l. a liq u o t s were taken from the sam ples number 4 a f t e r r e e x t r a c t io n .
39
compared w ith th e sam p les w h ich w ere n o t so t r e a t e d . Of
i n t e r e s t i s th e f a c t t h a t th e c h o l e s t e r o l c o n te n t o f th e
p o ta ssiu m h y d r o x id e - t r e a t e d hom ogenate was s l i g h t l y lo w er
th an th e n o n -h y d r o ly z e d and a l s o th e u r e a - t r e a t e d sa m p le s.
Of ev en more i n t e r e s t i s th e f a c t t h a t th e in c u b a te d sam p les
number f o u r y i e l d e d m ark ed ly more c h o l e s t e r o l than th e
u n in c u b a te d sa m p le s. The same i s tr u e t o a l e s s e r d eg ree o f
sam ples number t h r e e , i . e . , th e c h o l e s t e r o l c o n t e n t o f th e
in c u b a te d sam p les e x c e e d e d t h a t o f th e u n in c u b a te d sa m p le s.
The r e s u l t s o f r e e x t r a c t i o n o f th e t i s s u e r e s id u e s
o f sam p les number fo u r above are shown in T ab le VI, e x p e r i
m ent e l e v e n . The o r i g i n a l a l c o h o l - e t h e r e x t r a c t s were
d e c a n te d a f t e r th e r e s id u e s h ad b een se d im e n ted b y c e n t r i f u
g a t i o n . To the r e s id u e in e a c h f l a s k was added one m i l l i
l i t e r o f a one t o one s o l u t i o n o f e t h a n o l- a c e t o n e and t h r e e
d rops o f c o n c e n tr a te d h y d r o c h lo r ic a c i d , g i v in g a pH o f
ab ou t 3 . 0 . The f l a s k s were h e a te d a t 60°. C. f o r o n e - h a lf
hour and th en n e u t r a l i z e d t o about pH 7 .0 w it h a s a t u r a t e d
s o l u t i o n o f p o ta ssiu m h y d ro x id e in n i n e t y - f i v e p e r c e n t
e t h a n o l , f o l lo w in g w hich th e y w ere f i l l e d abou t tw o - t h ir d s
f u l l w ith a lc o h o l- a c e t o n e (one t o on e) and r e f lu x e d a t
5 5 ° t o 6 0 ° C. f o r o n e - h a lf h o u r. I t was foun d th a t abou t
0 .1 1 mg. a d d i t i o n a l c h o l e s t e r o l was p r e s e n t in th e f l a s k con
t a i n i n g th e u n in c u b a te d sam ple and 0 .2 2 mg. In th e f l a s k con
t a i n i n g th e in c u b a te d sa m p le. I f i t be assum ed t h a t a l l o f
th e c o lo r was due to c h o l e s t e r o l p r e s e n t and none due t o
humin fo r m a tio n , w hich a ssu m p tio n i s p o s s i b l y n o t j u s t i f i e d
ev en though no v i s i b l e pigm ent was d e t e c t e d , th en one m ust
c o n c lu d e t h a t a d d i t io n a l c h o l e s t e r o l was rem oved from th e
t i s s u e r e s id u e s d u rin g th e seco n d e x t r a c t i o n . Assum ing th a t
0 . 5 m l. o f a l c o h o l - e t h e r e x t r a c t rem ained in f l a s k s number
fo u r from th e o r i g i n a l e x t r a c t i o n a f t e r p o u r in g o f f th e
s u p e r n a ta n t, i t can be assum ed t h a t two 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 t w e n t y - f i v e m i l l i l i t e r f l a s k s rem oved by
th e f i r s t e x t r a c t i o n rem ain ed in ea ch f l a s k and w ould be
d eterm in ed in th e r e a d in g s made on th e seco n d e x t r a c t .
H owever, two p er c e n t o f th e c h o l e s t e r o l in th e u n in c u b a te d
f l a s k number fo u r a s d e ter m in e d in ex p erim en t te n w ould be
0 .0 2 9 m g ., and th e v a lu e 0 .1 1 mg. o b ta in e d on r e e x t r a c t i o n
i s s i g n i f i c a n t . In a s i m i l a r manner two 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 in c u b a te d f l a s k number fo u r d e ter m in e d in
ex p erim en t ten w ou ld be 0 .0 4 m g ., and th e 0*22. mg. fou n d on
r e e x t r a c t i o n i n d i c a t e s t h a t more s t e r o l was o b ta in e d on th e
se co n d e x t r a c t i o n o f th e r e s i d u e . I f ea ch f l a s k c o n ta in e d
o n e - s i x t h gram o f l i v e r i n i t i a l l y , th e amount r e e x t r a c t e d
e x p r e s s e d in term s o f m illig r a m s c h o l e s t e r o l p e r gram o f
l i v e r w ould be 0 .4 9 mg. p e r gram o f l i v e r f o r th e u n in c u b a te d
sam ple and 1 .1 mg. p er gram o f l i v e r f o r th e in c u b a te d f l a s k ,
s u b t r a c t in g th e amounts c a l c u l a t e d t o have rem ain ed in th e
f l a s k from the o r i g i n a l e x t r a c t i o n . Compared w it h th e amounts
41
e x t r a c t e d i n i t i a l l y , 8 .6 4 mg. p er gram and 1 2 .0 0 mg. p e r
gram f o r t h e u n in c u b a te d and in c u b a te d sam p les r e s p e c t i v e l y ,
t h e s e v a lu e s are r e l a t i v e l y i n s i g n i f i c a n t . M oreover, th e
r e s u l t s o b ta in e d do n o t le n d su p p o rt t o th e f a c t t h a t th e
’’in c r e a s e " in c h o l e s t e r o l in th e in c u b a te d sam ples number
fo u r in e x p e rim en t te n a r e due t o in a d eq u a te e x t r a c t i o n o f
th e u n in c u b a te d s a m p le s , f o r on r e e x t r a c t i o n more c h o l e s t e r o l
was a g a in o b t a in e d from th e r e s id u e s w h ich had been in c u b a te d .
T h u s, i t w ould appear t h a t t h e r e was an a c t u a l i n c r e a s e in
th e amount o f c h o l e s t e r o l o f sam p les number fo u r in e x p e r i
m ent te n upon in c u b a tio n o f th e h o m o g en a tes, an in c r e a s e n o t
e x p la in a b le as an a r t i f a c t due t o in c o m p le te e x t r a c t i o n .
H owever, i t i s d i f f i c u l t t o e x p la in why th e o th e r sam p les o f
th e same hom ogenate in the ex p erim en t d id n o t show a s i m i l a r
change in th e c h o l e s t e r o l c o n te n t a f t e r in c u b a t io n . The
tr e a tm e n t o f t h e s e v e r a l sam p les d i f f e r e d o n ly in th e manner
in w h ich th e s t e r o l was e x t r a c t e d .
F* C h o le s t e r o l D e te r m in a tio n .
The c h o l e s t e r o l c o n te n t s o f th e l i v e r hom ogenates
w ere d e ter m in e d b y a m o d ifie d S ch oen h eim er-S p erry-C h an ey p r o
c e d u r e . An a l i q u o t o f th e a l c o h o l - e t h e r e x t r a c t o f ea ch
sam ple was ta k e n w h ich was e s t im a t e d to c o n t a in betw een 0 .1
and 0 .9 mg. o f c h o l e s t e r o l . The a l i q u o t s were e v a p o r a te d to
d r y n e s s , and th en th e sam ples were d i s s o l v e d in a one to one
42
e th a n o l- a c e t o n e s o l u t i o n and s a p o n i f i e d w ith t h i r t y - t h r e e
p er c e n t aqueous p o ta ssiu m h y d r o x id e . The sam p les were
n e u t r a l i s e d w it h f i f t e e n p er c e n t a c e t i c a c i d , u s i n g p h e n o l-
p h t h a le in as th e i n d i c a t o r . The c h o l e s t e r o l was th e n
p r e c i p i t a t e d w it h d i g i t o n i n and a llo w e d t o s ta n d th r e e h ou rs
a t 3 7 ° C. The i n s o lu b l e d i g i t o n i d e was c e n t r if u g e d down,
and th e su p e r n a ta n t d e c a n te d . A f t e r th e p r e c i p i t a t e was
w ashed w ith anhydrous e t h e r and r e c e n t r i f u g e d , t h e s u p e r
n a ta n t was d is c a r d e d ; th e p r e c i p i t a t e was th e n d r ie d and
d i s s o l v e d in g l a c i a l a c e t i c a c i d . Three m i l l i l i t e r s o f
ch lo ro fo r m were added and th e c o lo r d e v e lo p e d by a d d it io n o f
one m i l l i l i t e r o f a one t o n in e s u l f u r i c a c i d - a c e t i c an h yd rid e
m ix t u r e . The c o lo r d evelop m en t was a llo w e d t o tak e p la c e a t
3 5 ° C. f o r e x a c t l y te n m in u te s , and th en f u r t h e r r e a c t i o n
was sto p p e d b y p l a c in g th e sam ples in an i c e b a th u n t i l r e a d .
The amount o f c h o l e s t e r o l i s d e ter m in e d from th e r e a d in g s o f
th e c o lo r d e n s i t y as o b ta in e d w it h th e K lett-S u m m erson p h o to
e l e c t r i c c o lo r im e t e r , u s i n g a f i l t e r w ith maximum t r a n s m is
s i o n a t 62.0 m i l l i - m i c r a . In m ost o f the work t r i p l i c a t e
a l i q u o t s o f l i v e r hom ogenate were p rep a red f o r th e u n in c u
b a te d and f o r th e in c u b a te d sa m p le s, and a f t e r e x t r a c t i o n o f
the- c h o l e s t e r o l , d u p lic a t e a l i q u o t s o f th e a l c o h o l - e t h e r
e x t r a c t were ta k e n from each o f th e hom ogenate sam p les f o r
t o t a l c h o l e s t e r o l d e t e r m in a t io n . T hus, in m ost c a s e s s i x
r e a d in g s f o r th e c h o l e s t e r o l c o n te n t were o b ta in e d f o r
43
e a ch hom ogenate s t u d i e d . The amount o f c h o l e s t e r o l in a l i
q u o ts was r e a d d i r e c t l y o f f a sta n d a r d cu rve p rep a red by th e
d e te r m in a tio n o f known amounts o f c h o l e s t e r o l .
M ention sh o u ld be made o f th e f a c t t h a t th e r e a re
s e v e r a l c r i t i c a l s t e p s in th e p ro ced u re w h ich may m ark ed ly
a l t e r th e r e s u l t s o b ta in e d . Many o f th e d is c r e p a n c ie s
o b ta in e d in th e e a r l y e x p e rim en ts were u n d o u b te d ly due t o
th e f a c t t h a t n o t enough c a re was e x e r c i s e d in th e a c t u a l
c h o l e s t e r o l d e t e r m in a t io n s . S in c e o n ly th e f r e e c h o l e s t e r o l
and n o t th e e s t e r i f i e d p o r t io n i s p r e c i p i t a t e d by d i g i t o n i n ,
i t i s im p ortan t in th e d e te r m in a tio n o f t o t a l c h o l e s t e r o l t o
make su re t h a t s a p o n i f i c a t i o n i s c o m p le te . One m ust a g i t a t e
th e m ix tu r e o f d i s s o l v e d c h o l e s t e r o l and p o ta ssiu m h y d ro x id e
f r e q u e n t l y t o in s u r e th o ro u g h m ix in g . F u rth erm ore, b e f o r e
th e d i g i t o n i n i s added i t i s im p o rta n t t h a t a l l sam p les be
made a c id w it h a c e t i c a c i d . Care m ust be e x e r c i s e d when th e
su p e r n a ta n t i s poured o f f th e i n s o l u b l e c h o l e s t e r o l d i g i t o -
n id e so t h a t none o f th e p r e c i p i t a t e i s l o s t . Then b e fo r e
th e p r e c i p i t a t e i s d i s s o l v e d in g l a c i a l a c e t i c a c id i t i s
v e ry im p ortan t th a t i t be c o m p le te ly d r y , f o r even s l i g h t
amounts o f w a te r p r e s e n t d u rin g th e develop m en t o f c o lo r
w i l l c o n s id e r a b ly a f f e c t th e r e a d in g s . F i n a l l y , o f c o u r s e ,
th e tem p era tu re o f th e sam ples and th e tim e d u r in g c o lo r
d evelop m en t m ust be r i g i d l y c o n t r o l l e d . A f t e r th e r e a c t i o n
has been sto p p e d by lo w e r in g th e tem p era tu re o f th e sam ples
44
t o 0 ° C ., th e c o l o r la s t a b l e f o r perhaps f o r t y - f i v e
m in u te s . There a p p ears to be some f a d in g i f th e r e a d in g s
a r e n o t made b e f o r e f o r t y - f i v e m in u te s a f t e r p la c in g th e
sam ples in th e i c e b a th .
CHAPTER IV
STABILITY OP PURE CHOLESTEROL U2JDER CERTAIN CONDITIONS
The d eg ree o f r e c o v e r y o f known .amounts o f pure
c h o l e s t e r o l when s u b j e c t e d t o th e above p roced u re f o r th e
d e te r m in a tio n o f t i s s u e c h o l e s t e r o l has b een i n v e s t i g a t e d b y
Marx (8 8 ) and b y N i e f t ( 8 9 ) . They fou n d t h a t r e c o v e r y was
e s s e n t i a l l y c o m p le te , i f c a re was e x e r c i s e d d u rin g th e d e
te r m in a tio n a t c e r t a i n c r i t i c a l p o in t s in th e p r o c e d u r e .
In th e c o u rse o f a tte m p tin g to im prove the p ro ced u re
f o r e x t r a c t i o n o f t i s s u e c h o l e s t e r o l , i t was a tte m p te d , a s
above r e p o r t e d , t o h y d r o ly s e th e t i s s u e p r o t e in s b e f o r e
e x t r a c t i o n o f th e l i p i d s in o rd er t o f r e e any c h o l e s t e r o l
w h ich m ig h t be bound to p r o t e i n . The h y d r o l y s i s was c a r r ie d
ou t w it h h y d r o c h lo r ic a c i d , p o ta ssiu m h y d r o x id e , and w ith
a c e t i c a c i d . Not knowing th e s t a b i l i t y o f c h o l e s t e r o l in th e
p r e se n c e o f t h e s e r e a g e n t s , i t was d e c id e d t o i n v e s t i g a t e t h e
d e g r ee o f r e c o v e r y o f pure c h o l e s t e r o l when s u b je c t e d t o th e
c o n d it io n s u s e d in th e h y d r o l y s i s .
In e a c h o f fo u r t w e n t y - f i v e m i l l i l i t e r f l a s k s was
p la c e d 3 .7 5 mg. o f pure c h o l e s t e r o l in one m i l l i l i t e r o f a
one t o one a lc o h o l- a c e t o n e s o l u t i o n . To one f l a s k th r e e
drops o f c o n c e n tr a te d h y d r o c h lo r ic a c i d were added, g i v i n g a
r e s u l t a n t pH o f ab ou t 3 . 0 . To th e se co n d f l a s k one m i l l i l i t e r
o f g l a c i a l a c e t i c a c id was ad d ed , g i v in g a pH o f about 4 . 0 .
To th e t h i r d and f o u r t h f l a s k s were added th r e e and f i v e
drops r e s p e c t i v e l y o f a s a t u r a t e d a l c o h o l i c s o l u t i o n o f
p o ta ssiu m h y d r o x id e , g i v i n g a pH o f ab ou t 9 . 0 in b o th f l a s k s .
A l l f o u r f l a s k s were th en h e a te d a t 60° c . f o r o n e - h a lf h o u r,
b rou gh t t o ab ou t pH 7 . 0 , and f i l l e d tw o -th ih d s f u l l w ith
a l c o h o l - a c e t o n e , f o l lo w in g w hich t h e y were r e f l u x e d a t 6 0 ° C.
f o r o n e - h a lf h o u r . (The f l a s k s c o n t a in in g a c id w ere n e u t r a l
i z e d w ith a l c o h o l i c p o ta ssiu m h y d r o x id e ; t h o s e c o n t a in in g
*
p o ta ssiu m h y d ro x id e were n e u t r a l i z e d w it h d i l u t e a c e t i c
a c i d . ) A l l f l a s k s were f i l l e d t o th e mark w it h a l c o h o l -
a c e t o n e , and a L iq u o ts were ta k e n f o r t o t a l c h o l e s t e r o l
d e te r m in a tio n . As can be se e n from th e d ata o f T ab le V II,
ex p erim en t e l e v e n , t h e r e was in c o m p le te r e c o v e r y o f th e
c h o l e s t e r o l in a l l fo u r f l a s k s . A n oth er sta n d a r d was run
w h ich had n o t been s u b j e c t e d to any su c h tr e a tm e n t p r io r t o
th e d e te r m in a tio n ; h e r e r e c o v e r y was c o m p le te . The r e c o v e r y
was p o o r e s t in th e c a se o f th e sam ple t r e a t e d w it h g l a c i a l
a c e t i c a c i d . H ow ever, th e l o s s in a l l f l a s k s was la r g e
enough to be s i g n i f i c a n t , i f su ch a l o s s a l s o o c c u r s when th e
t i s s u e c h o l e s t e r o l i s so t r e a t e d . Ho a tte m p ts have b een
made t o i n v e s t i g a t e th e problem f u r t h e r , and th e above work
was n o t r e p e a t e d .
TABLE VII
Experiment 11
EFFECTS OF POTASSIUM HYDROXIDE, HYDROCHLORIC ACID, AND GLACIAL ACETIC
. ACID UPON THE RECOVERY OF PURE CHOLESTEROL
F la sk
No.
C h o le ste r o l
Content
I n i t i a l H eated With
pH
K le t t
Readings
C h o le ste r o l
Content
F in a l
Per Cent
R ecovery o f
C h o le ste r o l
l a
b
3 .7 5 mg. C oncentrated HC1 3 .0 126
12V
3 .2 5 mg. 87
2a
b
3 .7 5 mg. G la c ia l A c e tic Acid 4 .0 94
93
2 .2 8 mg. 61
3a
b
3 .7 5 mg. S a tu ra ted A lc o h o lic
KO H (3 drops)
9 .0 128
125
3 .2 5 mg. 87
4a
b
3 .7 5 mg. S a tu ra ted A lc o h o lic
KOH (5 drops)
9 .0 121
120
3 .0 6 mg. 82
5 0 .3 0 mg. No Treatment 144 0 .3 0 mg. 100
2 m l. a liq u o t s were taken from the f l a s k s 1 to 4 (25 m l. f l a s k s ) ; th e K le tt Readings
r e p r e se n t a c h o le s t e r o l v a lu e 2 /2 5 th a t r ep o rted in the column la b e le d " C h o le ste r o l Content
F in a l" .
Sample 5 was a prepared known standard c o n ta in in g 0 .3 mg. c h o l e s t e r o l .
-Cl
CHAPTER V
EFFECT OF THE LENGTH OF CHOLESTEROL FEEDING AND THE SEX OF
THE ANIMAL UPON THE DEGREE OF RECOVERY OF CHOLESTEROL
FROM LIVER HOMOGENATE AFTER INCUBATION
An a ttem p t h as been made t o c o r r e l a t e th e d e g r ee o f
r e c o v e r y o f c h o l e s t e r o l from r a t l i v e r hom ogenate a f t e r in c u
b a t io n w ith th e l e n g t h o f tim e th e anim al was on th e h ig h
c h o l e s t e r o l d i e t , th e s e x o f th e a n im a l, and a l s o th e s i z e
o f th e l i v e r (T a b le V I I I ) . Such a c o r r e l a t i o n I s perhaps
n o t s i g n i f i c a n t in view o f th e f a c t s t h a t th e a n im a ls
compared were n o t a l l l i t t e r m ates and th e a g e s a t th e tim e
th e a n im a ls were s t a r t e d on th e c h o l e s t e r o l f e e d i n g were n ot
i d e n t i c a l . F u rth erm ore, th e c h o l e s t e r o l in ta k e was not
r i g i d l y c o n t r o l l e d , f o r th e a n im a ls were f e d th e d i e t ad
l i b i t u m .
I t i s im p o rta n t t o r e c o g n iz e t h a t th e v a lu e f o r th e
c h o l e s t e r o l c o n t e n t o f th e hom ogenates r e p o r te d in a l l th e
e x p e rim en ts in term s o f m illig r a m s o f c h o l e s t e r o l p er gram o f
l i v e r u s e d in th e p r e p a r a tio n o f th e hom ogenate i s n o t a
v a l i d in d e x o f th e c h o l e s t e r o l c o n te n t o f th e l i v e r i t s e l f .
As was shown in e x p e r im e n ts f i v e a n d . s i x , th e c h o l e s t e r o l con
t e n t o f a hom ogenate t h a t h a s been c e n t r if u g e d v a r ie s
I n v e r s e l y w it h th e l e n g t h o f tim e and (u n d o u b te d ly ) th e
sp eed o f th e c e n t r i f u g a t i o n . B ecau se o f th e v a r i a t i o n in
th e le n g t h o f tim e o f c e n t r i f u g a t i o n o f th e hom ogenates
TABLE V III
ATTEMPT TO CORRELATE THE LENGTH OF CHOLESTEROL FEEDING, THE SEX OF ANIMAL, AND THE
WEIGHT OF THE LIVER WITH THE DEGREE OF RECOVERY OF CHOLESTEROL AFTER INCUBATION
Mg. C h o les- Mg. C holes
Length o f Animal Wt. t e r o l Per t e r o l Per
Exp. C h o le ste r o l when L iv er L iv er Wt. v Gram L iv er
Animal Wt. U nincubated
Gram L iv er Per Cent
No. Sex F eeding S a c r if ic e d Wt. Incubated Change
9 M 0 days 215 gm. 7 .6 gm. 3 .5 % 1 .8 1 .9 +*6
9 M 29 22,0 1 2 .4 5 .6 2 8 .4 2 9 .3 * 3
12 M 31 233- 1 1 .5 4 .9 1 6 .9 1 5 .4 -9
3 M
56
w m m l
1 3 .9
m . m
8 .8 4 .4 -50
4 M 57 353 1 4 .2 4 .0 1 0 .9 2 .3 -79
12 M 71 340 1 6 .9 5 .0 2 3 .2 2 1 .0 -1 0
6 M 72 320 ----- p o o led (
1 9 .6
1 7 .1 -1 3
365
--------------
-----homogenate (
7 M 90 300 2 1 ,8 1 8 .2 -1 6
8 M 91 285 1 0 .2 9 .4 -8
9 M 148 325 1 7 .6 5 .4 1 4 .5 1 4 .3 -1
2 M 1502
-------------- -------------
1 8 .8 6 .6 -65
13 F 7 215 1 1 .4 5 .3
10 F 41 190 7 .9 4 .2 8 .6 1 2 .0 440
13 F 41 200 7 .8 3 .9 1 6 .9 1 7 .5 + 4
'Jp The o m itted v a lu e s in th e t a b le are due to the f a c t th a t some o f th e data was not
record ed in s e v e r a l o f th e ex p erim en ts.
2 The fe e d in g p e r io d was not known a c c u r a te ly in experim ent 2; i t was b e lie v e d to be
about 5 m onths.
< o
50
p rep a red in th e v a r io u s e x p e r im e n ts r e p o r t e d and th e o th e r
v a r ia t i o n s in th e p r e p a r a tio n o f th e h o m o g e n a te s, th e v a lu e s
f o r th e c h o l e s t e r o l c o n te n t b e f o r e in cu ib ation do n o t g iv e
r i s e t o a sm ooth or i n t e l l i g i b l e curve when p l o t t e d a g a in s t
th e l e n g t h o f c h o l e s t e r o l f e e d i n g .
Nor was t h e r e any c o r r e l a t i o n fo u n d b etw een th e
l e n g t h o f c h o l e s t e r o l f e e d in g and th e changes in th e c h o l e s
t e r o l c o n te n t o f th e hom ogen ates a f t e r in c u b a t io n , a s
d e ter m in e d in th e e x p e r im e n ts r e p o r t e d .
Of i n t e r e s t , b u t o f q u e s t io n a b le s i g n i f i c a n c e , i s th e
f a c t t h a t in a l l c a s e s where s t a t i s t i c a l l y s i g n i f i c a n t
"breakdowntr o f c h o l e s t e r o l was fou n d th e a n im a ls u s e d were
m a le s . In th e one in s t a n c e in w h ich a s i g n i f i c a n t in c r e a s e
in c h o l e s t e r o l r e s u l t e d a f t e r in c u b a tio n th e an im al u s e d was
a f e m a le .
CHAPTER VI
RESULTS OP ATTEMPTS TO DEMONSTRATE CHANGES IN THE AM OUNT
OP CHOLESTEROL IN HOMOGENATES AFTER INCUBATION
In an e a r l y e x p e rim en t i t was a tte m p te d to d eterm in e
w h eth er any d e s t r u c t io n o f c h o l e s t e r o l c o u ld he o b se r v e d
d u rin g in c u b a tio n o f hom ogenates w h ich had b een s t o r e d u n d er
d i f f e r e n t c o n d i t i o n s . I t was hoped t h a t su ch s t o r e d p r e p a
r a t i o n s would show a c t i v i t y , th e r e b y o b v ia t in g th e n e c e s s i t y
o f p r e p a r in g f r e s h t i s s u e e x t r a c t e a c h tim e an ex p erim en t
was r u n . The l i v e r s o f t h r e e m ale r a t s were hom ogenized as
p r e v io u s l y d e s c r i b e d , and a l i q u o t s o f th e p o o le d hom ogenate
were ta k en f o r im m ediate d e te r m in a tio n o f c h o l e s t e r o l ;
s i m i l a r a l i q u o t s w ere in c u b a te d f o r tw e n ty h ou rs a t 3 7 ° C.
A p o r t io n o f th e rem ain in g hom ogenate was s t o r e d in th e
r e f r i g e r a t o r one week w it h 1 : 4 0 ,0 0 0 added aqueous Z ep h ira n .
A t h i r d p o r t io n was s t o r e d in th e r e f r i g e r a t o r one week
w ith o u t added p r e s e r v a t i v e . F i n a l l y , th e rem aind er was
l y o p h i l i z e d f o r tw e lv e h ou rs and th e n s t o r e d in th e f r e e z i n g
compartment o f th e r e f r i g e r a t o r f o r one w eek. ..The s t o r e d
sam p les were th en removed and a l i q u o t s ta k e n f o r im m ediate
d e te r m in a tio n o f c h o l e s t e r o l ; s i m i l a r a l i q u o t s were in c u
b a te d tw e n ty h o u r s . (The l y o p h i l i z e d p o r t io n was made up to
i t s o r i g i n a l volume b e fo r e a l i q u o t s w ere t a k e n .) As th e
d a ta o f T ab le IX i n d i c a t e , th e u n s to r e d sam p les showed a
TABLE IX
Experiment 2
EFFECT OF STORAGE OF LIVER HOM OGENATE UNDER DIFFERENT CONDITIONS
UPON THE DEGREE OF RECOVERY OF CHOLESTEROL AFTER INCUBATION
Sample
S torage
C on ditions
Mean K le tt
Reading
I S .E . o f Mean
U nincubated
Mg. C h oles
t e r o l Per
Gram L iv er
U nincubated
Mean K le tt
Reading
+ S .E . o f Mean
Incubated
Mg. C h o les
t e r o l Per
Gram L iv er
Incubated
Per Cent
Change
A Not sto r e d 135 t 8 .7 1 8 .8
49 t 3 .9
6 .6 -65
Bl S tored 1 wk. in
r e f r ig e r a t o r 140 ib 2 .7 1 9 .8
142. + 3 .6
2 0 .2 + 2
2 Same 141 t 2 .8 1 9 .8 155 + 5 .5 2 2 .2 -P12
C S tored 1 wk, in
r e f r ig e r a t o r w ith
lr 4 0 ,0 0 0 Zephiran 129 ± 5 .4 1 7 .9 143 t 4 .0 2 0 .2 4-13
D L y o p h iliz e d and
s to r e d 1 wk. in
r e f r ig e r a t o r 1 5 6 't 3 .9 22;. 4 143 + 4 .0 2D.2 -1 0
cn
to
53
d e c r e a s e o f s i x t y - f i v e p er c e n t in c h o l e s t e r o l c o n te n t a f t e r
in c u b a t io n . In th e c a se o f each o f th e s t o r e d sam ples no
s i g n i f i c a n t a l t e r a t i o n in th e c h o l e s t e r o l c o n t e n t o f th e
hom ogenate was o b se r v e d a f t e r in c u b a t io n . I t i s tem p tin g to
c o n c lu d e t h a t u n d e r 'th e c o n d it io n s o f s t o r a g e th e l i v e r
p r e p a r a tio n s somehow l o s t t h e i r a b i l i t y t o d egrad e c h o l e s t e r o l.
I t was s u b s e q u e n tly a tte m p te d t o d eterm in e w h eth er
su ch "breakdown’1 o f c h o l e s t e r o l c o u ld be a c c e le r a t e d by
a g i t a t i n g th e hom ogenates In an oxygen atm osphere d u rin g in c u
b a t i o n . As th e d a ta o f ex p erim en t t h r e e , T a b le X, i n d i c a t e ,
th e hom ogenates th rou gh w h ich oxygen had been b u b b led p r io r
t o in c u b a tio n showed th e g r e a t e s t d e c r e a s e in c h o l e s t e r o l
c o n t e n t . D i f f i c u l t t o e x p l a i n , h o w ev er, i s th e f a c t t h a t
th e sam ples th rou gh w h ich n it r o g e n had b e e n b u b b led b e fo r e
in c u b a tio n showed e s s e n t i a l l y th e same d e c r e a s e in c h o l e s
t e r o l , w hereas th o s e s t o r e d in a c o n s ta n t h u m id ity chamber
f i l l e d w ith oxygen d u rin g in c u b a tio n showed no s i g n i f i c a n t
c h o l e s t e r o l l o s s . W hether or n o t th e p r e p a r a tio n s were
a g i t a t e d d u r in g in c u b a tio n in a c o n s ta n t sh a k in g d e v ic e had
no i n f lu e n c e upon th e c h o l e s t e r o l c o n t e n t s o f th e in c u b a te d
s a m p le s. ( I t sh o u ld be rem arked t h a t th e n it r o g e n was n o t
in tr o d u c e d in su ch a f a s h io n t h a t a l l o f th e a i r was d i s
p la c e d from th e t u b e s , i . e . no d ou b le s t o p - c o c k arrangem ent
was u s e d . )
TABLE X
Experim ent 3
EFFECT OF AGITATION AND THE PRESENCE OF OXYGEN DtJRING INCUBATION
. OF LIVER HOM OGENATE UPON THE RECOVERY OF CHOLESTEROL
Sample
C on d ition s
Daring J
In cu b ation
Mean K le tt
Reading
S .E . o f Mean
U nincubated
Mg. C h oles- Mean K le tt
t e r o l Per Reading
Gram L iv er t S .E . o f Mean
U nincubated Incubated
Mg. C h oles
t e r o l Per
Gram L iv er
Incubated
Per
Cent
Change
A Not in cu b ated
( c o n tr o l) 66 t 3 .0 8 .8 5
------- ------
B Incubated under
oxygen in c o n sta n t
hu m id ity chamber ----- 54 t 7 .0 7 .2 6 -18
C Oxygen bubbled
through homogenate
b e fo r e in c u b a tio n ----- 34 t 6 .3 4 .4 3 -50
D Oxygen bubbled
through homogenate;
in cu b ated in c o n sta n t
shaking d e v ic e 34 t 1 .9 4 .4 3 -50
E N itrogen bubbled
through homogenate
b e fo r e in c u b a tio n
mm mm «
39 i 4 .7 5.08 -43
F N itrogen bubbled
through homogenate;
in cu b a ted in c o n sta n t
sh akin g d e v ice
w . tm
39 1 3 .4 5 .0 8 -43
55
The a n im a ls u s e d in m ost o f th e work w ere c h o l e s t e r o l
f e d a n im a ls , i t b e in g assum ed t h a t i f th e l i v e r were ca p a b le
o f d e g r a d in g c h o l e s t e r o l su ch a p r o c e s s would b e a c t i v a t e d
by e x c e s s f e e d in g o f th e s t e r o l . I t was h o p ed , h ow ever,
t h a t l i v e r p r e p a r a tio n s from norm al r a t s m ig h t show some
a c t i v i t y . T h e r e fo r e , a com parison was made o f th e d eg ree o f
r e c o v e r y o f c h o l e s t e r o l from th e in c u b a te d l i v e r hom ogenates
p r e p a r ed from a norm al r a t , from a r a t on th e h ig h c h o l e s
t e r o l d i e t fo u r w eeks (tw e n ty -n in e d a y s ) , and from an anim al
on th e d i e t f o r f i v e months (148 d a y s ) . As can be se e n from
th e d a ta o f T ab le X I, no s i g n i f i c a n t a l t e r a t i o n o f th e
c h o l e s t e r o l c o n te n t o f any o f th e hom ogenates o b ta in e d a f t e r
in c u b a tio n was fo u n d . For t h i s r e a s o n , no c o n c lu s io n s may
be drawn r e g a r d in g th e a b i l i t y o f l i v e r hom ogenates from th e
norm al r a t t o a l t e r t h e i r c h o l e s t e r o l c o n te n t on in c u b a t io n ,
e x c e p t t h a t u n d er th e c o n d it io n s o f t h i s e x p e rim en t no su ch
a l t e r a t i o n was d e m o n str a b le .
In th e p r o c e s s o f a tte m p tin g t o answ er th e q u e s tio n
why ap p aren t "breakdown1 1 or " s y n t h e s is " o f c h o l e s t e r o l
o c c u r r e d in some e x p e rim en ts and n o t in o t h e r s , i t was con
j e c t u r e d t h a t perhaps c e r t a i n l a b i l e c o f a c t o r s were an
i n t e g r a l p a r t o f th e p r o c e s s b u t were d e s tr o y e d u n d er c e r t a i n
c o n d i t i o n s . I t was f u r t h e r s u g g e s te d a s a p o s s i b i l i t y t h a t
by a l t e r i n g th e c o n c e n t r a t io n s o f su ch c o f a c t o r s in th e
hom ogenate one m igh t e f f e c t a p rep on d eran ce o f e i t h e r th e
TABLE XI
Experim ent 9
EFFECT OF LENGTH OF CHOLESTEROL FEEDING UPON THE DEGREE OF RECOVERY
OF CHOLESTEROL FR O M LIVER HOM OGENATE AFTER INCUBATION
Length of
C h o le ste r o l
F eeding
Mg, C h o le ste r o l Per
Gram L iv er jh’S .E . of Mean
Unincuhated
Mg. C h o le s te r o l Per
Gram L iv er |r S .E . o f Mean
Incubated
Per Cent
Change
0 days 1 .9 0 t ' 0 .0 8 1 .9 0 ± 0 .0 3 4-4
29 2 8 .4 t 0 .8 2 9 .3 t 1 .1 4- 3
148 1 4 .7 t 0 .2 1 4 .1 jf 0 ,4 -4
0 1
0 1
57
d e s t r u c t i v e or s y n t h e t i c r e a c t i o n . In T ab le X I I , exp erim en t
t w e lv e , th e r e s u l t s o f su ch an attem p t a r e sum m arized. One
hom ogenate was p rep a red from a m ale r a t on a h ig h c h o l e s
t e r o l in ta k e f o r fo u r w eeks ( t h i r t y - o n e d a y s ) , in th e manner
p r e v io u s l y d e s c r ib e d . A. se co n d hom ogenate was p rep a red u s in g
th e l i v e r from a m ale r a t on h ig h c h o l e s t e r o l in ta k e f o r te n
weeks (s e v e n t y -o n e d a y s ) . Two m i l l i l i t e r s o f 0 .8 8 m olar
s u c r o s e (w ith o u t added p o ta ssiu m c h l o r i d e ) p e r gram o f l i v e r
w ere u s e d in p r e p a r a tio n o f th e h o m o g en a tes. A f t e r c e n t r i f
u g a t io n and r e h o m o g e n iz a tio n o f th e s u p e r n a t a n t s , 0 .5 m i l l i
l i t e r a l i q u o t s o f e a c h p r e p a r a tio n were t r a n s f e r r e d t o
t w e n t y - f i v e m i l l i l i t e r v o lu m e tr ic f l a s k s , some o f w h ich were
t o be e x t r a c t e d im m e d ia te ly f o r c h o l e s t e r o l and th e rem aind er
t o be in c u b a te d . To some o f b o th th e f l a s k s t o be e x t r a c t e d
im m e d ia te ly and th o s e t o be in c u b a te d was added 0 . 5 m i l l i l i t e r
o f d i s t i l l e d w a te r . To th e r em a in in g f l a s k s ( c o n t r o l s and
in c u b a t e s ) 0 .5 m i l l i l i t e r o f a s p e c i a l s o l u t i o n ( s o l u t i o n A)
was add ed . T h is s o l u t i o n was so p rep a red t h a t on d i l u t i o n
one to o n e , a s in th e e x p e r im e n t, th e f o l l o w i n g c o n c e n t r a t io n s
w’er.e o b ta in e d in th e h o m o g e n a te s;
M o la r ity
Magnesium s u l f a t e
P o ta ssiu m c h lo r id e
1 - m a lic a c id
P hosp hate b u f f e r (pH 7 .4 )
A d en osin e t r ip h o s p h a te
Cytochrome £
Coenzyme I
0 .0 0 0 2 5
0 .0 0 2 5
0 .0 0 0 0 2 5
0.01
0.0001
10“6
4 .6 x 1 0 “4
TABLE X II
Experim ents 12;., 13
EFFECTS OF ADDED COFACTORS UPON THE RECOVERY OF CHOLESTEROL
FRO M LIVER HOM OGENATE AFTER INCUBATION
Exp.
No.
Length o f
C h o le ste r o l
F eeding
C ofactors
Addedi
Mean K le tt
Reading
t S .E . o f Mean
U nincubated
Mg. C h oles
t e r o l Per
Gram L iv er
U nincubated
Mean K le t t
Reading
± S .E . o f Mean
Incubated
Mg. C h oles
t e r o l Per Per
Gram L iv e r Cent
Incubated Change
12 31 days None 110 ± 1 . 0 1 6 .9 101 ± 3 .5 1 5 .4 - 9
S o lu tio n A
107 f 1 .3 1 6 .5 97 ± 2 .6 1 5 .0 - 9
71 None 144 ± 2 .0 2 3 .2 133 ± 0 . 8 2 1 .0 -1 0
S o lu tio n A 144 ± 0 .5 2 3 .2 134 ± 0 . 9 2 1 .4 — 8
13 7 days None 306 £ 3 .5
___ 2
296 + 4 .4
S o lu tio n B 302 £ 2 .9
w m m m w
300 ± 9 .0
------ — -
S o lu tio n C 296 £ 6 .7
------
286 ± 9 . 6
41 None 104 £ 1 .6 1 6 .9 106 ± 0 .7 1 7 .5 + 4
S o lu tio n B 105 * 1 .8 1 7 .2 106 ± 0 .7 1 7 .5 + 2
S o lu tio n C 106 - k 0 .4 1 7 .5 106 ± 0 .4
1 7 .5 0
1 For com p osition o f c o fa c to r s o lu t io n s se e t e x t o f t h e s i s .
2 S in ce the a liq u o t s f o r c h o le s t e r o l d eterm in a tio n from the 7 day animal were much to o
l a r g e , th e read in gs f e l l beyond the l in e a r p o r tio n o f th e stan dard curve and th e
c h o le s t e r o l c o n te n ts could n ot be c a lc u la t e d w ith a ccu ra cy .
oi
oo
59
As can be se e n from th e d a ta o f T a b le X I I , th e amount o f
c h o l e s t e r o l r e c o v e r e d a f t e r in c u b a tio n from th e hom ogenates
o f b o th th e fo u r week and th e t e n week anim al w ere th e same
w h eth er or n o t th e above d e s c r ib e d c o f a c t o r s o l u t i o n was
ad d ed . The s l i g h t d e c r e a s e in c h o l e s t e r o l o b ser v ed i n a l l
sam p les a f t e r in c u b a tio n ap p ears s i g n i f i c a n t . W hether or
n o t t h i s r e p r e s e n t s a c t u a l breakdown o r m e r e ly an a r t i f a c t
due t o e x t r a c t i o n i s d i f f i c u l t to d e te r m in e . The t i s s u e
r e s id u e s were n o t r e e x tr a c te d .. H ow ever, a c c o r d in g t o th e
work o f R ic e (9 0 ) a lr e a d y r e f e r r e d t o , th e m ethods u s e d in
t h i s in s t a n c e f o r e x t r a c t i o n ( r e f l u x i n g o f th e hom ogenate a t
6 0° C. f o r o n e - h a l f hou r w it h a l c o h o l - e t h e r ) a r e e f f e c t i v e
in rem oving v i r t u a l l y a l l o f th e c h o l e s t e r o l .
E xperim ent t h i r t e e n , T ab le X I I , was co n d u cte d In a
s i m i l a r m anner. H om ogenates were p rep a red as in th e e x p e r i
ment above from th e l i v e r s o f two a n im a ls , one o f w h ich had
b een on a h ig h c h o l e s t e r o l d i e t se v e n d a y s , th e o th e r f o r
f o r t y - o n e d a y s . C o n tro l and in c u b a te d sam p les w ere ta k e n
from ea ch hom ogen ate. To o n e - t h ir d o f th e c o n t r o l s and
in c u b a te s o f e a c h hom ogenate was added d i s t i l l e d w a te r . To
a n o th e r t h i r d was added a s o l u t i o n ( s o l u t i o n B) g i v in g th e
same f i n a l c o n c e n tr a t io n o f c o f a c t o r s a s in th e p r e v io u s
e x p e r im e n t. (In t h i s e x p e r im e n t, h o w ever, a n o th e r sam ple o f
ATP and o f cytochrom e _ c o f known a c t i v i t y w ere u s e d . ) To
th e rem a in in g o n e - t h ir d o f th e f l a s k s was added a c o f a c t o r
s o l u t i o n ( s o l u t i o n C) w h ich gave th e f o l l o w i n g f i n a l con
c e n t r a t i o n s in th e h o m o g e n a te s:
In o t h e r w o rd s, th e c o n c e n tr a tio n s o f ATP and cytochrom e c.
w ere in c r e a s e d by a f a c t o r o f te n o v e r t h a t o f t h e f l a s k s
f o r t i f i e d w it h s o l u t i o n B. In t h i s i n s t a n c e a g a in th e
d eg ree o f r e c o v e r y o f c h o l e s t e r o l from th e hom ogenates a f t e r
in c u b a tio n was th e same w h eth er or n o t th e c o f a c t o r s o l u t i o n s
were add ed . T h is t im e , h o w ev er, no s i g n i f i c a n t d i f f e r e n c e s
were fo u n d in th e c h o l e s t e r o l c o n te n t s o f t h e c o n t r o l and
in c u b a te d sa m p le s.
One f u r t h e r f i n d in g o f s i g n i f i c a n c e , w h ich has a lr e a d y
b een r e p o r te d in th e s e c t i o n on m e th o d s, b u t w h ich m e r it s
f u r t h e r c o n s id e r a t io n i s t h a t o f e x p e rim en t t e n , T ab le VI.
As s t a t e d p r e v i o u s l y , th e r e was a s i g n i f i c a n t in c r e a s e in
th e c h o l e s t e r o l c o n te n t o f th e in c u b a te d hom ogenates number
f o u r , an i n c r e a s e w h ich cannot be r e a d i l y a t t r i b u t e d t o
in a d eq u a te e x t r a c t i o n o f th e u n in c u b a te d sa m p le s.
M o la r ity
Magnesium s u l f a t e
P o ta ssiu m c h lo r id e
1 -m a lic a c id
P hosp hate b u f f e r (pH 7 . 4 )
A d en o sin e tr ip h o s p h a te
Cytochrome o _
Coenzyme I
0 .0 0 0 2 5
0 .0 0 2 5
0 .0 0 0 0 2 5
0.01
0 .0 0 1
1 0 ‘ 5 „
4 . 6 x 10"4
CHAPTER V II
DISCUSSION
A ttem p ts t o i n t e r p r e t th e above or any s i m i l a r d a ta
m ust be made o n ly w it h c a r e f u l c o n s id e r a t io n o f th e a c c u r a c y ,
s e n s i t i v i t y , and l i m i t a t i o n s o f th e m ethods u s e d f o r ob
t a i n i n g th e d a t a . In th e work u n d er c o n s i d e r a t io n , i t b e a r s
em p hasis t h a t th e m ethod u s e d f o r th e d e te r m in a tio n o f
“c h o l e s t e r o l ” i s n o t s p e c i f i c f o r t h a t s t e r o l . In g e n e r a l,
t h e r e b e in g a few e x c e p t i o n s , any s t e r o l w ith a f r e e h y d r o x y l
group a t carbon atom t h r e e , c i s w it h r e s p e c t to th e m eth yl
group on carbon t e n , and h a v in g a d o u b le bond b etw een carbon
atoms f i v e and s i x w i l l be d e t e c t e d by th e S ch oen h eim er-
S p erry m eth od . For p r a c t i c a l p u r p o s e s , th e o n ly su ch s t e r o l
p r e s e n t n a t u r a l l y in anim al t i s s u e s in d e t e c t a b l e amounts i s
c h o l e s t e r o l . H ow ever, in a tte m p tin g t o d em o n stra te
“d e s t r u c t i o n ” o f c h o l e s t e r o l in v i t r o by t i s s u e p r e p a r a tio n s
i t i s im p o rta n t t o r e a l i s e th a t any a p p a ren t l o s s o f th e
s t e r o l as d eterm in ed b y th e above m ethod m ig h t be due t o
r e l a t i v e l y m inor ch an ges in th e s t e r o i d n u c le u s , th u s
r e n d e r in g th e s t e r o l n o n - d e t e c t a b le b y th e d i g i t o n i d e p r e
c i p i t a t i o n a n d /o r th e L ieberm ann-B urchard r e a c t i o n . Such a
change in th e m o le c u le w o u ld , h ow ever, be s i g n i f i c a n t , o f
c o u r s e , f o r th e anim al organism i s h i g h l y d is c r im in a t in g in
i t s r e sp o n s e t o v a r io u s s t e r o l s , i . e . , two s t e r o l s w h ich
62.
a p p a r e n tly d i f f e r o n ly s l i g h t l y in s t r u c t u r e may p la y v a s t l y
d i f f e r e n t r o l e s in th e p h y s io lo g y o f th e o rg a n ism .
Are th e changes in c h o l e s t e r o l c o n te n t o f th e hom oge-
n a te s a f t e r in c u b a t io n r e p o r te d a b o v e , t h e n , s i g n i f i c a n t ?
Many, i f n o t th e m a j o r i t y , o f such changes a r e n o t . Some
a re u n d o u b te d ly due to in a c c u r a c ie s in th e d e te r m in a tio n o f
th e s t e r o l , p a r t i c u l a r l y i n th e e a r l y e x p e r im e n ts .
A d m itte d ly , n o t enough a t t e n t i o n was g iv e n t o c e r t a i n c r i t i
c a l s t e p s in th e p r o c e d u r e . A nother so u rc e o f e r r o r and
v a r i a t i o n in th e r e s u l t s was th e la c k o f u n if o r m it y o f th e
hom ogenates in some e x p e r im e n ts , as was borne out by th e
i n v e s t i g a t i o n s o f t h i s p rob lem . I t was r e c o g n iz e d t h a t so
lo n g as a c c u r a te ch eck s c o u ld n o t be o b ta in e d on i d e n t i c a l
a l i q u o t s o f th e same h om ogen ate, i t was f u t i l e t o a ttem p t t o
d em o n stra te any a l t e r a t i o n s in th e c h o l e s t e r o l c o n t e n t a f t e r
in c u b a t io n . In th e a tte m p t to a v o id su ch v a r i a t i o n s in th e
r e s u l t s o b ta in e d from i d e n t i c a l a l iq u o t s i t was f u r t h e r
r e a l i z e d t h a t in a d eq u a te e x t r a c t i o n o f th e s t e r o l m igh t be a
f a c t o r . The work b y R ic e a lr e a d y m en tio n ed and some o f th e
d a ta p r e s e n te d above w ould ap p ear t o p r e c lu d e th e p o s s i b i l i t y
o f in a d eq u a te e x t r a c t i o n a c c o u n tin g f o r a l l o f th e r e s u l t s
o b t a in e d . The r e s u l t s o f th e s to r a g e e x p e r im e n t, T ab le IX,
do n o t le n d t o r e a d y e x p la n a t io n as b e in g m e r e ly a r t i f a c t s
o f e x t r a c t i o n . The c h o l e s t e r o l c o n t e n ts o f th e u n in c u b a te d
s a m p le s , b o th th o s e s t o r e d and th o s e n o t s t o r e d , were
63
e s s e n t i a l l y th e sam e. The in c u b a te d sam p les w h ich had n o t
been s t o r e d , h o w ev er, showed much l e s s c h o l e s t e r o l th a n th e
in c u b a te d hom ogenates w h ich had b een s t o r e d . I f one were t o
h
e x p la in t h e s e d i f f e r e n c e s on th e b a s i s o f v a r ia t i o n s in
e x t r a c t a b i l i t y , th en he w ould have t o assume t h a t in c u b a t io n
o f a hom ogenate c a u se s a d e c r e a s e in th e e x t r a c t a b i l i t y o f
c h o l e s t e r o l , a d e c r e a s e -which d oes n o t o c cu r i f th e sam ple
h a s b een p r e v i o u s l y s t o r e d a t 0 ° C. An a l t e r n a t i v e e x p la n a
t i o n w h ich i s , a p r i o r i , more p l a u s i b l e i s t h a t l i v e r homoge
n a t e , w h ich In th e f r e s h s t a t e i s c a p a b le o f d e g r a d in g
c h o l e s t e r o l , l o s e s t h i s a b i l i t y on s t o r a g e .
F u rth erm o re, i f a l l o f th e a p p a r e n tly s i g n i f i c a n t
ch an ges in th e c h o l e s t e r o l c o n te n t a f t e r in c u b a tio n w ere t o
be e x p la in e d a s a r t i f a c t s o f e x t r a c t i o n , th e n one w ou ld be
l e d t o assum e th a t in exp erim en t th r e e th e b u b b lin g o f
n it r o g e n and oxygen th ro u g h th e hom ogenates p r io r t o in c u b a
t i o n r e n d e r e d th e c h o l e s t e r o l l e s s e x t r a c t a b l e , an a ssu m p tio n
w h ich i s v e r y u n l i k e l y . In ex p erim en t te n a s i g n i f i c a n t
in c r e a s e in th e c h o l e s t e r o l c o n te n t o f some o f th e in c u b a te d
sam p les was n o t e d . A gain a r e we t o assume th a t in t h i s ca se
th e s t e r o l was more e a s i l y e x tr a c t e d a f t e r th e t i s s u e had
been in c u b a ted ? On r e e x t r a c t i o n o f th e t i s s u e r e s id u e s o f
t h e s e sam p les i t was a g a in fou n d t h a t more c h o l e s t e r o l was
o b ta in e d from th e in c u b a te d sa m p le, th u s d i s c r e d i t i n g su ch
an e x p la n a t io n .
64
The one ex p erim en t done on th e e f f e c t s o f a c id s and
a l k a l i . ’ upon th e r e c o v e r y o f pure c h o l e s t e r o l r a i s e d some
i n t e r e s t i n g and im p o rta n t q u e s t io n s as t o th e v a l i d i t y o f
th e m ethods u se d f o r c h o l e s t e r o l d e t e r m in a t io n s , q u e s t io n s
w h ich have n o t b een f u r t h e r i n v e s t i g a t e d . The d a t a , w hich
i n d ic a t e d d e s t r u c t io n or a l t e r a t i o n o f pure c h o l e s t e r o l upon
tr e a tm e n t w ith h y d r o c h lo r ic a c i d , a c e t i c a c i d , or p o ta ssiu m
h y d r o x id e , i s n o t r e a d i l y r e c o n c il e d w ith th e known f a c t s .
Schoenheim er and B reu sch (6 3 ) r e p o r te d e s s e n t i a l l y co m p lete
r e c o v e r y o f known amounts o f c h o l e s t e r o l t r e a t e d w it h sodium
h y d r o x id e un der more v ig o r o u s c o n d it io n s o f tem p era tu re and
a l k a l i n i t y than done in th e above e x p e r im e n t. F u rth erm o re,
i n th e m ethod f o r d e te r m in a tio n o f c h o l e s t e r o l u s e d a b o v e ,
th e c h o l e s t e r o l i s r o u t i n e l y d i s s o l v e d in g l a c i a l a c e t i c
a c id a t 60° C. b e f o r e th e c o lo r r e a g e n t i s ad d ed , and
u n p u b lis h e d work by Marx (8 8 ) i n d i c a t e s t h a t r e c o v e r y o f
known amounts o f c h o l e s t e r o l c a r r ie d th r o u g h th e above
d e te r m in a tio n i s v i r t u a l l y c o m p le te .
O ther p o s s i b l e e x p la n a t io n s o f th e ch an ges foun d
a f t e r in c u b a tio n w ou ld in c lu d e b a c t e r i a l s y n t h e s i s or d e
s t r u c t i o n o f th e s t e r o l d u rin g in c u b a tio n and ch an ges in th e
volume o f th e h o m ogen ate, w h ich w ould be r e f l e c t e d as
v a r i a t i o n s in th e c o n c e n tr a tio n o f th e s t e r o l in th e a l i q u o t s
u s e d f o r d e t e r m in a t io n . The form er p o s s i b i l i t y was n o t i n
v e s t i g a t e d b e c a u se o f th e f a c t t h a t th e m icr o o rg a n ism s known
65
t o be c a p a b le o f d e s t r o y in g c h o l e s t e r o l a r e n o t commonly
fo u n d as co n ta m in a n ts u n d er th e c o n d it io n s u s e d in th e
p r e p a r a tio n and in c u b a tio n o f th e h o m o g e n a te s. The p o s s i
b i l i t y o f volume changes was n o t p r e c lu d e d ,in th e e a r l i e r
e x p e r im e n ts , one t o e i g h t , f o r a f t e r in c u b a tio n in a
" c o n s ta n t h u m id ity chamber” th e sam p les were e x t r a c t e d w ith
te n m i l l i l i t e r s o f s o l v e n t , i . e . , th e y w ere n o t made up t o a
d e f i n i t e volum e. U n d o u b te d ly , th e r e was s l i g h t d i l u t i o n o f
t h e s e hom ogenates d u r in g in c u b a t io n , w h ich w ould be r e f l e c t e d
a s a d e c r e a s e in c h o l e s t e r o l c o n c e n tr a t io n a s compared w ith
th e u n in c u b a te d sa m p le s. H owever, t h i s would n o t a cco u n t
f o r th e d e c r e a s e s n o te d in e x p e r im e n ts two and th r e e f o r
some o f th e in c u b a te d sam p les showed a much g r e a t e r d e c r e a s e
in c h o l e s t e r o l than o th e r s and a l l w ould be d i l u t e d t o th e
same d e g r e e d u rin g in c u b a tio n b y th e d i s t i l l i n g o f th e w a te r
in th e c o n s ta n t h u m id ity chamber i n t o th e h om ogenate. The
d e c r e a s e in c h o l e s t e r o l c o n c e n tr a t io n b ro u g h t about b y such
d i l u t i o n w ould be o f a much s m a lle r o r d e r o f m agnitud e th an
any o f th e d e c r e a s e s c o n s id e r e d s i g n i f i c a n t . F u rth erm ore,
in e x p e r im e n ts n in e and f o l l o w i n g , su ch volume changes were
o b v ia t e d b y m aking ea ch sam ple up t o t w e n t y - f i v e m i l l i l i t e r s
w it h s o l v e n t a f t e r in c u b a t io n .
A d m itte d ly few d e f i n i t e c o n c lu s io n s can be drawn on
th e b a s i s o f th e work d on e. I f th e c o n c e n tr a t io n changes
n o te d be n o t a r t i f a c t s but r e p r e s e n t a c t u a l d e s t r u c t io n and
66
s y n t h e s i s o f s t e r o l or even ch an ges w h ich ren d e r s t e r o l s n o t
d e t e c t a b l e and d e t e c t a b l e r e s p e c t i v e l y b y th e m ethods u s e d
f o r c h o l e s t e r o l d e t e r m in a t io n , th e n i t m igh t be q u e s tio n e d
w h eth er th e p r o c e s s e s a r e e n z y m a t ic a lly c o n t r o l l e d .
W in t e r s t e in e r and B ergstrom have r e p o r te d a u t o - o x i d a t i o n o f
c h o l e s t e r o l in th e a b se n c e o f any t i s s u e p r e p a r a tio n w it h
r e s u l t a n t p r o d u c ts w h ich w ould n o t be d e t e c t e d in th e
S ch o en h eim er-S p erry m ethod f o r c h o l e s t e r o l . Such o x id a t io n
to o k p la c e r a p i d l y a t 8 5 ° C. and was fou n d t o p r o c e e d even a t
room te m p e r a tu r e , a lth o u g h a t a much s lo w e r r a t e , in v o lv in g
s e v e r a l d a y s . C o n c e iv a b ly , b y some su ch p r o c e s s some s t e r o l s
m igh t be so a l t e r e d t h a t th e y w ould be c o n v e r te d from com
pounds n o t d e t e c t e d t o th o s e d e t e c t e d b y th e S ch oen h eim er-
S p erry m eth od. The l i t t l e work t h a t has b een done i n v e s t i
g a t i n g th e n a tu r e o f th e changes r e p o r te d in t h i s t r e a t i s e
a p p ears to i n d i c a t e t h a t th e y a r e n o t e n z y m a tic .
Work b y L i p s e t t (8 7 ) showed t h a t in a few i n s t a n c e s ,
’’d e s t r u c t i o n 1 * o f c h o l e s t e r o l in r a t l i v e r hom ogenates s i m i l a r
t o t h a t r e p o r te d above to o k p la c e even a f t e r th e t i s s u e
p r e p a r a tio n had b een t r e a t e d w it h h e a v y m e t a l s . F u rth erm ore,
work by S ch otz (9 1 ) on th e tim e r e l a t i o n s h i p s o f su ch b r e a k
down i n d i c a t e s t h a t a p p a r e n tly l i t t l e change in th e s t e r o l
c o n te n t o c cu rs d u rin g th e f i r s t f i v e hou rs o f in c u b a t io n ,
w h ich i s h a r d ly c h a r a c t e r i s t i c o f an e n z y m a t ic a lly c o n t r o l l e d
r e a c t i o n . I t i s p o s s i b l e t h a t sim u lta n e o u s s y n t h e s i s and
67
d e s t r u c t io n occu r, o f c o u r s e ; i f th e r a t e s o f b o th p r o c e s s e s
w ere i d e n t i c a l , no n e t change in th e s t e r o l c o n c e n tr a t io n
w ould be d e t e c t e d . I t i s c o n c e iv a b le t h a t su ch m igh t ta k e
p la c e d u rin g th e f i r s t f i v e h o u r s , and t h e r e a f t e r ' t h e
d e s t r u c t i v e r e a c t i o n m igh t p red o m in a te. Such an e x p la n a tio n
r e s t s upon im p r o b a b le , i f n o t u n t e n a b le , a s s u m p tio n s .
An im p o rta n t q u e s t io n w h ich p r e s e n t s i t s e l f i s why
can n ot th e r e s u l t s o b ta in e d in th e e a r l i e r w ork, i n d i c a t i n g
d e s t r u c t io n o f c h o l e s t e r o l , be d u p lic a t e d i f th e y w ere n o t
a r t i f a c t s p rod u ced by th e m eth od s. There a r e s e v e r a l p o s s ib le
e x p la n a t io n s . S in c e th e a n im a ls were n o t a l l l i t t e r m ates
th e in d iv id u a l v a r ia t i o n in th e a n im a ls m ig h t p la y an
im p ortan t r o l e . A gain i t i s p o s s i b l e t h a t c e r t a i n f a c t o r s
e s s e n t i a l to an e n z y m a tic r e a c t io n m igh t be d e s tr o y e d un der
th e c o n d it io n s o f some o f th e e x p e r im e n ts , even though i t
was a tte m p te d t o k eep th e c o n d it io n s f a i r l y c o n s ta n t from
ex p erim en t t o e x p e r im e n t. In d eed , su ch a p o s s i b i l i t y was
s u g g e s t e d b y th e s to r a g e ex p erim en t a lr e a d y d i s c u s s e d .
W hat, th e n , may one c o n c lu d e as a r e s u l t o f t h i s stu d y?
M erely t h a t ’i t has b een d em o n stra ted t h a t r a t l i v e r homoge
n a t e s in a few i n s t a n c e s have shown s i g n i f i c a n t a l t e r a t i o n s
in t h e i r c h o l e s t e r o l c o n t e n ts a f t e r in c u b a t io n . These -
changes a re n o t r e a d i l y e x p la in e d a s a r t i f a c t s produced by
th e m ethods em p loyed . F i n a l l y , th e d i e t m ig h t p la y an
im p o rta n t p a r t in th e p r o c e s s ; t h e r e may have b een v a r ia t i o n s
68
in th e d i e t s p rep a red d u rin g th e c o u r se o f th e e x p e r im e n t s ,
v a r ia t i o n s u n a v o id a b le b e c a u se o f th e f a c t t h a t c e r t a i n
c o n s t i t u e n t s were p ro cu red from d i f f e r e n t m a n u fa c tu r e r s , No
a ttem p t h a s been made t o a s s a y th e d i e t u s e d f o r c e r t a i n
a c c e s s o r y d i e t a r y f a c t o r s su ch a s l i p o t r o p i c s u b s ta n c e s and
th e v it a m in s . H owever, a tte m p ts t o r e p la c e any su ch l a b i l e
f a c t o r s by f o r t i f y i n g th e hom ogenates w it h c e r t a i n s u b s ta n c e s
known to be n e c e s s a r y f o r in te r m e d ia te f a t and c a rb o h y d r a te
m eta b o lism d id n o t l e a d t o any d em o n stra b le s t e r o l breakdown
or s y n t h e s i s .
CHAPTER V III
SUM M ARY
I t h a s been a tte m p te d t o d em o n stra te w h eth er s i g n i f i
c a n t changes in th e c h o l e s t e r o l c o n te n t o f r a t l i v e r homoge
n a te s occu r d u rin g in c u b a tio n a t 3 7 ° C. E a r ly work gave
i n c o n s i s t e n t r e s u l t s w h ich have been a s c r ib e d in p a r t t o
e r r o r s in th e te c h n iq u e f o r c h o l e s t e r o l d e t e r m in a t io n , non
u n if o r m it y o f th e hom ogenates u s e d , an d , p o s s i b l y , in a d eq u a te
e x t r a c t i o n o f c h o l e s t e r o l from th e t i s s u e s . T hese
i n c o n s i s t e n c i e s were s u b s e q u e n tly a v o id e d by more c a r e f u l
a t t e n t i o n t o m eth o d s, b e t t e r h o m o g en izin g ap p a ra tu s and
te c h n iq u e f o r p r e p a r a tio n o f th e h o m og en ate, and by an
im proved m ethod o f e x t r a c t i o n .
The amount o f c h o l e s t e r o l r e c o v e r e d from r a t l i v e r
hom ogenate a f t e r in c u b a t io n f o r s i x t e e n t o tw e n ty h o u rs was
in some (b u t l e s s than a m a j o r it y o f ) in s t a n c e s m ark ed ly
l e s s than t h a t fo u n d in th e u n in c u b a te d c o n t r o l s . I t i s
d i f f i c u l t t o e x p la in th e r e s u l t s e n t i r e l y on th e b a s i s o f. an
a r t i f a c t produ ced by th e m ethods em ployed. In one in s t a n c e
t h e r e was a s t a t i s t i c a l l y s i g n i f i c a n t in c r e a s e in th e amount
o f c h o l e s t e r o l r e c o v e r e d from th e hom ogenate a f t e r in c u b a tio n
a s compared w ith t h a t o b ta in e d from th e u n in c u b a te d c o n t r o l ,
w h ich a g a i n , i f an a r t i f a c t , i s o f unknown n a t u r e . In con
t r a s t t o t h e s e o b s e r v a t i o n s , many e x p e rim en ts showed no
70
a l t e r a t i o n in th e c h o l e s t e r o l c o n t e n t o f th e hom ogenates
a f t e r in c u b a t io n . No c o r r e l a t i o n was fou n d betw een th e
l e n g t h o f th e c h o l e s t e r o l f e e d in g or th e l i v e r w e ig h t and
th e d e c r e a s e or in c r e a s e fou n d in th e c h o l e s t e r o l o f th e
l i v e r hom ogenate a f t e r in c u b a t io n , A ttem p ts to a c c e l e r a t e
c h o l e s t e r o l “d e s t r u c t i o n ” or S y n th e s i s ” by th e a d d it io n o f
s e v e r a l f a c t o r s known t o be e s s e n t i a l t o c e r t a i n s t e p s in
th e in te r m e d ia r y m eta b o lism o f f a t s and ca rb o h y d r a tes were
u n s u c c e s s f u l .
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^ElvGrcfty of S o u t t o n C ollfem lo (LDbrow

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Creator Adams, Willard P (author)

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

Degree Master of Science

Degree Program Biochemistry

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

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

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Advisor Marx, Walter (committee chair), [illegible] (committee member), Visser, Donald (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-776599

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Legacy Identifier EP41301.pdf

Dmrecord 776599

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Rights Adams, Willard P.

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Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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