University of Southern California Dissertations and Theses

The extraction and isolation of phytosterol from avocado oil

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The extraction and isolation of phytosterol from avocado oil

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Content The E x t r a c t i o n and I s o l a t i o n
o f P h y t o s te r o l
from
Avocado O il
S 3
A T h esis
P r e s e n te d to th e D epartm ent o f C h em istry
U n iv e r s ity o f S o u th e rn C a l i f o r n i a
In p a r t i a l f u l f i l l m e n t
o f th e
R eq u irem en ts f o r th e
D egree o f M a ste r o f A r t s .
By
G-eorge A . G andara
Ja n u a ry 1 9 , 1933
UMI Number: EP43808
All rights reserved
INFORMATION TO ALL USERS
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OiaseftatiGtt PibfeMng
UMI EP43808
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This thesis, written under the direction of the
candidate’s Faculty Committee and approved hy
all its members, has been presented to and ac
cepted by the Council on Graduate Study and
Research in partial fulfillment of the require
ments for the degree of
Date... J .a n u a r y ,.l 9 3 3 ..........
Faculty Committee
PrpfsssprW eatherby
Chairman
P r o fe s. s.Q.r. S tab le r ..........
3 * ^
ecretary
Dean
P r o fe sso r L ife
CONTENTS
Page
I* I n t r o d u c t i o n .........................................................•. 1
S t e r o l s ............................................ .... 3
I I . D iv is io n I . ................................................................ . . 4
E x p e rim e n ta l w o rk .......................... 4
T h e o ry ........................................................ 4
P ro c ed u re *. . . .............. 6
R e s u l t s ................... 9
A n a l y s i s .................................. 11
T e s ts f o r th e p h y t o s t e r o l ....................... 12
I I I . D iv is io n I I .................................. 15
N u t r i t i o n E x p e rim e n t .................................. 15
T h eo ry * ............ 15
V ita m in s ........................ 16
V itam in D............................... 18
E x p e rim e n ta l.............. 20
R a ts S t o c k . ...................... 21
T ab le o f W eights * ...............................*. 23
A n a ly s is o f th e c u r v e s ....................... . . . 27
IV . C o n c lu s io n s .............................................. 30
B ib lio g r a p h y ................................................ 35
Introduction
The avocado grow ing i n d u s t r y i n S o u th e rn C a l i f o r
n ia has i n c r e a s e d i n a marked way i n th e l a s t few y e a rs r e
s u l t i n g i n a s e r i e s o f r e s e a r c h e s upon th e p r o p e r t i e s o f
th e avocado f r u i t .* 1 -
A t th e p r e s e n t tim e th e p r o p e r t i e s o f t h i s f r u i t
a r e more and more s t u d i e d i n o r d e r t o e x te n d th e a p p l i c a
tio n s o f th e d i f f e r e n t b y -p ro d u c ts from i t . Among th e s e
l a s t ones we can f i n d t h a t th e avocado i s p ro d u c in g n o t
o n ly e d ib le p ro d u c ts b u t a ls o b y -p ro d u c ts w hich a r e b e in g
u se d i n th e making o f t o i l e t a r t i c l e s * I t i s p ro b a b le t h a t
a f t e r more r e s e a r c h work has been done t h a t many o th e r a p p l i
c a tio n s w i l l be fo u n d .
Prom th e v ie w -p o in t o f n u t r i t i o n th e avocado i s
c o n s id e re d one o f th e b e s t foods b e cau se i t i s b e lie v e d to
c o n ta in most o f th e n e c e s s a ry v ita m in s f o r body grow th
and m a in te n a n c e .
The e x t r a c t i o n and I s o l a t i o n from avocado o i l
o f p h y t o s t e r o l , w hieh i s th e p r e c o u r s e r o f v ita m in D, i s th e
o b j e c t o f t h i s e x p e r im en tal vfork,
1 . G o o d rich , B e rth a L o u is e . M aster T h e s is , U. S . C. 1930
*The d e te r m in a tio n o f v ita m in s A i n avocado o i l . *
L am b ert, A lgy L . , Mastetf T h e s is , U. S . C ., 1929.
" E x tr a c t i o n and u t i l i z a t i o n o f avocado o i l . *
M cH affie, A r lin e E li z a b e th , M a ste r T h e s is , U. S . C ., 1930
*The n u t r i t i o n a l v a lu e o f avocado s e e d .*
And many o t h e r s .
2
B e sid e s t h i s e x p e rim e n ta l work b e lo n g in g to t h e
A p p lie d C h e m istry , a n o th e r e x p e rim e n t has been made i n th e
b io c h e m ic a l f i e l d , w ith th e p u rp o se o f p ro v in g th e a n t i -
r i c k e t i c p r o p e r t i e s o f th e s e s t e r o l s a f t e r th e y have been
tra n s fo rm e d i n t o v ita m in s D by i r r a d i a t i o n from a m ercury
v a p o r lam p.
S t e r o l s
The word s t e r o l ^ s i g n i f i e s a s o l i d a l c o h o l , and
d ep en d in g on th e o r i g i n , i s c a l l e d a l s o c h o l e s t e r o l o r
p h y t o s t e r o l ; o h o l e s t e r o l b e in g th e s o l i d , u n s a p o n if ic a b le
a lc o h o l from an im al f a t s , and p h y t o s t e r o l th e name g iv e n
f o r th e same k in d o f a lc o h o ls from v e g e ta b le o r i g i n .
w hich a r e s t e r o l s from a n im al o r i g i n and p h y t o s t e r o l s th o s e
w hich a r e s t e r o l s o f v e g e ta b le o r i g i n . B oth s a t u r a t e d and
u n s a t u r a t e d s t e r o l s o c c u r i n n a tu r e b u t th e fo rm er f o r t h e
m ost p a r t , a r e p r e s e n t i n much l a r g e r q u a n t i t i e s o f th e un
s a t u r a t e d ty p e ; e r g o s t e r o l i s now th e b e s t known s t e r o l o f
v e g e ta b le o r i g i n .
The p o s s ib l e form u la f o r s t e r o l i s :
C £ 7 H 4 4 Q, b u t t h i s fo rm u la re sp o n d s f o r p h y t o s t e r o l a s
w e ll as f o r c h o l e s t e r o l . The g r a p h ic fo rm u la f o r c h o l
e s t e r o l i s :
L ew kow itsch^has s u g g e s te d c a l l i n g z o o s t e r o l s th o s e
CHOH
1 . M orse, A p p lie d B io c h e m is try , 1925.
2 . Ja m ie so n , P a ts and V e g e ta b le o i l s , 311, 1932.
4 .
D i v i s i o n i
E x p e rim e n ta l Work
Theory
I f we r e c a l l th e c o m p o sitio n o f f a t s and o i l s
from a n im al o r from v e g e ta b le o r i g i n , we w i l l f in d t h a t
th e y a r e m ix tu re s o f e s t e r s o f th e f a t t y a c id s w ith g l y
c e r i n e . These e s t e r s can he l i q u i d s o r s o l i d s , a c c o rd in g
to th e number o f c arb o n a to m s; t h e h i g h e r number o f c a r
bon atom s c o rre s p o n d in g t o th e s o l i d f a t s .
T hese e s t e r s have th e known p r o p e r ty o f b e in g
v e ry e a s i l y s a p o n i f ie d by th e s t r o n g a l k a l i s , p ro d u c in g
a so ap and th e f r e e g l y c e r o l . B oth so a p an d g l y c e r o l a r e
s o lu b le i n w a te r w h ile t h e s t e r o l s a r e n o t , t h i s p r o p e r ty
b e in g made u se o f i n th e e x t r a c t i o n and. i s o l a t i o n o f s t e r
o l s . The s t e r o l s a re v e ry s o lu b le i n t h e e s t e r s w hich
compose t h e m ix tu re known as f a t o r o i l , so i f t h e f a t i s
decom posed by means o f a s tr o n g a l k a l i , i t w i l l le a v e th e
s t e r o l s i n a c o n d itio n o f b e in g i s o l a t e d .
I n o r d e r to a v o id t h e n e c e s s i t y o f s a p o n if y
i n g th e w hole f a t , s t e r o l s can be s e p a r a t e d by c h i l l i n g th e
w hole o i l a t 0 C. u n t i l no more s o l i d m a tte r h as s e t t l e d .
The e x p erim e n t h as shown t h a t t h i s c h i l l i n g n o t
o n ly s o l i d i f i e s t h e s t e r o l s b u t a g r e a t d e a l o f s t e a r i n .
Some o f t h e s t e r o l s p a ss i n s o l u t i o n d u rin g th e f i l t r a
t i o n o r s e p a r a t i o n o f th e s o l i d m a tte r i f t h e p ro p e r te m p e r
a t u r e has n o t been m a in ta in e d d u rin g t h e o p e r a t i o n . The
s o l i d m a tte r o b ta in e d th r o u g h t h i s m ethod c o n ta in s th e
s t e r o l s w hich can be e x t r a c t e d a f t e r s e v e r a l tr e a tm e n ts
w ith a l k a l i *
I n o r d e r t o know w hich i s th e b e s t way t o sep*-
a r a t e th e s t e r o l from th e o il* two d i f f e r e n t e x t r a c t i o n s
w ere perform ed*
The f i r s t e x t r a c t i o n was made by s a p o n i f i c a t i o n
o f t h e s o l i d m a t t e r w hich was o b ta in e d by c h i l l i n g ; an d
t h e o t h e r one was made by d i r e c t s a p o n i f i c a t i o n o f th e
whole o i l .
E x p e rim e n ta l Work
P ro c e d u re
A. S a u o n i f i c a t i o n o f t h e C h i ll e d P o r t i o n o f O i l ,
A q u a n t i t y o f avocado o i l o f 900 grams was
p la c e d i n a w ide-m outhed b o t t l e , and th e whole was k e p t
f o r 6 0 horn’s i n a r e f r i g e r a t i o n box a t a te m p e ra tu re o f
0 t o 5 0* A f t e r t h a t tim e a s o l i d se d im e n t was o b s e rv e d .
T h is sed im en t was s e p a r a te d by f i l t r a t i o n i n an a p p a ra tu s
w hich was f i x e d i n su c h a way as t o k eep t h e same low
te m p e ra tu re d u r in g t h e o p e r a t i o n . • The f i l t r a t i o n was
made th ro u g h a p ie c e o f s i l k t i s s u e p la c e d on a f u n n e l,
an d i n o r d e r t o g e t a more r a p i d f i l t r a t i o n th e vacuum
pump was u s e d . A c i r c u l a r p ie c e o f co pper s h e e t was
p la c e d o n t h e fu n n e l a s a p r o t e c t i o n f o r t h e s i l k t i s s u e .
The s o l i d m a tte r s e t t l e d by c h i l l i n g shows th e
a p p e a ra n c e o f g re e n f l a k e s t w hich m e lt e a s i l y a t a tem p
e r a t u r e h ig h e r th a n 5 0 . g o in g a g a in i n t o s o l u t i o n i n th e
same o i l from w hich i t had been s e p a r a t e d . S o , i n o r d e r
to g e t th e m ost com plete s e p a r a t i o n , th e f i l t r a t i o n a p p a r
a tu s has b to be m a in ta in e d a t a te m p e ra tu re lo w e r th a n
5 0 .
The s o l i d m a t t e r was p la c e d i n an e rle n m e y e r
f l a s k and 30 c c . o f a b s o lu te a lc o h o l w ere added and m ixed.
To t h i s s o l u t i o n was added a c o n c e n tr a te d a l k a l i s o l u t i o n
(5 0 ^ sodium h y d ro x id e s o l u t i o n i n w a t e r ) . The m ix tu re
was b o i l e d f o r 30 m in u tes and a r e f l u x c o n d e n se r was u se d ,
C ausing th e s a p o n i f i c a t i o n o f t h e re m a in in g o i l . 1
A f t e r c o o lin g , 50 c c . o f e t h e r w ere added! and
m ixed, and c a r e f u l l y t r a n s f e r r e d t o a 5 0 0 c c . s e p a r a t o r y
f u n n e l . The f l a s k was r i n s e d w ith two 50 c c . p o r tio n s
o f e t h e r ; th e s e a n d th e fo rm e r s o l u t i o n were m ixed. The
s a p o n i f i c a t i o n f l a s k was a g a in washed w ith a d i l u t e s o l
u t i o n o f NaOH and p ou red i n t o t h e s p p a r a to r y f u n n e l, avoid-,
in g and e m u lsio n . A f t e r th e l i q u i d s were s e p a r a te d i n
l a y e r s , t h e so a p l a y e r was drawn off. The volume o f e t h e r
has t o he k e p t a t a c o n s ta n t volum e, th u s r e p l a c i n g th e
e v a p o ra te d e t h e r .
The e t h e r l a y e r was w ashed tw ic e w ith th e d i l u t e
s o l u t i o n o f NaOH (1 .1 2 $ ) an d a t l a s t w ith d i s t i l l e d w a te r ,
u n t i l t h e w ashings d id n o t show any a l k a l i n e r e a c tio n #
The w ashings w ith d i l u t e a l k a l i n e s o l u t i o n a r e
u sed w ith th e p u rp o se o f p r e v e n tin g th e fo rm a tio n o f a c i d
soaps w hich can n o t be rem oved from th e e t h e r .
The e th e r s o l u t i o n i s p la c e d i n an e rle n m e y e r
f l a s k and rem oved by d i s t i l l a t i o n on a w a te r b a th , u s in g
a d i s t i l l i n g t r a p t o a v o id any l o s s . I f any w a te r o r a l
co h o l rem a in s i n th e f l a s k , t h i s can be rem oved by l e a v in g
i t on th e w a te r - b a th f o r some tim e.-
B. S a p o n i f i c a t i o n o f th e Whole O i l .
The seco n d p ro c e d u re c o n s i s t s o f th e same sa p o n -
i f l c a t i o n b u t t h e wh o le o i l i s u s e d , 2
1 . Ja m ie so n , F a ts and V e g eta b le ' G i l s , 5^-0, 1932.
2 . L each, Food i n s p e c t i o n and a n a l y s i s , 522, 1920
The w eighed sam ple o f o i l i s p la c e d i n an e r l e n -
m eyer f l a s k and a n a l c o h o l i c s o l u t i o n o f p o ta s siu m h y d ro
x id e i s added { 2 0 0 c c . o f 2.0% s o lu tio n ) * and t h e m ix tu re
i s h o i le d f o r 40 m inutes* u s in g a lo n g g l a s s tu b e a s an
a i r condenser* When s t i l l warm th e p ro d u c t i s t r a n s f e r r e d
t o one. l i t e r s e p a r a t o r y f u n n e l. The s a p o n i f i c a t i o n f l a s k
i s r i n s e d w ith 400 c c , d i s t i l l e d w a te r . When t h e m ix tu re
i s c o o l, 5 0 0 c c . o f e t h e r a r e added* th e n sh ak en v i g o r
o u s ly and a llo w e d t o s ta n d u n t i l t h e two l a y e r s a r e v e ry
w e ll s e p a ra te d *
The soap s o l u t i o n i s drawn o f f and th e e t h e r
s o l u t i o n i s washed w ith two o r t h r e e p o r tio n s o f e t h e r ,
u s in g 2 5 0 c c . e ac h tim e , and a l l th e s e w ash ing s a r e added
t o t h e f i r s t e t h e r s o lu tio n *
The fether i s rem oved by d i s t i l l a t i o n on-a
w a te r b a th as i n th e o t h e r p ro c e d u re .
I f some f a t rem ain s i n th e r e s i d u e from th e
t t h e r s o l u t i o n , a new p o r t i o n o f a b o u t 2 0 c c . o f t h e a l c o
h o l i c s o l u t i o n i s added and b o i l e d f o r a few m inutes* and
t h e s t e p s a r e fo llo w e d a s b e f o r e .
9 .
R e s u lts
F o r th e f i r s t p ro ce d u re w hich I c a l l t h e * c h i l l
in g p ro c e d u re * 900 g r s . o f o i l w ere u se d and from i t ab o u t
2 5 g r s . o f s o l i d m a t t e r was o b ta in e d , th e secon d tim e i t
was p e rfo rm e d . 8 g r s * were o b ta in e d th e f i r s t tim e , g iv in g
a r e s u l t o f 0 . 0 ? 5 ^ t o 0 .1 2 9 7 grams o f u n s a p o n if ic a b le m a t t e r .
These two f i g u r e s o b ta in e d i n two d i f f e r e n t
c h i l l i n g s p rove t h a t a p ro p e r te m p e r a tu re f o r th e f i l t r a t i o n
h as t o be k e p t . The f i r s t tim e th e s o l i d m a tte r was f i l t
e re d o f f .on t h e u s u a l f i l t e r i n g a p p a ra tu s f o r h o t f i l t r a t -
io n s , ch an g in g , o f c o u rs e , th e b a th w ith fcce and s o l i d CO 2 »
b u t as th e a p p a r a tu s i s v e ry open th e te m p e ra tu re does n o t
r e a c h th e f u n n e l . The se co n d tim e th e f i l t e r i n g a p p a ra tu s
was f i x e d i n a d i f f e r e n t way and th e o i l w h ile i t was f i l t
e r in g th ro u g h th e fu n n e l had a te m p e r a tu re below 5 C.>
c o n se q u e n tly a v o id in g h a v in g th e s o l i d m a tte r go back i n t o
s o l u t i o n .
In t h i s f i l t r a t i o n th e c o ld m ix tu re was i n c lo s e
c o n ta c t w ith t h e fu n n e l and an i c e - s a l t m ix tu re was u s e d .
T h is f i l t r a t i o n c o u ld be p e rfo rm ed i n a r e f r i g e r
a t o r b u t th e te m p e r a tu re s h o u ld be k e p t lo w e r th a n 0 C ,,
u s in g a c o ld m ix tu re and f i x i n g th e f i l t e r i n g a p p a ra tu s
so t h a t i t w i l l c o n ta in a g r e a t d e a l o f th e o i l ; o p e n in g
o f t h e d o o r s h o u ld be a v o id e d as much a s p o s s i b l e ’ so a s to
k eep th e c o n s ta n t low te m p e r a tu r e . The open a p p a ra tu s w hich
was u se d , and t h e c o n ta c t o f th e c o ld m ix tu re w ith th e fu n
n e l , made i t e a sy t o k e ep a c o n s ta n t te m p e ra tu re and t o f i l t e r
s m a ll am ounts o f t h e o i l a t t h e same tim e* T his o b ta in e d
good r e s u l t s from th e vacuum pump and a l s o h a s te n e d f i l t
r a t i o n .
T h e . r e s u l t s i n b o th e x t r a c t i o n s w ere a s fo llo w s
A , C h i l l i n g p ro c e d u re :
O il u sed b o th tim e s 900 g r s . U n s a p o n ific a b le m a t t e r
0 .0 7 5 $ t o 0 .1 2 9 7 $
B. D ir e c t s a p o n i f i c a t i o n p ro c e d u re :
O il u se d th e f i r s t tim e 5 g r s . U n s a p o n ific a b le m a tte r
O il u se d th e 2n d. tim e 20 g rs* 2 .0 6 2 $ t o 3 .1 6 $
A n a ly s is o f t h i s d a ta *
As we can s e e i n th e r e s u l t s o f th e two d i f f e r e n t
p ro c e d u re s , t h e r e i s a p p r e c ia b le d i f f e r e n c e i n th e p e r c e n t
a g e . I f we c o n s id e r th e c o m p o sitio n o f t h e u n s a p o n if ic a b le
m a tte r w hich i s a m ix tu re o f h ig h ,m o le c u la r w eig h t a lc o h o ls
and h ig h h y d ro ca rb o n s w hich a r e n o t a t t a c k e d by a l k a l i s ,
and a ls o th e p h y t o s t e r o l which i s v e ry low i n p e r c e n ta g e , , ^
i t i s e a s f l y u n d e rs to o d t h a t by u s in g th e f i r s t c h i l l i n g
p ro c e d u re i t i s p o s s i b l e t o o b t a i n a more co m plete sap o n
i f i c a t i o n ; i n o t h e r words i t i s p o s s ib le to o b t a i n - a more
c o n c e n tr a te d a c t i v e m a t t e r , a s w e ll a s a p u re a c t i v e m a t t e r .
1 . Ja m ie so n , V e g e ta b le F a ts and O i l s , 311, 1932.
T e s ts f o r th e P h y t o s te r o l
C h o le s te r o l and p h y t o s t e r o l a r e ,s u p p o s e d t o be
d e r i v a t i v e s o f t h e phem antrene group* h a v in g th e .s a m e g ro u p s
I n t h e i r s id e c h a in s and b e in g is o m e re s , so u n d er c e r t a i n
r e a g e n ts a s a c e t i c a n h y d rid e and s u l f u r i c a c i d th e y p ro d u ce
C o n d en satio n r e a c tio n s * and g iv e th e same c o lo r r e a c t i o n s *
A t e s t w hich g iv e s good r e s u l t s * i s th e form o f t h e c r y s t a l s
o b ta in e d from an a l c o h o l i c s o l u t i o n . W hile t h e c h o l e s t e r o l
c r y s t a l s a r e o f a v e ry w ide sh a p e , th e p h y t o s t e r o l c r y s t a l s
a r e n e e d le s o f ro m b o id a l form o r narrow plates.-*- These
c r y s t a l s som etim es a re jo in e d fo rm in g a c h a r a c t e r i s t i c s t a r .
T h is form o f c r y s t a l l i z a t i o n can be s e e n c l e a r l y i n th e p i c
tu re * ( s e e page- )
Some a u th o r s d is c u s s t h e p o s s i b i l i t y o f p r e c i p
i t a t i n g p h y t o s t e r o l s w ith d i g i t o n i n . ^ Sherman sa y s t h a t
e r g o s t e r o l can be p r e c i p i t a t e d i n th e same manner and t h a t
t h i s p h y t o s t e r o l can be i r r a d i a t e d w ith good r e s u l t s w h ile
c h o l e s t e r o l can n o t be i r r a d i a t e d . The p h y t o s t e r o l from th e
u n s a p o n if ic a b le m a tte r was s e p a r a te d by means o f d i g i t o n i n ,
and on lo o k in g fo rw a rd i n t h i s p a p e r, we w i l l f in d t h a t we
g o t p h y t o s t e r o l w hich c o u ld be i r r a d i a t e d .
The d i g i t o n i n t e s t s c o n s i s t s i n t r e a t i n g th e
u n s a p o n if ic a b le m a t t e r w ith a 1% d i g i t o n i n a l c o h o l i c s o l u t
i o n . P h y t o s te r o l g i v e a w h ite p r e c i p i t a t e a f t e r h e a tin g ;
1 . L each, -Food i n s p e c t i o n and a n a ly s is * 524, 1920.
2 . Sherm an and S m ith , V i t a m i n s ,.1932 .
from t h i s p r e c i p i t a t e can be o b ta in e d th e p h y t o s t e r o l
a c e t a t e by t r e a t i n g t h i s p r e c i p i t a t e w ith an e x c e ss o f
a c e t i c a n h y d rid e ( 5 o r 6 cc.) and b o i l i n g f o r a few min
u t e s . A f t e r p o u rin g t h i s s o l u t i o n i n w a te r t h e p h y t o s t e r
o l a c e t a t e c r y s t a l l i z e s o u t and can be s e p a r a t e d by a
sim p le f i l t r a t i o n . The p r e c i p i t a t e a f t e r w ashed and d r i e d ,
i s t r e a t e d w ith e t h e r w hich d i s s o l v e s t h e a c e t a t e s e p a r a t
in g i t from th e d i g i t o n i n . The e t h e r s o l u t i o n i s a llo w e d
t o e v a p o ra te slo w ly and th e c r y s t a l s a re o b ta in e d on th e
w a lls o f th e c r y s t a l l i n g d i s h . These c r y s t a l s a re lo n g
n e e d le s w hich a d h e re i n blanches t i g h t l y t o th e w a l l s , i t
b e in g v e ry d i f f i c u l t to remove them w ith a k n i f e .
The m i l t i n g p o in t o f th e a c e t a t e o b ta in e d was
118 C .; 1 2 5 .6 b e in g th e t r u e m e ltin g p o i n t , 1 b u t depend-,
in g on many f a c t o r s su c h a s th e d ry n e s s o f th e c r y s t a l s ,
a lth o u g h a m e ltin g p o in t o f 117 C ., i s c o n s id e re d a p o s i
t i v e t e s t f o r p h y t o s t e r o l s T h e m e ltin g p o in t f o r t h e
a c e t a t e s i s su p p o sed t o be 1 2 5 .6 c . t o 137 C ., b u t C. E .
B i l l s and E. M. H oneyw ell have r e p o r te d ^ t h a t th e m e ltin g
p o in t * i s o f no p r a c t i c a l s i g n i f ic a n c e * b e ca u se i f i s
e x c e e d in g ly s e n s i t i v e t o d i f f e r e n c e s i n m o is tu re c o n te n t,
e s p e c i a l l y f o r e r g o s t e r o l a c e t a t e .
I n th e a b se n c e o f c h o l e s t e r o l and r e s i n s , th e
c o lo r t e s t s a r e a p p l i c a b l e a s a means o f i d e n t i f y i n g
p h y t o s t e r o l . Two r e a c t i o n s w ere made and b o th w ith p o s i
t i v e r e s u l t s .
_____________ The f i r s t one c a l le d L ie b e rm a n ’s r e a c t i o n ^ c o n -
1 , 1&2 L each, Pood i n s p e c t i o n and a n a l y s i s , 526, 1 92 0.
3 . Ja m ie so n , V e g e ta b le P a ts and O i l s , 315, 1932.
4 . Ja m ie so n , V e g e ta b le F a ts and O i l s , 312, 1932.
s i s t s o f t r e a t i n g a p h y t o s t e r o l s o l u t i o n i n a c e t i c anhyd
r i d e w ith 3 o r 5 d ro p s o f c o n c e n tr a te d s u l f u r i c a c i d ; a
v i o l e t p in k c o lo r d e v e lo p s , g iv in g a p o s i t i v e t e s t .
The B u rch ard m o d if ic a tio n 1 c o n s i s t s i n d i s
s o lv in g a sm a ll p o r t io n o f th e compound w hich i s g o in g
t o he t e s t e d , i n 2 c c . o f c h lo ro fo rm , and ad d in g t o t h i s
s o l u t i o n 2 0 d ro p s o f a c e t i c a n h y d rid e and two drops o f
s u l f u r i c a c i d ; t h e v i o l e t - r e d d i s h c o lo r changes t o b lu e
o r g r e e n . T his t e s t was a ls o a p p lie d t o p h y t o s t e r o l from
avocado o i l and t h e r e s u l t was d i s t i n c t l y p o s i t i v e .
Prom t h e p o s i t i v e t e s t s o b ta in e d , th e m e ltin g
p o i n t , and t h e sh ap e o f t h e c r y s t a l s , we can conclude
t h a t p h y t o s t e r o l has b een o b ta in e d i n a more o r l e s s p u re
s t a t e .
The p e rc e n ta g e o b ta in e d was 0 .0 7 5 $ , w hich i s
v e ry n e a r l y a p u re compound.
We m ust r e c a l l t h a t th e s t e r o l i s a m ix tu re o f
d i f f e r e n t p h y t o s t e r o l s , th e p e rc e n ta g e o f each one i n a
m ix tu re b e in g so low t h a t i t i s a lm o st im p o s s ib le to
s e p a r a t e them .
1 . Ja m ie so n , V e g e ta b le P a ts and O i l s , 312, 1932.
151
DI.VI SIO H IX
N u t r i t i o n E xperim ent
Theory
The human l i f e a s w e ll a s t h e an im al l i f e i s
a r e s u l t o f th e f u n c t i o n o f t h e d i f f e r e n t o rg an s w hich form
t h e "body. The "body i s a p e r f e c t m achine w hich n eed s d i f f
e r e n t s u b s ta n c e s f o r l i v i n g . These s u b s ta n c e s a r e c a l l e d ,
i n th e o r d in a r y la n g u a g e , fo o d . The fo o d s u p p lie s th e
en erg y f o r w ork, and i f i t does n o t c o n ta in t h e e le m e n ts
r e q u i r e d , th e body becomes s u b j e c t to v a r io u s d i s e a s e s w hich
g iv e e v id e n c e o f t h e i r a b s e n c e .
The en erg y s u p p lie d by t h e fo o d i t s e l f i s n o t
enough t o m a in ta in l i f e ; i t i s a l s o n e c e s s a r y th a t' th e fo o d
c o n ta in th e s u b s ta n c e s known a s v i t a m i n s .
I f s p e c i a l d i e t i s g iv e n t o an e x p e rim e n ta l a n i
mal w hich la c k s a c e r t a i n v ita m in d i s e a s e w i l l d e v e lo p . The
o b j e c t o f t h i s e x p e rim e n ta l work was t o d e v e lo p a v ita m in
D d e f i c ie n c e d is e a s e i n r a t s , known a s r i c k e t s , an d t o p ro v e
th e e x is ta n c e o f v ita m in D i n i r r a d i a t e d p h y t o s t e r o l from
avocado o i l by fe e d in g i t to th e s e same r a t s a s a c u r a t i v e .
1 . Sherm an, Pood and N u t r i t i o n , 1932,
Sherm an & S m ith , V ita m in s, 1931.
J.. B io l. C h e m istry , 51, 4 l — 4 7 .
J . B i o l, C h em istry , 76, 256-6 1 .
Vitamins
A v ita m in i s a food s u b s ta n c e o f unknown chem
i c a l c o m p o sitio n e x e r t i n g a n o rm a liz in g i n f lu e n c e upon
n u t r i t i o n , m ain ten a n ce and g ro w th . They a r e known o n ly
by t h e e f f e c t s , o r by th e d e f e c t s p roduced i n th e body
when t h e r e i s an i n s u f f i c i e n t amount o r a b se n c e o f them
i n th e d i e t .
Up t o th e p r e s e n t tim e th e v ita m in s known a r e
as f o llo w s :
V itam in A: i s f a t s o l u b l e . I t i s fou nd i n m o s t'o f th e
g re e n v e g e t a b l e s , p l a n t s and l e a v e s . I t r e s i s t s h e a t , can
be o x id iz e d , and s u b s ta n c e s w hich c o n ta in i t can be d r i e d
w ith o u t d e s t r u c t i o n o f th e v ita m in . T his v ita m in i s a n t i -
x e ro p h th a lm ic ; i t prom otes g ro w th ; a b se n c e o f t h i s v ita m in
pro d u ces c e s s a t i o n o f g ro w th , and th e d e f i c ie n c y r e s u l t s i n
a t r o p h i c c o n d itio n s , p ro d u ce s anem ia, te n d e n c y t o i n f e c t i o n ,
r e s p i r a t o r y d i s e a s e s , e t c .
V ita m in D: i s f a t s o l u b l e and i s h e a t s t a b l e . I t i s th e
a n t i r a c h i t i c v i ta m in . I t i s found i n fo o d s c o n ta in in g c a l
cium and p h o sp h o ro u s, and i s s t o r e d i n f i s h and l i v e r o i l s .
I t i s f r e q u e n t ly a s s o c i a t e d w ith v ita m in A. I t re s e m b le s
c h o l e s t e r o l , has no t o x i c i t y i n l a r g e am ounts and can be
r e p la c e d by i r r a d i a t i o n w ith a l i g h t wave o f 2900 A l e n g t h .
The a b sen c e o f t h i s v ita m in p ro d u ces r i c k e t s ; a d e f i c ie n c y
p ro d u ces r i c k e t s , l o s s o f a p p e t i t e , and a lim e n ta r y d i s t u r b
an ces .
V itam in E: i s f a t s o l u b l e . I t i s th e r e p r o d u c tiv e v ita m in .
I t i s w id e ly d i s t r i b u t e d i n a lm o st e v e ry fo o d . I t i s s o lu b le
i n a c e to n e s and can be p r e c i p i t a t e d by l e a d a c e t a t e i n
n e u t r a l a c i d s o l u t i o n s . The a b sen c e o f t h i s v ita m in p ro
duces la c k o f l a c t a t i o n and o f norm al growth*
V itam in B: i s w a te r s o lu b le and i s w id e ly d i s t r i b u t e d .
I t i s c o n ta in e d i n m ilk , l e a f y v e g e t a b l e s , c i t r u s f r u i t s ,
"v •
to m a to e s, egg y o lk , g la n d u la r o rg a n s , c e r e a l s and y e a s t .
I t i s more s t a b l e t o h e a t th a n i s v ita m in A. I t i s s t a b l e
i n weak a c i d s , and p r e v e n tiv e e f f e c t s a r e produced by v e ry
sm a ll am ounts o f i t . T his v ita m in i s a n t i n e u r i t i c and a l
so prom otes grow th* The a b sen ce o f t h i s v ita m in p ro d u ces
b e r i - b e r i , p o l y n e u r i t i s ; i t s d e f i c ie n c y p ro d u ces l o s s o f
a p p e t i t e , w eakness, anem ia, edema, c o n s t i p a t i o n , c o l i t i s ,
and c a r d ia c d e p r e s s io n .
V itam in C: i s w a te r s o l u b l e . I t i s a n t i s c u r b u t i c . I t i s
found i n raw cab b ag e, g ra p e f r u i t , p e p p e rs , raw s p in a c h ,
raw t u r n i p s , and most v e g e ta b le s and a c i d f r u i t s . I t i s
v e r y s u s c e p t i b l e t o h e a t and i s d e s tr o y e d on s t a n d in g . The
a b se n c e o f t h i s v ita m in p ro d u ces s c u rv y , l o s s o f e n e rg y ,
s a llo w com plexion, rheum atism pains- and m en tal d is o r d e r s *
Vitamin D
V itam in D, a s i t i s known, has th e m arked a c t i o n
on th e c a l c i f i c a t i o n o f t h e h o n e s, t h i s p r o p e r ty "being u se d
t o prove i t s e x is te n c e i n i r r a d i a t e d p r o d u c ts .
P h y t o s te r o ls can be tra n s fo rm e d i n t o v ita m in D
by i r r a d i a t i o n w ith a m ercury v a p o r lam p , i f t h i s p ro d u c t
w hich i s supposed to c o n ta in th e v ita m in D i s g iv e n to r i c k -
e t i c a n im a ls , a f t e r a c e r t a i n p e rio d o f tim e , t h e r e i s mar
ked e v id e n c e o f i n c r e a s e d c a l c i f i c a t i o n o f th e bones*
R ic k e ts i s a d is e a s e r e s u l t i n g from a la c k o f
v ita m in D i n th e body. I t has b een p ro v en t h a t a body i n
s p i t e o f a s u f f i c i e n t amount o f c alciu m and p h o sp h o ro u s,
makes no d e p o s it o f them i n th e bones, b e ca u se o f a l a c k o f
v ita m in s u p p ly ; so i f t h e body i s a r t i f i c i a l l y s u p p lie d
w ith v ita m in D, any r i c k e t s p r e s e n t w i l l be c u re d , o r i t s
developm ent p r e v e n te d .
T his p r o p e r ty i s c a u se d , i t i s b e lie v e d , by th e
i n c r e a s e d number o f ions^-df c a lc iu m and phosphorous in" th e
l i q u i d s su rro u n d in g th e b o n e. The calciu m and phosphorous
s a l t s can n o t a c c o m p lish th e phenomenon o f c a l c i f i c a t i o n
due t o t h e l a c k o f s u f f i c i e n t i o n s , th u s p ro d u c in g th e d i s
e a s e ,
I f v ita m in D i s p r e s e n t i n th e body, a lth o u g h
t h e r e may n o t be s u f f i c i e n t calciu m and phosphorus i n th e
fo o d , c a l c i f i c a t i o n w i l l ta k e p la c e by a b s o rb in g th e c a lc iu m
1 . Sherm an, Pood an d n u t r i t i o n , 1932.
and phosphorus from th e t i s s u e s u n t i l th e norm al concen
t r a t i o n i s reach ed * T his r e s u l t s i n a l o s s o f w e ig h t from
th e body*
E x p e rim e n ta l
P r e p a r a t io n o f th e I r r a d i a t e d P ro d u c t
F o r t h i s ex p erim en t t h e p ro d u c t o f t h e e x t r a c t
io n from avocado o i l c o n s i s t i n g o f p h y t o s t e r o l c r y s t a l s ,
p u r i f i e d s e v e r a l tim e s by d i s s o l v i n g i n a b s o lu te a lc o h o l
and s u c c e s s iv e c r y s t a l l i z a t i o n , was u s e d .
The p h y t o s t e r o l was w eighed o u t and e m u ls if ie d
i n d i s t i l l e d w a te r . The w eig h t o f p h y t o s t e r o l was 0 .2 9 7
g r s . and th e volume o f w a te r u sed was 15 c c . The s t r e n g t h
o f th e e m u lsio n was c a l c u l a t e d t o c o n ta in 0 . 0 0 1 gram f o r
each d ro p .
The e m u lsio n was p la c e d i n a sh a llo w d i s h i n
o r d e r t o have a v e ry t h i n film o f a b o u t one m il l im e te r ,
and was s u b m itte d t o t h e a c t i o n o f a m ercury v a p o r la m p 1 ,
a t t h e d i s t a n c e o f 16 i n c h e s . The i r r a d i a t i o n l a s t e d 20
m in u te s , and e v e ry f o u r o r f i v e m in u tes th e em u lsio n was
sh ak en u p , so th e i r r a d i a t i o n would be v e ry c o m p le te .
A f t e r th e i r r a d i a t i o n , th e em u lsio n was k e p t i n a d a rk
g l a s s b o t t l e i n o r d e r t o p re v e n t th e a c t i o n o f t h e l i g h t .
1 . Lamp u se d : Cooper H ew itt M ercury V a p o r Q u a rtz Lamp.
R a ts S to c k
The e x p e rim e n t had t o b e g in by b r in g in g th e
r a t s to a p ro p e r d e g re e o f r i c k e t s d e v elo p ed by means
o f a s p e c i a l d i e t . The experim ent was s t a r t e d w ith
f o u r gro u p s o f r a t s :
Group I N e g a tiv e C o n tro l w ith 2 r a t s .
Group I I P o s i t i v e C o n tro l w ith 3 r a t s .
Group i l l R i e k e ti c w ith .3 r a t s .
Group IV R i e k e ti c w ith 3 r a t s .
In o r d e r t o g e t r i c k e t s i n th e r a t s o f th e
gro u p s 1 - 2 - 3 - 4 , th e y w ere fe d d u rin g e i g h t weeks w ith
th e b a s a l diet-*- o f S te en b o c k and B lack #2965 o f th e
fo llo w in g fo rm u la :
y e llo w co rn m e a l . . . . . ' .............................76 p a r t s
w heat g l u te n ^0-50% p r o te in ^ ' 2 0 p a r t s
calciu m c a r b o n a te 3 p a r t s
sodium c h l o r i d e . 1 p a r t
T o ta l * .................100 p a r t s .
Each gro u p was p la c e d i n a s e p a r a t e cage and
k e p t i n good s a n i t a r y c o n d itio n s .
T here was g iv e n t o e a c h r a t 10 grams o f th e
d i e t d a i l y and p le n ty o f w a te r . The windows o f th e room
i n w hich t h e r a t s .w e r e k e p t were c o v e re d w ith brown p a p e r
i n o r d e r t o a v o id any a b s o r p tio n o f v ita m in D from th e
1 . Sherm an & S m ith , The V ita m in s, 316, 1931
2* The f i r s t week a com m ercial g lu te n was used f o r t h e d i e t ,
b u t t h e r e s u l t s w ere n o t s a t i s f a c t o r y . Thfe n e x t' week th e
d i e t was made w ith g l u t e n p re p a re d from f l o u r by w ashing
o u t m ost o f th e s t a r c h , d ry in g a t 1 0 0 °C . and g ra in in g -;
a s f i n e as pow der.
s u n l i g h t . The room was k e p t a t a r e a s o n a b ly c o n s ta n t
te m p e ra tu re and a d e q u a te ly v e n t i l a t e d .
A utopsy showed slow developm ent o f r i c k e t s
p ro b a b ly a s t h e r a t s had been one week on norm al d i e t
c o n ta in in g cod l i v e r o i l b e fo re b e in g s t a r t e d on th e
e x p e rim e n t.
A f t e r e ig h t weeks s u f f i c i e n t r i c k e t s had
d e v e lo p e d to s t a r t on th e t e s t d i e t s .
S u b se q u e n t d i e t was a s f o l l o w s t
Group I N e g a tiv e c o n tr o l B a s il d i e t a lo n e .
Group I I P o s i t i v e c o n tr o l B a s il d i e t & 1 d ro p cod l i v e r o i l .
Group I I I R i c k e t i c c o n tr o l B a s il d i e t & 2 d ro p s o f
i r r a d i a t e d p h y t o s t e r o l ( . 0 0 2 g r . )
Group I ? R i c k e t i c B a s il d i e t & 1 d ro p o f
i r r a d i a t e d p h y t o s t e r o l C .Q O igr.)
23
T able o f W eights
Group I - N e g a tiv e C o n tro l
D ates T1 T2
O c t, 27 61 69
Nov# 3 60 72
Nov. 15 57 75
Nov. 22 56 78
Nov. 28 61 84
Dec. 2 60 85
D ec. 8 55 70
Dec. 16 54 67
Dec. 26 70 83
J a n . 4 70 90
J a n . 8 71 8 8
J a n . 13 71 95
J a n . 16 71 102
O U P
■ C 6 . g£.
24
T able o f W eights
Group I I - P o s i t i v e C o n tro l
D ates T3 T4 T5
O c t. 27 62
67
56
Nov. 3 58
67 52
Nov. 15 6 1 71 56
Nov. 22 6 6 8 8
57
Nov* 28
67 83 63
Dec. 2
67
82 60
Dec. 9 6 0 73 56
Dec. 16 67
80 62
Dec. 26 81 94 6 8
J a n . 4 80 90 6 6
J a n . 8 85
94 72
J a n . 13 84
95 72
J a n . 16 8 6 94 72
i Ke
rn
ws
itn
25 #
T ab le o f 'W eights
Group I I I « R ic k e ts
D ates T6 T7 T8
O c t. 27
63 51 51
Nov. 3 62 57 48
Nov. 15
65
54 49
Nev. 2 2
65 53 49
Nov. 28 72 60 54
Dec. 2 72 58 54
D ec. 9 6 6 48 48
Dec. 16
63 46 46
D ec. 26 77
62 48
J a n . 4 83 6 8 52
J a n . 8 83 69 53
J a n . 13 83
70
55
J a n . 16
85
72
57
Jpa
V * - '
26
T ab le o f W eights
Group IV** Ri o h ets
D ates T9 T10 T il
O c t. 2? 72 59 53
Nov, 3 6 6 62
65
Nov, 15 74 60
63
Nov, 22' 76 75 6 8
Nov, 28 84 83 73
Dec, 2 8 6 85
72
Dec. 9 74 70 60
Dec, 16
72 63 59
Dec. 26
77 61 62
J a n . 4 85 67 67
J a n . 8 84
« # « ■ »
67
J a n . 13
87
70
J a n . 16 85 70
r s e . r m t j s .
t o o
'M
' M l a t
7 5 "
A n a ly s is o f th e C u rv e s.
As we have s a i d b d fo re , th e b e g in n in g o f th e b io
ch em ical ex p erim en t was s t a r t e d by o b ta in in g r i c k e t s on a l
b in o r a t s and th e n c u rin g them by fe e d in g i r r a d i a t e d phy-
t o s t e r o l .
Group I - N e g ativ e c o n tr o l
Two h e a lth y r a t s were s u b m itte d to th e S te e n b lg c k
d i e t #2965, th e fo rm u la o f w hich i s g iv e n i n th e d i v i s i o n I I .
T h is d i e t i s supposed to produce r i c k e t s a f t e r t h r e e weeks
b u t i n t h i s e x p e rim e n t i t took ab o u t e ig h t weeks b e cau se th e
r a t s had p re v io u s ly been fe d w ith a norm al d i e t and cod l i v e r
o i l , w hich allo w ed them t o s t o r e a l a r g e amount o f v ita m in D
i n t h e i r b o d ie s .
As soon as r i c k e t s a p p e a re d , th e r a t s began t o lo o s e
i n w e ig h t a s shown by t h e c u rv e . A f t e r two weeks more th e
r a t s s t a r t e d g a in in g w e ig h t, showing t h a t i n s p i t e o f t h e v i t a
min D d e f ic ie n c y grow th was p o s s ib l e .
T here were f i v e r a t s i n th e group a t t h e b e g in n in g
o f t h e e x p e rim e n t. A f t e r th e t h i r d week one was k i l l e d so as
t o d e te rm in e th e d e g re e o f r i c k e t s . A n o th er was k i l l e d two
weeks l a t e r f o r th e same p u rp o se , and a n o th e r d u rin g th e e i g h t t h
w eek. These a u to p s ie s showed t h a t r i c k e t s had d ev elo p ed o n ly
a f t e r th e s e v e n th w eek. The cu rves p l o t t e d were s i m i l a r f o r
a l l f i v e r a t s . Two r a t s were l e f t f o r n e g a tiv e c o n t r o l .
W hile th e r a t s were on th e v ita m in D f r e e d i e t i t
can he o b se rv ed from th e g ra p h t h a t th e y d id n o t fo llo w a
r e g u l a r l y i n c r e a s in g o r d e c r e a s in g c u rv e , b u t t h a t t h e r e w ere
sudden c h a n g e s. These changes can be c o n s id e re d a r e s u l t o f
th e a lim e n ta r y d is tu r b a n c e s w hich a r e c a u se d by th e la c k o f
th e n e c e s s a ry v ita m in s , o t h e r th a n v ita m in D.
Group I I - P o s i t i v e c o n t r o l .
If^w e fo llo w th e cu rv e s as f a r a s th e c i r c l e s ,
w hich i n d i c a t e a change i n th e a lim e n ta r y p r o c e s s , we w i l l
se e t h a t ' th e cu rv e s o b ta in e d from th e n e g a tiv e c o n tr o l anim
a l s a r e a l i k e . T his f a c t shows t h a t th e r a t s o f a l l th e
groups w ere s u f f e r i n g th e same a lim e n ta ry , d i s t u r b a n c e s .
The p o s i t i v e c o n tr o l group was composed o f f i v e
r a t s , one o f w hich was k i l l e d a f t e r th e t h i r d week, and th e
o t h e r a f t e r th e s e v e n th week, so as to d e te rm in e th e d e g re e
o f r i c k e t s . T h ree r a t s were l e f t f o r th e c o n tin u a tio n o f th e
e x p e rim e n t,
A f t e r th e s e v e n th week, w hich i s i n d i c a t e d on th e
p l o t by a c i r c l e , one d ro p o f cod l i v e r o i l was added to th e
d i e t . The cu rv e s show t h a t t h e r e was a norm al b u t sm a ll i n
c re a s e i n w e ig h t a f t e r t h e n i n e t h w eek. The cu rv es do n o t
show sudden b re a k s , w hich i n d i c a t e s t h a t th e r a t s were g e t t i n g
b a ck t o a norm al c o n d itio n .
Group I I I - R ic k e ts . C u ra tiv e g ro u p .
T his g ro u p was composed o f f i v e r a t s , two o f w hich
were k i l l e d ; one a f t e r th e t h i r d week and th e o th e r a f t e r th e
s e v e n th w eek. Three r a t s w ere l e f t f o r t h e re m a in d e r o f th e
e x p e rim e n t.
The cu rv es up to th e c i r c l e s show t h a t r i c k e t s
d ev elo p ed w h ile th e r a t s were on a v ita m in D f r e e d i e t o n ly .
The curve a f t e r th e c i r c l e i n d i c a t e s a change i n th e g e n e r a l
c o n d itio n o f th e r a t s . We can se e a d is a p p e a ra n c e o f th e
a lim e n ta r y d is tu r b a n c e s and a norm al i n c r e a s e i n w e ig h t. The
cu rv e s a sc e n d slo w ly b u t n o rm a lly .
T h is group was fe d w ith two drops o f i r r a d i a t e d
p h y t o s t e r o l i n a d d i t i o n t o th e v ita m in D f r e e d i e t .
Group IV - R i c k e t s . C u ra tiv e g ro u p .
T his g ro u p can be o b se rv e d i n th e same m anner as
g r o u p 'I I I . The o n ly d i f f e r e n c e betw een them i s t h a t t h i s
group was f e d a f t e r t h e s e v e n th week w ith one d ro p o f i r r a d
i a t e d p h y to s te r o l#
The r e s u l t s , a s we can se e on t h e c u rv e s , w ere a l
m ost t h e same a s th o s e o b ta in e d w ith group I I I .
IV-. C o n c lu s io n s ,
The cu rv es show a com plete change i n th e c o n d itio n
o f h e a l t h i n groups I I , I I I , and IV a f t e r th e y were fe d w ith
cod l i v e r o i l and i r r a d i a t e d p h y t o s t e r o l r e s p e c t i v e l y .
The a u to p s y o f th e r a t s i s a b s o l u te ly n e c e s s a r y i n
o r d e r t o f in d o u t i f th e r e s u l t s o b ta in e d a s shown by th e
cu rv es a re i n a c c o rd a n c e w ith th e r e s u l t s o b se rv e d d i r e c t l y
from th e b o n e s.
As we know, v ita m in D has a re m a rk a b le a c t io n on
th e c a l c i f i c a t i o n o f th e bone a s can be s e e n by o b s e rv in g
th e new c a l c i f i c a t i o n i n th e same bo n e. T his c a l c i f i c a t i o n
was caused by th e a d d i t i o n o f v ita m in D to th e d i e t . The
c a l c i f i c a t i o n can be d e te c te d by th e e x p e rim e n t c a l le d * th e
l i n e t e s t . *
L in e t e s t : The a c t io n o f th e v ita m in D in c r e a s e s
th e number o f calciu m and phosphorus io n s d e p o s ite d on th e
bone and t h i s d e p o s it cau se s th e c a r t i l a g e t o become a n o r
mal h a rd b one. I t a l s o prom otes norm al grow th o f th e bone
and I n c r e a s e s th e w id th ' o f th e w a lls o f th e bo ne. In
r i c k e t i c a n im als i t can be se e n t h a t th e c a r t i l a g e o c c u p ie s
a l a r g e wide s e c t i o n i n th e bone betw een th e e p ip h y s is and th e
d ia p h y s is , w hich i s c a l l e d m e ta p h y s is .
The m etap h y sis s e c t i o n i s found o n ly i n r i c k e t i c
bones and i s a r e s u l t o f th e la c k o f c a l c i f i c a t i o n . W herever
t h i s pheunomenon e x i s t s r i c k e t s r e s u l t .
A f t e r fe e d in g w ith d i e t s o r s u b s ta n c e s r i c h i n
v ita m in D, th e m eta p h y sis o f th e hone d i s a p p e a r s , t h e w ide
c a r t i l a g e s e c t i o n becomes v e ry narrow and c a l c i f i c a t i o n ta k e s
p la c e b eh in d th e narrow ed c a r t i l a g e . 3 -
T h is l i n e o f c a l c i f i c a t i o n can be o b se rv ed and th e
e f f e c t s m easured by th e use o f t h e ' c o n tr o l a n im a ls .
1 . J o u r , .of B i o l. Chem.; 68, 8 2 1 -6 4 , (1926)
Technic
The t i b i a o f t h e k i l l e d an im al i s c u t t r a n s -
v e r s a l l y , o p p o s ite t o th e knobs o f th e bone. I t i s th e n
imm ersed i n a 1 0 $ form aldehyde s o l u t io n f o r 24 h o u r s . The
*
p e rio d o f tim e must be no l e s s th a n 24 h o u rs a s t h e bones
w i l l n o t be s o f t enough. A f te r t h i s , th e bones a r e washed
3 o r 4 tim e s w ith d i s t i l l e d w a te r and imm ersed i n a c e to n e
f o r t h r e e m in p te s— sh a k in g them s l i g h t l y d u rin g t h i s tim e .
The a c e to n e i s washed o u t w ith 3 ofc 4 p o r tio n s o f d i s t i l l e d
w a te r and th e n th e bones a r e imm ersed f o r t h r e e m in u tes i n
a 2$ s i l v e r n i t r a t e s o l u t i o n . The e x c e ss o f s i l v e r n i t r a t e
i s washed o u t w ith 3 o r 4 p o r tio n s o f d i s t i l l e d w a te r and th e n
exposed f o r 3 m in u tes to th e a c t i o n o f s u n l i g h t .
The bones a r e k e p t i n w a te r w h ile im m ediate o b s e r
v a tio n s o f them a r e made u n d e r a b in o c u la r m ic ro sc o p e .
The r e s u l t s o b ta in e d w ith th e bones from th e e x p e r
im e n ta l r a t s were as fo llo w s
Group I - N e g ativ e c o n tr o l -f. h e a lin g
Group I I - P o s i t i v e c o n tr o l +4-4"+- h e a lin g
Group I I I - C u ra tiv e group f- -t+ - h e a l in g
Group IV - C u ra tiv e g roup 4-4-4- h e a l in g
These r e s u l t s have been t r a n s l a t e d a c c o rd in g to
th e m ethod g iv e n by C. E . B i l l s and Me D onald: -f- h e a l in g i s
c o n s id e re d a n e g a tiv e t e s t ;+ + h e a l in g , +44 h e a l in g , a n d -t+ ff h e a l
in g a r e c o n sid e re d po s i t i v e t e s t s i n t h e g ra d u a l c u rin g o f r i c k
1 . B i l l s , G. E . and Me D onald; P . G .; J o u r , o f B. Ch. 6 8 : 8 2 1 -
832; 1926.
33
e ta *
These r e s u l t s can n o t he ta k e n as a c o n c lu s iv e
p o s i t i v e ones b ecau se o f th e c o n d itio n o f th e r a t s u sed f o r
t h e e x p e rim e n t. They were v e ry h e a lth y and t h e r e f o r e I t was
im p o s s ib le to o b t a i n a c o n d itio n o f s e v e re r i c k e t s i n them .
The p o s i t i v e c o n tr o l showed ++-M - h e a l in g as m ight
be e x p e c te d .
S in c e th e n e g a tiv e c o n tr o l showed h e a l in g , due
t o i n s u f f i c i e n t r a c h i t i c d i e t , th e o t h e r groups may be com
p a re d w ith t h i s and i f a g r e a t e r d e g re e o f h e a lin g i s ob
s e rv e d , i t may be re g a rd e d t h a t th e d i e t was a n t i - r a c h i t i c
t o th e e x te n t t h a t th e s e t e s t s show s u p e r i o r h e a lin g t o th e
n e g a tiv e c o n t r o l .
B oth o f th e t e s t d i e t s showed an in te r m e d ia r y con
d i t i o n o f h e a lin g , o r h e a l in g . T his i s p ro o f t h a t th e
d i e t s were a n t i - r a c h i t i c t o a re a s o n a b le d e g re e . I t was d i f f
i c u l t t o d i s t i n g u i s h betw een th e r e s u l t s o f th e two t e s t r a t
i o n s . F u r th e r work i s recommended i n w hich b o th th e young
r a t s and m oth ers be p la c e d on a r a c h i t i c d i e t i n o r d e r t o p ro
duce i n a more s e v e re d e g re e .
3 4 .
Summary
J , P h y to s te r o l $ as p re p a re d from avocado o i l and i d e n t
i f i e d by q u a l i t a t i v e t e s t s .
I I . The p h y t o s t e r o l was i r r a d i a t e d and by means o f t e s t
e x p erim e n ts on r a c h i t i c r a t s , was shown to have a n t i
r a c h i t i c p r o p e r ty .
B ib lio g r a p h y
Chem. A b s t r a c t s ; 20, 62, (1926)
Chem. A b s t r a c t s ; 20,. 9 3 2 , (1926)
Jam ieso n ; V e g e ta b le O ils and F a t s , (1932)
J o u r , o f B i o l. Chem.; 51, 4 1 -5 0 , (1922)
J o u r , o f B i o l. Chem.; 6 8 , 8 2 1 -6 4 , (1926)
J o u r , o f B i o l. Chem.; 76, 251-64, (1928)
J o u r , o f B i o l. Chem.; 90, 61 9-36, (1931)
L each; Food I n s p e c tio n and A n a ly s is , U. S . C .,( l 9 2 0 )
M aster T h e s is ; P h y t o s te r o l o f C o tto n S eed S t e a r i n , U. S . C.
(1927)
M a ste r T h e s is ; The A n t i r a c h i t i c E f f e c t o f I r r a d i a t e d Whole
C o tto n se e d O i l , e t c . , U. S . C ., (1926)
M aster T h e s is ; E x tr a c t i o n and U t i l i z a t i o n o f Avocado O i l ,
U. S . C .(19 29)
M aster T h e s is ; The A n t i r a c h i t i c V alue o f I r r a d i a t e d P h y t o s te r o l,
U. S . C ., (1929)
M orse; A p p lie d B io - c h e m is tr y ,(1 9 2 5 )
O il and F at I n d u s t r i e s ; 426, J u ly (1928)
Sherm an; Food and N u t r i t i o n , (1932)
Sherman & S m ith ; The V ita m in s, (1931)

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Asset Metadata

Creator Gandara, G. A (author)

Core Title The extraction and isolation of phytosterol from avocado oil

Degree Master of Arts

Degree Program Chemistry

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

Tag agriculture, food science and technology,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Weatherby, Leroy S. (committee chair), Life, Andrew C. (committee member), Stabler, Laird J. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c20-352468

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Identifier EP43808.pdf (filename),usctheses-c20-352468 (legacy record id)

Legacy Identifier EP43808.pdf

Dmrecord 352468

Document Type Thesis

Rights Gandara, G. A.

Type texts

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

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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