The site of conversion of carotene to vitamin A.

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Content THE SITE OF CONVERSION OF CAROTENE TO VITAMIN A
A D is s e r ta ti o n
P r e s e n te d t o
th e F a c u lty o f th e G rad u ate S ch o o l
U n iv e r s ity o f S o u th e rn C a l if o r n ia ,
I n P a r t i a l F u lf illm e n t
o f th e R eq u irem en ts f o r th e D egree
D o cto r o f P h ilo so p h y
by
F re d Hugh M attso n
Ju n e 1948
UMI Number: DP21538
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t ) 0 / c s » - ^4 fi i
71 his dissertation, written by ’ T V ' ^
...................r a E D .H U a H .M T T S p N ................................
under the. guidance of hX S. Faculty Committee
on Studies, and approved by all its members, has
been presented to and accepted by the Council
on Graduate Study and Research, in partial ful
fillment of requirements for the degree of
D O C T O R O F P H I L O S O P H Y
H. W. PATMORE
Secretary
D a te June...l948...........
C om m ittee on Studies
Chairman
TABLE OF CONTENTS
CHAPTER PAGE
I . INTRODUCTION................................................................... 1
I I . HISTORICAL REVIEW OF THE LITERATURE . . ... 2
I d e n t i f i c a t i o n o f C a ro te n e . ............................. 2
I d e n t i f i c a t i o n o f V itam in A ....................... 4
P r o p e r t i e s o f C aro ten e and V itam in A . . . . 6
S te re o is o m e ris m o f th e C a ro te n o id s and
V itam in A . . . . ............................ 7
B io lo g ic a l R e la tio n s h ip o f C aro ten e and
V itam in A . . . . . . 9
M ethods o f A n a ly s is f o r V itam in A . . . . , 10
M ethods o f A n a ly s is f o r C a ro te n o id s . . . .' 1§
A b so rp tio n o f C aro ten e and V itam in A . . . . 17
F u n c tio n o f C aro ten e and V itam in A ......... 19
S to ra g e and D is tr ib u t io n o f C aro ten e and
V itam in A . . . . . . . . . . 20
E x c re tio n o f C aro ten e and V itam in A . . . . 22
M echanism o f C o n v ersio n o f C aro ten e to V ita
min A . . . . . . . . . . . . . . . . . . 22
The S i t e o f C o n v ersio n o f C aro ten e to
V itam in A ♦
24
i i
CHAPTER PAGE
I I I . EXPERIMENTAL............................................................................. 29
M a te r ia ls . . . . . . . . . . ............................. . 29
S o lv e n ts . . . . . . . . . . . . 29
E reb s~ R in g er P h o sp h a te B u ffe r . . . . . . . 30
C aro ten e S o lu tio n s ................................. 31
V itam in A S o lu tio n s . . . . . . . . . . . . 32
A nim als . . . . . . . . . . . . . .................... 33
P ro c e d u re f o r In Vivo E x p e r i m e n t s ........................ 34
T reatm en t o f A nim als and T i s s u e s ................... 34
A n a ly s is o f T i s s u e s ..................................................... 34
C a lc u la tio n s . . . . . . . . . . . . . . . 35
p ro c e d u re f o r I n V itr o E x p e r i m e n t s .................... 3?
P r e p a r a tio n o f T is s u e s . . . . . 37
A n a ly s is o f M edia and T is s u e s . . . . . . . 40
C a lc u la tio n s . . . . . . . .................................. 43
IV . RESULTS........................................................................... 45
I n Vivo E x p e r im e n ts ............................ 45
The E f f e c t o f th e A d m in is tra tio n o f C o tto n
se e d O i l ....................................................................... 45
The D isa p p e a ra n c e o f V itam in A from V ario u s
O rgans .................................. 47
The R ate o f A ppearance o f V itam in A in th e
I n t e s t i n a l T ra c t and th e L iv e r a f t e r
F eed in g C a ro te n e . . . . . . . . . . . . 49
i i i
C HAPTER PAGE
The N atu re o f th e L iv e r P igm ent . . ... 51
Q u a li ta ti v e I d e n t i f i c a t i o n o f V itam in A
i n th e I n t e s t i n a l T ra c t a f t e r F eed in g
C a ro te n e ........................ 54
I n V itro E x p e r im e n ts ................................................. . 57
Tween a s th e C aro ten e S o l v e n t ........ 57
© ioxane a s th e C aro ten e S o lv e n t .. .. . 50
A cetone a s th e C aro ten e S o lv e n t .......... 61
Aqueous C aro te n e S o l . ................................... 61
Use o f I n t e s t i n a l Homogenate and Added
A denosine T r i p h o s p h a t e * . . 65
V. D ISC U SSIO N .................................................................. 6 6
V I. STOM ARY ............................. 73
BIBLIOGRAPHY . . . » 74
LIST OF TABLES
TABLE PAGE
1 . R e la tiv e B io lo g ic a l A c t i v i t i e s o f Some Members
o f S te re o iso m e ric C a ro te n e S e ts . ...................................11
2. S ta n d a r d iz a tio n o f C a r r - P r ic e R e a c tio n
f o r V itam in A .......................................... 56
3 . S ta n d a r d iz a tio n o f C a r r - P r ic e R e a c tio n f o r
C a r o t e n e .............................................................. .... . , . , 38
4 . V itam in A R e c o v e r y ............................. . ................................ 42
5 . The "C a ro te n e " and "V itam in A" C o n ten t o f th e
L iv e r and I n t e s t i n a l T r a c ts o f v ita m in A-
B e p le te d R a ts a t V a rio u s P e r io d s a f t e r Stom ach-
T ubing w ith C o tto n se e d O il . . . . . . . . . . . 46
6 . The V itam in A C o n te n t o f V a rio u s T is s u e s o f R a ts
S upplem ented w ith a S in g le Dose o f H igh P o te n c y
F is h -L iv e r O il C o n c e n tra te and th e n M a in ta in e d
on a V itam in A -F ree D ie t ................... 48
7 . The C aro ten e and V itam in A C o n te n t o f th e L iv e rs
and I n t e s t i n a l T r a c ts o f V itam in A -D epleted
A nim als a t V ario u s I n t e r v a l s a f t e r Stom ach-
T ubing w ith a S o lu tio n o f C a ro te n e in O il . . . SO
8 . The A b so rp tio n V alu es o f ^ - C a h o te n e and th e
L iv e r P igm ent ....................................... . . . . . . . . 52.
V.
TABLE PAGE
9 . The A b so rp tio n V alues o f V itam in A A lc o h o l and
th e F lu o re s c e n t M a te r ia l o f th e I n t e s t i n a l
T ra c t . . . . . . . ................... . . . . . . . . 55
10. Tween a s th e C aro ten e S o lv e n t . . . . . . . . . 58
11. A eetone a s th e C aro te n e S o lv e n t . . . . . . . . 62
1 2 . A queous C aro te n e S o l .................................................... 62
1 3 . Aqueous C a ro te n e S o l .................................................... 64
14. I n t e s t i n a l Homogenate and A denosine T rip h o s p h a te 6 6
LIST OF GRAPHS
GRAPH PAGE
1 . The A b so rp tio n Curve o f (3 -C a ro te n e and th e
L iv e r P ig m e n t...................................................................... 53
2 . The A b so rp tio n C urves o f V itam in A A lc o h o l
and th e F lu o re s c e n t M a te r ia l o f th e I n t e s t i n a l
T r a c t ................................................................................................ 56
CHAPTER I
INTROUJCTION
A n y 's ta n d a rd te x tb o o k , c o n ce rn ed w ith v ita m in s , a s c r ib e s
th e s i t e o f c o n v e rs io n o f e a ro te n e t o v ita m in A t o th e l i v e r .
I t i s a u n iq u e s i t u a t i o n t h a t su ch a book w i l l u s u a ll y r e f e r to
s e v e r a l e x p e rim e n ts by v a r io u s w e ll-re c o g n iz e d w o rk e rs who
have been u n a b le t o c o n firm t h i s h y p o th e s is . A s e a r c h o f th e
l i t e r a t u r e o f th e p a s t tw e n ty y e a rs r e v e a ls many p a p e rs th e
r e s u l t s o f w hich a r e d i f f i c u l t t o e x p la in on th e b a s i s t h a t
th e l i v e r i s th e o rg a n s o le l y r e s p o n s ib le f o r t h i s c o n v e rs io n .
The c l a s s i c a l e x p e rim e n t, w hich i s th e fo u n d a tio n o f th e id e a ,
i s t h a t o f Thomas Moore (7 8 ), On th e b a s is o f th e p a p e rs pub
l i s h e d , a p p a r e n tly o n ly a few w o rk ers a p p e a r to have q u e s tio n e d
th e v a l i d i t y o f th e c o n c lu s io n s ,
Edwin S ex to n (1 0 0 ), a fo rm er g ra d u a te s tu d e n t o f t h i s
d e p a rtm e n t, a tte m p te d a r e - i n v e s t i g a t i o n o f th e p ro b lem . On
th e b a s is o f h is e x p e rim e n ts* he was u h a b le to e s t a b l i s h t h a t th e
l i v e r was th e r e s p o n s ib le o rg an ; a lth o u g h he had no d e f i n i t e
p r o o f , he was le d to advance th e h y p o th e s i s .th a t th e c o n v e r s io n ■
to o k p la c e i n th e i n t e s t i n a l w a ll.
In th e p r e s e n t t h e s i s , w ork s lo n g t h i s same l i n e h as
b e e n c o n tin u e d and s u b s t a n t i a l e v id e n c e i s o f f e r e d t h a t th e
i n t e s t i n a l w a ll i s a t l e a s t one o f th e s i t e s o f c o n v e rs io n o f
c a ro te n e to v ita m in A i n th e r a t .
CHAPTER I I
HISTORICAL REVIEW OF THE LITERATURE
The l i t e r a t u r e on c a ro te n e and v ita m in A i s v e ry e x te n
s i v e . Each y e a r s e e s th e a d d i tio n o f s e v e r a l h u n d red new p a p e rs .
F or t h i s re a s o n i t i s n e c e s s a ry to l i m i t t h i s h i s t o r i c a l d i s
c u s s io n t o th o s e p a p e rs w hich a r e co n ce rn ed w ith th e f a c t o r s
r e l a t e d t o th e c o n v e rsio n o f c a ro te n e to v ita m in A. T hus, th e
m ethods m en tio n ed a r e th o s e w hich a r e o f o u ts ta n d in g h i s t o r i c a l
i n t e r e s t o r w hich w ere d i r e d t l y c o n ce rn ed w ith th e e x p e rim e n ta l
p ro c e d u re s u s e d . S im ila r ly , a com plete re v ie w o f th e d i s t r i b u t i o n
o f c a ro te n e an d v ita m in A in th e a n im al and v e g e ta b le kingdom s
w ould be im p r a c tic a b le . O nly th o s e p a p e rs w hich may p o s s ib ly
th ro w l i g h t on th e p r e s e n t pro b lem a r e m e n tio n e d .
I d e n t i f i c a t i o n o f C aro ten e
W ackenroder (115) i n 1831 r e p o r te d th e i s o l a t i o n o f a
ru b y - r e d c r y s t a l l i n e m a te r ia l from c a r r o t s w hich he named c a ro
t i n . T here i s no f u r t h e r r e f e r e n c e to t h i s m a te r ia l u n t i l 1907
when W i l l s t S t t e r and M ieg (120) showed i t to be a h y d ro carb o n
w ith th e e m p ir ic a l fo rm u la C4 Q Hgg. A lm ost a n o th e r two d ecades
p a sse d u n t i l th e work o f K a rre r e t a l . ( 5 1 ) , Z e c h m e iste r (1 2 8 ),
and Kuhn and Brockman (64) d e f i n i t e l y e s ta b l is h e d th e s t r u c t u r e
o f B - c a r o te n e a s :
Work c a r r i e d on a t t h i s same tim e d e m o n stra te d th e ex
is te n c e o f compounds c l o s e l y r e l a t e d to p - c a r o te n e and u s u a ll y
found in c lo s e a s s o c ia t io n w ith i t . Thus i n 1931 K a rre r and
h i s a s s o c ia t e s (51) e s t a b l i s h e d th e s t r u c t u r e o f a compound
w hich th e y named c* - c a r o t e n e ;
H
S im i la r l y , Kuhn and Broekmann (6 2 ) added th e compound 2 f-c a ro
te n e whose s t r u c t u r e th e y showed to b e:
T hese compounds w ere s h o r t l y fo llo w e d by th e d is c o v e ry
o f l u t e i n o r x a n th o p h y ll (5 6 ):
, C .
H3C CH3
HgC C-^Q-G=G-C^-C=(?-^=G-C-C-C=C~C-e-C^Q-CH 0H2
H H H H H H H H H E
/ \ CH3 UHg CHg VH3 / \
h3<\ ,CH 3
A
H3 CCH3
f t
/ \ H3 0 /JHg
C
c h 3 v,H 3 uh3 qh3 / \
I
HOCH C-CH3 HgC-C HCOH
4.
an d c ry p to x a n th in (6 5 ):
SsH H 3 C G%
G C
/ \ CHg GHg GHg CHg / \
HgC C-C=C-C=C-CsC-6 =G-C_C-G=.G-CzC-e=-6 -GzC-C c h 2
I I H H H H H HHHH HHH H H
HoC C-CH« H3 C-C HCOH
\ / \ /
c c
H2 H2
B e s id e s th e s e compounds a t l e a s t f i f t y o th e r s have been
i s o l a t e d . The s t r u c t u r a l fo rm u la s o f o n ly th e s e few have been
g iv e n s in c e th e y a re th e o n ly ones o f g r e a t im p o rtan ce in t h i s
s tu d y .
I d e n t i f i c a t i o n o f V itam in A.
Cod l i v e r o i l h a s b e e n u sed a s a t h e r a p e u ti c a g e n t f o r
many c e n t u r i e s , th e o r i g i n o f i t s u s e ly in g i n 'p r a c t i c a l f o l k
l o r e . The f i r s t s c i e n t i f i c d e s c r ip t io n o f th e symptoms w hich
we know to d a y to be due to a d e f ic ie n c y o f v ita m in A was a r e - ’ -
p o r t in 1904 o f x e ro p h th a lm ia a s an ep id em ic d is e a s e in J a p a n .
The e x p e rim e n ta l work o f O sborne and M endel (8 5 -8 7 ) and
McCollum and D avis (6 9 -7 3 ) betw een 1913 and 1915 e s t a b l is h e d
th e p re s e n c e o f a grow th f a c t o r , " f a t s o lu b le A ," in b u t t e r
and cod l i v e r o i l . In 1914 McCollum and D avis (74) showed
t h i s m a te r ia l to be in th e n o n - s a p o n if ia b le f r a c t i o n o f b u t t e r .
I t was n o t u n t i l 1931 t h a t K a rre r (5 4 ) i s o l a t e d v ita m in A in
s u f f i c i e n t p u r i t y t o s u g g e s t i t s fo rm u la to b e:
5.
h 3c , P %
H2e C~CH= CH-C- CH“ CH> CH* U=CH" CHgOH
A
/ \ oh3 ch3
V
g - c h 3
Hg
In su b se q u e n t work (55) he o f f e r e d p ro o f o f tn e c o r r e c tn e s s o r
th e fo rm u la and i t was f u r t h e r co n firm ed by th e w ork o f H e il-
b ro n (4 0 ).
A ll o f th e p re e e e d in g work h ad b een done on m a te r ia ls
i n w hich th e s o u rc e o f th e v ita m in A was m a rin e f i s h e s . Work
on f r e s h w a te r f i s h e s (26) showed th e p re se n c e o f a compound
s im i l a r to b u t n o t i d e n t i c a l w ith v ita m in A. T h is compound
h a s been g iv e n th e name v ita m in Ag. Two s t r u c t u r a l fo rm u la s
have b een s u g g e s te d :
HgC C-CHg
V
HgQ pHg
c h 3 gh3
h2u C~CH=uH~C=uH~ uH=oH- c =CH-uHg0H
H G fl-CE
C
H
The l a t t e r in w hich th e r e a r e two d o uble bonds i n th e (3 -io n o n e
r i n g i s p ro b a b ly th e e o r r e e t one (8 1 ).
W ith in th e p a s t few y e a rs th e s u c c e s s f u l s y n th e s is o f
v ita m in A h a s b een r e p o r te d by s e v e r a l a u th o rs ( 4 ,7 6 ,8 4 ) , :© f
a l l t h e s e , th e one t h a t h a s e x c ite d th e m ost i n t e r e s t in t h i s
c o u n try , a lth o u g h d e t a i l s o f th e m ethod have n o t been g iv e n ,
i s t h a t o f Cawley ( 1 4 ) . The i n t e r e s t h a s b een m a in ly econom ic
s in c e i t i s c la im e d th e m ethod i s p r a c t i c a b l e co m m ercially .
I f t r u e , t h i s w i l l be o f g r e a t im p o rta n c e in view o f th e in
c r e a s in g s h o rta g e o f sh a rk l i v e r o i l .
P r o p e r ti e s o f o a ro te n e and V itam in A
From th e fo rm u la s f o r th e c a r o te n o id s and v ita m in A,
t h e i r s t r u c t u r a l s i m i l a r i t y can r e a d i l y be se e n . T h is i s
e s p e c i a l l y tr u e f o r /3 - c a r o te n e w hich on h y d r o ly tic c le a v a g e
o f th e c e n t r a l d o uble bond w ould y i e l d two m o le c u le s o f
v ita m in A.
B ecause o f t h i s co rre sp o n d e n c e in s t r u c t u r e , a s im i
l a r i t y in c h em ica l and p h y s ic a l p r o p e r t i e s w ould be e x p e c te d ;
such i s th e c a s e . Common p r o p e r t i e s w hich th e y s h a re a re
s t a b i l i t y to a l k a l i , l a b i l i t y to a c id and oxygen, and s o lu
b i l i t y in o rg a n ic s o lv e n ts .
The p re s e n c e o f an a le o h o l group a s in c ry p to x a n th in
o r v ita m in A o r two a lc o h o l g ro u p s a s i n l u t e i n g iv e s th e
compounds d i f f e r e n t i a l s o l u b i l i t y In a lc o h o l s o lu ti o n s . Thus
l u t e i n can be s e p a r a te d from (3 - c a r o te n e i n t h a t l u t e i n i s
* ? .
h y p o p h asic betw een 8 S p e r c e n t e tn y l a lc o h o l and p e tro le u m
e t h e r w h ereas ^ - c a r o t e n e i s e p ip h a s ic ( 9 ).
In 1906 T sw ett (109) in tro d u c e d ch ro m ato g rap h y . I t was
f i r s t u se d e x te n s iv e ly in th e stu d y o f th e c a r o te n o id p ig m e n ts.
By p ro p e r te c h n ic th e v a r io u s compounds d ev elo p w e ll m arked
bands and th u s can be re c o v e re d in th e p u re s t a t e , i t i s p o s
s i b l e t o a p p ly t h i s t o o l to v ita m in A s in c e i t can be v is u a l iz e d
by th e f lu o r e s c e n c e w hich i t d e v e lo p s in u l t r a v i o l e t l i g h t .
m th e s e c ti o n on M ethods o f A n a ly s is th e s p e c t r a l
c h a r a c t e r i s t i c s o f th e s e compounds a r e d is c u s s e d .
S te re o iso m e ris m o f th e C a ro te n o id s and V itam in A
From th e s t r u c t u r e o f c a ro te n e and v ita m in A i t i s
ob v io u s t h a t s te re o is o m e rs may e x i s t (6 0 ). The f i r s t s e p a r a tio n
o f such an iso m er was s u c c e s s f u lly a c c o m p lish e d by G illa m and
E l E id i (3 1 ). T h is f i e l d h a s b een e x te n s iv e ly in v e s t ig a t e d by
Z e c h m e iste r. A r e c e n t re v ie w a r t i c l e (127) by t h i s a u th o r
g iv e s an e x c e lle n t d is c u s s io n o f th e th e o ry and p r a c t i c e o f
s te re o is o m e ris m . The m ethods u se d to b r in g a b o u t t h i s change
a r e ; a) th e rm a l is o m e r iz a tio n in s o lu ti o n ; b ) th e rm a l i s o
m e riz a tio n a c h ie v e d by m e ltin g c r y s t a l s ; c) is o m e r iz a tio n
in d u c ed by io d in e c a t a l y s i s ; o r d) by a c id c a t a l y s i s ; e) p h o to
is o m e r iz a tio n .
P a u lin g (90) h as ad v an ced th e th e o ry t h a t th e number
o f iso m e rs i s c o n t r o l le d by th e r e l a t i o n o f th e d is ta n c e s o f
th e m e th y l g ro u p s r e l a t i v e t o th e double bonds o f th e con-
8 .
ju g a te d c h a in . In th e fo llo w in g fo rm u la th e s t e r i c a l l y e f f e c
t i v e d o u b le bonds a re in d i c a te d by number:
H2
A
H2q 0 -(CH3 ) 2 CHg
I | H H H H H CHg H' CHg HC C >
HoC C ,C ,C V ,C ,G ,C . >CX ,C. ,C.
8 \ * S/ V v V-V V V' V V V , i 'C H g
N f H CHj H CHg H H H H H | I *
■ c h 3 ( 0% ) g 0 CHg
\ /
3 5 5 7 9 C
H2
F o r - c a r o te n e th e r e a r e 20 p o s s ib le s te r e o is o m e r s . To d a te
o n ly a few have b een i s o l a t e d b u t so f a r th e s e conform to th e
advanced h y p o th e s is .
T h at s im i la r s te re o is o m e rs o f v ita m in A sh o u ld e x i s t
i s s u g g e s te d b y i t s s t r u c t u r a l fo rm u la . In th e fo llo w in g f o r
m ula th e s t e r i c a l l y e f f e c t i v e d o uble bonds a r e in d ic a te d by
num ber:
H3C CHg
K
/ \ CHg CHg
H2C C - CH=CH~ C=c h -c h = c h - C - CH~ CH20H
3 5
H2c y J - C H g
C
H2
On t h i s b a s i s 4 s te re o is o m e rs a r e p o s s i b le . The f i r s t o f t h e s e ,
th e c i s - t r a n s iso m e r, was i s o l a t e d l a s t y e a r by Robeson and Bax
t e r ( 9 8 ) . On th e b a s is o f t h e i r w ork i t i s co n c lu d e d t h a t th e
v ita m in A o f f i s h l i v e r o i l u s u a lly c o n s is ts o f 35 p e r c e n t o f
t h i s c i s - t r a n s iso m e r, th e re m a in d e r b e in g th e a l l - t r a n s form*
B io lo g ic a l R e la tio n s h ip o f C a ro te n e and V itam in A
The e a r l y work on th e f a c t o r known a s " f a t s o lu b le A"
was g r e a t l y co n fu se d by th e f a e t t h a t t h i s m a te r ia l was r e p o r te d
by some to be a s s o c ia te d w ith th e y ello w m a tte r (c a r o te n e ) o f
v e g e ta b le s and o th e r s c laim e d i t f o r th e c o l o r l e s s m a te r ia l
fo u n d in f i s h l i v e r o i l s . T h is p roblem was f i n a l l y r e s o lv e d
when Moore (77) d e m o n stra te d by fe e d in g e x p e rim e n ts t h a t y e llo w
c a ro te n e was c o n v e rte d in t o e o l o r l e s s v ita m in A in th e an im al
body. T h is was f u r t h e r co n firm ed by th e s p e c tro p h o to m e tric
w ork o f C apper (15) who d e m o n stra te d th e a p p e a ra n c e o f v ita m in
A i n th e l i v e r o f v ita m in A - d e f ic ie n t r a t s f e d c a r o te n e .
S u b seq u en t work h a s shown t h a t t h i s c o n v e rs io n to v ita m in
A i s lim ite d to c e r t a i n c a r o te n o id s . The g e n e r a l c o n c lu s io n can
be drawn t h a t in o rd e r f o r a c a ro te n o id t o h a v e b i o l o g i c a l a c t i
v i t y a t l e a s t p a r t o f th e m o lecu le m ust have a s t r u c t u r e i d e n t i
c a l w ith t h a t o f v ita m in A e x c e p t f o r th e te r m in a l a lc o h o l g ro u p .
The fo llo w in g t a b l e sum m arizes th e p r e s e n t know ledge a s t o th e
p o te n c y o f th e v a rio u s im p o rta n t c a ro te n o id s ( 1 1 2 ).
Compound B io lo g ic a l a c t i v i t y
o c -c a ro te n e
/^ - c a r o te n e
y - e a r o te n e
1
2
e ry p to x a n th in
l u t e i n
1
0
10.
Worlc in th e p a s t few y e a rs h a s d e m o n stra te d t h a t th e
iso m e ric form o f th e c a ro te n o id i s a ls o o f g r e a t im p o rta n c e .
D euel and Z e c h m e iste r have c o lla b o r a te d on a s tu d y o f many o f
th e s e . T ab le I , g iv e n in one o f t h e i r p a p e rs (2 2 ) , o f f e r s a
com parison o f th e r e l a t i v e e f f e c t s o f is o m e r iz a tio n .
Kemmerer (59) i n a s im i la r in v e s t i g a t i o n h a s o f f e r e d
th e i n t e r e s t i n g f a c t t h a t th e r e i s is o m e ric re a rra n g e m e n t
a f t e r in g e s tio n o f n e o - /3 - c a r o te n e U in to /^ - c a r o t e n e and n eo -
P ^-carotene B, F u tu re work e l u c id a t in g th e r e l a t i v e im p o rtan ce
o f i n t e s t i n a l is o m e ric re a rra n g e m e n t w i l l be o f g r e a t i n t e r e s t .
M ethods o f A n a ly s is f o r V itam in A
The a n a l y t i c a l m ethods f o r v ita m in A a re one o f th e m ost
d is c o u ra g in g f e a t u r e s o f w orking in t h i s f i e l d , xhe la c k o f a
r a p i d , a c c u r a te m ethod f o r th e d e te rm in a tio n o f m in u te am ounts
o f th e v ita m in a c c o u n ts in no sm a ll way f o r th e slow ad vances
made i n th e s tu d y o f i t s p h y s io lo g ic a l r o l e . I n s p i t e o f th e
e x te n s iv e work in th e f i e l d o f m eth o d s, th e p ro c e d u re s u se d
to d a y a r e e s s e n t i a l l y th o s e in tro d u c e d tw e n ty y e a rs ag o . These
f a l l in t o th e th r e e g e n e r a l c l a s s i f i c a t i o n s b i o l o g i c a l , c h e m ie a l,
and p h y s ic a l.
From a t h e o r e t i c a l s ta n d p o in t th e b i o l o g i c a l m ethod i s
th e b e s t , s in c e th e im p o rta n t f a c t o r in any v ita m in i s how i t
r e a c t s p h y s io lo g ic a lly . F or t h i s re a s o n th e U n ite d S ta t e s
P harm acopoeia m ethod i s b a se d on a r ig o r o u s ly c o n t r o l le d b io -
TABLE I
RELATIVE BIOLOGICAL ACTIVITIES OF SOM E M EM BERS
OF STEREOISOMERIC CABOTEI® SETS
P ro b a b le Con- P ro v ita m in A
S e t Member f i g u r a t i o n o f p o te n c y
c i s compounds
o ^ -c a ro te n e a l l - t r a n s
neo U
neo B
(3 - c a r o te n e a l l - t r a n s
neo U
neo B
Y - c a ro te n e a l l - t r a n s
p ro
9-mono- c i s
5 ,9 ( o r 6 ,9 ) di*
c i s
53
13
16
3-mono- c i s
5 , 6 - d i - c i s
100
38
53
3 ,5 ,7 ,9 ,1 1 -
28
44
12.
l o g i c a l a s s a y . The in h e re n t e r r o r s in th e m ethod have r e c e n tly
b een e x te n s iv e ly d is e u s s e d b y G ridgem an { 3 7 ). One g r e a t d is
ad v a n ta g e o f th e m ethod l i e s i n th e lo n g p e r io d o f tim e r e q u ir e d
t o c a r r y o u t th e a s s a y . S in c e c a ro te n e i s c o n v e rte d in t o v i t a
m in A by th e t e s t a n im a l, th e m ethod does n o t r e a d i l y le n d i t
s e l f to d is ti n g u is h in g betw een th e p ro v ita m in A and v ita m in A
i t s e l f , when b o th a re p r e s e n t s im u lta n e o u s ly .
T h at f a c t o r s o th e r th a n v ita m in A may a l t e r th e re s p o n s e
o f th e t e s t a n im a ls i s now re c o g n iz e d . Thus Hickman e t a l
(4 1 , 43) fo llo w in g th e e a r l i e r w ork o f D avies and Moore (18)
have shown t h a t th e l e v e l o f to c o p h e ro l in th e e x p e rim e n ta l
d i e t w i l l m a rk e d ly a f f e c t th e r e s u l t s . The to c o p h e r o ls f u n c tio n
a s a n t i- o x id a n t s b o th in th e i n t e s t i n a l t r a c t and in th e a n i
mals* t i s s u e s . T h is p i c t u r e i s f u r t h e r c o m p lic a te d by th e
r e p o r t o f P a t r i c k and Morgan (89) t h a t th e r e i s an u n re c o g n iz e d
n u t r i e n t in y e a s t and soybean p h o s p h a tid e s w hich i s n e c e s s a ry
f o r th e p ro p e r u t i l i z a t i o n o f v ita m in s E and Ai
A s im i la r a n t i- o x id a n t e f f e c t h a s been a t t r i b u t e d to
x a n th o p h y ll by Sherman (104) f o r th e u t i l i z a t i o n o f c a ro te n e
by th e r a t . Q u ite th e o p p o s ite e f f e c t i s r e p o r te d by K e lle y
and Day (5 8 ) who found t h a t th e a d d itio n o f la r g e am ounts o f
l u t e i n to th e c a ro te n e supp lem en t i n t e r f e r e d w ith th e d e p o si
t i o n o f l i v e r v ita m in A. T h is a c t io n th e y a t t r i b u t e d to a
c o m p e titio n betw een th e l u t e i n and ^ - c a r o t e n e f o r th e enzyme
r e s p o n s ib le f o r th e c o n v e rs io n o f c a ro te n e to v ita m in A.
13.
B ecause o f th e s e f a c t o r s e x te n s iv e work h a s b een c a r r i e d
o u t on th e developm ent o f s a t i s f a c t o r y p h y s ic a l and ch em ical
m ethods o f t e s t i n g f o r v ita m in A.
The c o n ju g a te d d o uble bond sy stem o f v ita m in A f u n c tio n s
a s a chrom ophore group and g iv e s i t th e p r o p e r ty o f s e l e c t i v e
a b s o r p tio n o f l i g h t a t a p p ro x im a te ly 328 m /^ (8 0 ). T h is a b s o rp -
t i p n maximum w i l l v a ry d epending on th e s o lv e n t u se d ( 6 6 ).
B ecause o f th e im pure v ita m in A s o lu tio n s fo rm e rly a v a i la b l e
a s a s ta n d a r d , th e v a lu e f o r th e e x t in c ti o n c o e f f i c i e n t c o u ld
n o t be d e te rm in e d a c c u r a te l y . By common c o n se n t a v a lu e o f
1« £
®lcm 2000 i s u se d in t h i s c o u n try . A r e c e n t c o l la b o r a t iv e
t e s t u s in g c r y s t a l l i n e v ita m in A a c e ta t e w i l l p ro b a b ly b rin g
a b o u t a change o f t h i s v a lu e t o a p p ro x im a te ly 1800 (1 1 3 ). I t
i s o b v io u s t h a t any m ethod w hich u s e s a c o n v e rs io n f a c t o r
b a se d on a p r a c t i c a l a v e ra g e can n o t be to o a c c u r a te . H owever,
f o r p u rp o se s o f com parison such a f a c t o r i s s a t i s f a c t o r y .
The p h y s ic a l m ethod i s f u r t h e r lim ite d b y th e f a c t t h a t
v ita m in A i s n o t th e o n ly m a te r ia l t h a t a b so rb s l i g h t a t 328 m ^ ,
The e x tra n e o u s a b s o r p tio n v a r i e s from o i l to o i l and in one
c a s e was shown to b e a s h ig h a s 40 p e r c e n t (1 7 ). H ow ever, i t
i s p o s s ib le t o overcom e t h i s a s v ita m in A i s fo u n d i n th e non-
s a p o n if ia b le f r a c t i o n .
I n a d d itio n t h e r e a r e o th e r m a te r ia ls in th e n o n -sa p o n -
i f i a b l e f r a c t i o n t h a t a ls o ab so rb l i g h t . One group o f compounds
o f f e r in g d i f f i c u l t i e s a r e th e c a r o te n o id s . B oth is o m e r iz a tio n
(129) and o x id a tio n (125) o f th e s e w i l l ca u se an in c r e a s e in
14
th e amount o f l i g h t th e y a b so rb i n th e u l t r a v i o l e t ra n g e .
F o r tu n a te ly i t i s p o s s ib le to c o r r e c t f o r t h i s s in e e i t h as
been fou n d e m p ir ic a lly t h a t in th e m ethods u s u a ll y a p p lie d
th e y w i l l a t t a i n an e q u ilib riu m a t w hich x .1 3 = ®328*
The to c o p h e ro ls i n t e r f e r e in a s i m i l a r f a s h io n , in t h a t
t h e i r o x id a tio n p ro d u c ts show an in c re a s e d a b s o r p tio n in th e
n e a r u l t r a - v i o l e t . T h is d i f f i c u l t y h a s been overcom e in th e
c a se o f m a rg a rin e by th e m ethod o f Awapara e t a l ( 5 ) .
Iv e n when a l l th e s e f a c t o r s a r e ta k e n in t o a cc o u n t
th e r e i s s t i l l an a b s o rp tio n o f l i g h t in th e u l t r a v i o l e t by
o th e r com pounds. T h is i s e s p e c i a l l y t r u e when one i s w orking
w ith t i s s u e s (1 0 0 ). I f r a t h e r la r g e am ounts o f v ita m in A
a r e p r e s e n t, t h i s f a c t o r becom es n e g l ig ib l e and can be ig n o re d .
I n g e n e r a l, th e n , one may sa y t h a t i t i s n o t p o s s ib le
t o a p p ly th e p h y s ic a l m ethod t o m a te r ia ls c o n ta in in g sm a ll
am ounts o f v ita m in A a s th e n o n s p e c if ic a b s o r p tio n th e n be
comes a p p r e c ia b le , r e l a t i v e to t h a t o f th e v ita m in i t s e l f .
I n 192b C a rr and P r ic e (13) r e p o r te d th e developm ent
o f a b lu e c o lo r w ith a maximum a t 620 m ^w hen v ita m in A was
t r e a t e d w ith a c h lo ro fo rm s o lu ti o n o f antim o n y t r i c h l o r i d e .
T h is m ethod h a s b een e x te n s iv e ly s tu d ie d and h a s fo u n d w ide
u s e . A r e e e n t co m p a ra tiv e s tu d y o f th e b i o l o g i c a l , s p e c tr o -
p h o to m e trie and C a r r - P r ic e r e a c t i o n by O ser ( 8 8 ) shows t h a t
th e r e s u l t s o b ta in e d by th e C a r r - P r ic e r e a c t i o n a g re e more
15.
c l o s e l y w ith th e b i o l o g i c a l v a lu e th a n do th e s p e c tro p h o to -
m e tr ie v a lu e s .
U n fo rtu n a te ly th e C a r r - P r ic e r e a c tio n i s n o t s p e c i f i c
f o r v ita m in A. T hus, c a ro te n e a ls o d e v e lo p s a b lu e c o lo r , th e
maximum how ever b e in g a t 590 m /(. The c o lo r d e v elo p ed d i f f e r s
from t h a t o f v ita m in A in t h a t i t i s much more s t a b l e . W ith
a v ita m in A s o lu ti o n th e c o lo r b e g in s to fa d e in a few se c o n d s,
w h ereas w ith c a ro te n e th e c o lo r p e r s i s t s f o r two o r t h r e e
m in u te s (6 1 ).
S t e r o l s to o d ev elo p a c o lo r w ith antim o n y t r i c h l o r i d e ,
and t h i s r e a c tio n i s th e b a s i s o f a t e s t f o r v ita m in D ( 8 2 ) .
The r e a c tio n i s d i f f e r e n t in t h a t i t ta k e s s e v e r a l m in u te s to
re a c h th e maximum i n t e n s i t y and i s amber in c o lo r r a t h e r th a n
b lu e .
Numerous o th e r c o lo r r e a c tio n s o f v ita m in A have b een
s u g g e s te d . Of t h e s e , t h a t o f S o b e l (105) u s in g a c t iv a t e d
g ly c e r o l d i- c h lo r o - h y d r in a p p e a rs t o have th e g r e a t e s t p o s s i
b i l i t y o f g e n e ra l a c c e p ta n c e .
In s e e k in g a m ethod f o r th e p re s e n t e x p e rim e n ts a l l
th e v a r io u s m ethods w ere c o n s id e re d and t r i e d . The C a r r - P r ic e
r e a c tio n was f i n a l l y chosen a s th e b e s t m ethod. When w orking
w ith sm a ll am ounts o f v ita m in A i t h as th e m arked ad v a n ta g e
o f a h ig h e x t i n c t i o n c o e f f i c i e n t , b e in g a lm o st fo u r tim e s
t h a t o f th e p h y s ic a l m ethod. I n te r f e r e n c e by c a ro te n e can
16.
be c o r r e c te d by p ro p e r s ta n d a r d i z a t io n . M oreover, th e r a p id
fa d in g o f th e c o lo r o f a v ita m in A s o lu ti o n o f f e r s a t tn e same
tim e a q u a l i t a t i v e t e s t .
H etnods o f A n a ly s is f o r C a ro te n o id s
F or th e a n a ly s is o f c a ro te n e th e s ame m ethods a re a v a i l
a b le . Where one i s co n ce rn ed w ith a n a l y s i s f o r t o t a l c a r o te n o id s ,
few d i f f i c u l t i e s a r e o f f e r e d . H owever, i f one i s i n t e r e s t e d in
a s p e c i f i c pigm ent th e p ro c e s s o f s e p a r a tio n i s te d io u s .
The b io l o g ic a l m ethod i s a p p lic a b le w here th e c a ro te n o id
i s one o f th e p ro v ita m in s A. In f a c t , th e i n t e r n a t i o n a l s ta n d a rd
f o r th e b i o l o g i c a l a s s a y f o r v ita m in A chosen by th e Perm anent
Com m ission on B io lo g ic a l S ta n d a r d iz a tio n o f th e H e a lth O rg an i
z a t io n o f th e L eague o f N a tio n s was a sam ple o f /? - c a r o te n e .
Even to d a y one I n t e r n a t i o n a l U n it o f v ita m in A i s d e fin e d by
th e b i o l o g i c a l p o te n c y o f 0 .6 y o f ^ - c a r o t e n e . The b io lo g i
c a l m ethod f o r c a ro te n e i s e x a c tly th e same as t h a t f o r v ita m in
A (111) and s u f f e r s from th e same d is a d v a n ta g e s .
The m ethods in u s e to d a y f o r th e i s o l a t i o n o f th e e a ro -
te n o id s a re b a s e d on th e m ethod o f W i l l s t a t t e r and S t o l l (1 2 1 ).
I n t h e i r p ro c e d u re th e m a te r ia l u n d e r s tu d y was e x t r a c te d w ith
a c e to n e and th e p ig m en ts th e n t r a n s f e r r e d to e th y l e t h e r . The
c h lo ro p h y lls w ere rem oved by s a p o n if i c a ti o n w ith m e th y l a lc o
h o li c p o ta s h . T h is p ro c e d u re h a s b een m o d ifie d by G u ilb e rt (38)
17.
so t h a t th e i n i t i a l s te p i s s a p o n ir i e a ti o n . P e te r s o n , Hughes
and Freem an (91) p r e f e r th e u se o f p e tro le u m e t h e r to e th y l
e t n e r f o r e x t r a c t i o n .
I f i s o l a t i o n o f th e in d i v id u a l p igm ents i s d e s ir e d , i t
i s u s u a ll y n e c e s s a ry t o r e s o r t to chro m ato g rap h y . S in c e th e
in t r o d u c ti o n o f th e a b s o r p tiv e m ethod by T sw e tt, i t h a s been
u se d t o ad v a n ta g e and im proved te c h n iq u e s have been d e v elo p ed
by a g r e a t many w o rk ers in th e f i e l d , u f th e s e , r e f e r e n c e
h e re i s g iv e n o n ly to S t r a i n (106) and Z e e h m e iste r (1 2 6 ) s in c e
t h e i r te c h n iq u e s w ere u se d in th e p r e s e n t problem .
F or th e q u a n t i t a t i v e d e te rm in a tio n o f th e f i n a l sam ple
e i t h e r a c o lo r im e tr ic o r a s p e c tro p h o to m e tric m ethod may be
u s e d . F rap s (30) in a d is c u s s io n o f th e c o lo r im e tr ic m ethods
recommends th e u se o f a 0 .1 p e r c e n t p o ta ss iu m d ic h ro m ate
s o lu ti o n a s a s ta n d a r d . The m onograph o f P e te rs o n , Hughes
and Payne (91) d is c u s s e s th e s p e c tro p h o to m e tric d e te rm in a tio n .
When th e n e c e s s a ry equipm ent i s a v a i l a b l e , th e l a t t e r i s th e
p r e f e r r e d m ethod.
A b so rp tio n o f C a ro te n e and V itam in A
The a b s o r p tio n o f c a ro te n e i s m ore v a r ia b le th a n t h a t
o f v ita m in A. Ahmad (3 ) in 1931 d e m o n stra te d t h a t th e p re s e n c e
o f f a t i s n e c e s s a ry f o r th e p ro p e r a b s o r p tio n o f c a r o te n e .
Shaw and D euel (102) f e d r a t s v a ry in g am ounts o f c a ro te n e and
showed th e r a t e o f a b s o r p tio n o f c a ro te n e to be p r o p o r tio n a l
18.
to th e dose g iv e n .
G reaves and Schm idt (35) in e x p e rim e n ts on e h o le d o -
eh o c o lo sto m iz e d r a t s d e m o n stra te d th e n e c e s s i t y o f b i l e r o r
th e a b s o r p tio n o f c a r o te n e , The p re s e n c e o f b i l e was found
to be h e l p f u l b u t n o t n e c e s s a ry f o r th e a b s o r p tio n o f v i t a
m in A. I r v i n (4 7 ) w orking w ith i n t e s t i n a l lo o p s found sodium
d e so x y c h o la te t o be th e m ost e f f e c t i v e b i l e s a l t in pro m o tin g
th e a b s o r p tio n o f c a r o te n e .
The a b s o r p tio n o f v ita m in A re a c h e s a maximum in th r e e
to f i v e h o u rs (1 6 ). T h is i s c h a r a c te r iz e d by a r i s e in th e
l e v e l o f b lo o d v ita m in A and h as b een e s ta b l is h e d a s a c l i n i
c a l t e s t f o r i n t e s t i n a l a b s o r p tio n . R u s s e ll (99) in e x p e r i
m ents on c h ic k e n s fo u n d t h a t th e a b s o r p tio n o f v ita m in A was
th e same on norm al and low f a t r a t i o n s . An im proved a b s o rp tio n
o f v ita m in A in human s u b je c t s when l e c i t h i n i s added to th e
supplem ent h a s b een o b se rv e d by A d le rs b e rg ( 2 ) .
Low ered a b s o r p tio n o f v ita m in A and c a ro te n e i n human
s u b je c ts h a s b een r e p o r te d i n tu b e r c u lo s is ( 1 2 ) , s p ru e ( 1 ) ,
c a t a r r h a l ja u n d ic e ( 1 1 ) , and in G la rd ia la m b lia i n f e c t i o n s (5 7 ).
R eifm an, H allm an and D euel (97) fo u n d th e r a t e o f
a b s o r p tio n o f v ita m in A t o be p r o p o r tio n a l to th e c o n c e n tra
t i o n o f th e a d m in is te re d m a t e r i a l . By th e u s e o f f l u o r e s
c e n t m ic ro s c o p ic e x a m in a tio n , P opper (92) h a s o b se rv e d t h a t
th e a b s o r p tio n ta k e s p la c e by th e ly m p h a tic r o u t e . Y itam in A
19.
i s a p p a r e n tly c a r r i e d b y n e u t r a l f a t from th e e p i t h e l i a l c e l l s
t o th e l a c t e a l s . In t h i s c o n n e c tio n L overn and M orton ( 6 ?)
have o f f e r e d th e h y p o th e s is t h a t in c e r t a i n s p e c ie s o f f i s h
v ita m in A e s t e r s may a s s i s t i n th e rem oval o f f a t from th e
tu n i c a p r o p r ia .
F u n c tio n o f C a ro te n e and V itam in A
T here i s a s y e t no p ro o f t h a t c a ro te n e f u n c tio n s in
th e v e r t e b r a t e s o th e r th a n a s a p r e c u r s o r o f v ita m in A. W ith
( 1 2 2 ) in r e c e n t y e a rs h as q u e s tio n e d t h i s h y p o th e s is ; how ever,
th e e v id e n c e he o f f e r s can be a s e a s i l y e x p la in e d by o th e r
c a u se s (1 4 1 ), A ssum ing th e o r i g i n a l s ta te m e n t o f t h i s p a r a
g ra p h t o be t r u e , i t i s o n ly n e c e s s a ry th e n to d is c u s s th e
f u n c tio n o f v ita m in A.
In s p i t e o f th e f a c t t h a t v ita m in A was one o f th e f i r s t
grow th f a c t o r s d is c o v e re d , t h e r e i s l i t t l e known to d a y a s to
i t s e x a c t f u n c tio n . I t s r e l a t i o n s h i p to v i s u a l p u rp le h a s
b e e n w e ll e s ta b l is h e d b y th e work o f Wald (1 1 6 ). B ut t h e r e
i s a s y e t no e x p la n a tio n f o r th e g ro s s symptoms o f v ita m in A-
d e f ic ie n c y . An i n t e r e s t i n g exam ple o f th e d i f f i c u l t y in t h i s
f i e l d i s fou n d i n th e p a p e rs o f lo l b a e h (123) and M ellan b y (75)
on th e e f f e c t o f v ita m in A -d e fic ie n e y on bone d ev elopm ent.
W olbach a t t r i b u t e s th e m a lfo rm a tio n to a r r e s t e d grow th w h ereas
M ellanby a t t r i b u t e s i t to o v e rg ro w th . Theye a r e in agreem ent
t h a t th e n e rv o u s m a n if e s ta tio n s o f v ita m in A -d e fic ie n c y a r e due
go.
to t h i s bone m a lfo rm a tio n .
The a l t e r a t i o n s in e p i t h e l i a l s t r u c t u r e s , such a s th e
a tro p h y o f th e e p ith e liu m co n ce rn ed , r e p a r a t i v e p r o l i f e r a t i o n
o f b a s a l c e l l s and d i f f e r e n t i a t i o n o f th e new p ro d u c t in to a
s t r a t i f i e d k e r a t i n i z i n g e p ith e liu m , may be a m a n if e s ta tio n
s im i la r to th o s e changes o c c u rrin g in th e bo n es (1 8 4 ). A
rem ote cau se i s in d ic a te d by th e f a c t t h a t th e symptoms o f
v ita m in A -d e fic ie n c y o c c u r in p a r t in t i s s u e s t h a t do n o t
c o n ta in v ita m in A even in th e h e a lth y a n im a l. An a l t e r n a t i v e
e x p la n a tio n o f t h i s phenomenon c o u ld be t h a t th e v ita m in A
i s m e ta b o liz e d so r a p i d l y by such t i s s u e s t h a t i t s p re se n c e
can n o t be d e te c te d .
S to ra g e and D is tr ib u t io n o f C aro ten e and V itam in A
The e a r l y work o f Moore (7 8 ) d e m o n stra te d th e l i v e r
a s th e im p o rta n t o rg an o f s to r a g e f o r v ita m in A. The fo llo w in g
t a b l e g iv e s th e d i s t r i b u t i o n o f v ita m in A in v a r io u s t i s s u e s
o f th e r a t (1 1 8 ).
O rgan o r t i s s u e I , U. p e r gram o f
m o is t t i s s u e
liver
h e a r t
kidney
m u scle
lu n g
brain
7 .6
012
2 .7
0 .7
5 .3
a d r e n a ls
0
4 9 .
Although in higher animals the carotenoid pigments
a p p a r e n tly have no known f u n c tio n o th e r th a n t h e i r c o n v e rs io n
to v ita m in A, s t i l l th e y a r e s to r e d , i n c e r t a i n s p e c ie s , even
i f th e y do n o t p o s s e s s t h i s p o t e n t i a l i t y . T h is s to r a g e does
n o t e n t i r e l y depend on th e c a ro te n o id in g e s te d , b u t a l s o th e r e
i s a v a r ia tio n .b e tw e e n s p e c ie s a s to th e p a r t i c u l a r c a r o te n o id
s to r e d . In g e n e r a l, mammals seem to s t o r e th e c a ro te n e s
w h ereas fow l s to r e th e c a r o te n o ls . Thus Moore (79) h a s shown
th e pigm ent in l i v e r and m ilk f a t o f cows t o be m a in ly c a ro
te n e , a lth o u g h th e r e i s a v a r i a t i o n in th e d eg ree o f p ig m en ta
t i o n d ep en d in g upon th e b re e d . B a n e rje s and D a tta (7 ) r e
p o r te d t h a t n e i t h e r c a ro te n e n o r x a n th o p h y ll can be d e te c te d
in th e m ilk f a t o r b lo o d o f g o a ts even when e x c e s s iv e q u a n t i t i e s
o f th e s e a r e fe d . The same h o ld s t r u e f o r r a t s . In hedgehogs
th e fe e d in g o f c a ro te n e c a u se s a m arked in c re a s e in th e c a ro
te n e c o n te n t o f th e serum and l i v e r (1 0 7 ). Je n se n and W ith
(4 8 ) i n an e x te n s iv e s tu d y o f th e v ita m in A and c a r o te n o id
c o n te n t o f th e l i v e r s o f S3 d i f f e r e n t mammals b e lo n g in g to
21 s p e c ie s , 4 r e p t i l e s o f 2 s p e c ie s , and in 8 human specim ens
showed t h a t th e r e was c o n s id e ra b le s p e c ie s v a r i a t i o n in th e
amount o f c a ro te n o id p r e s e n t. C a ro te n e was found i n con
s id e r a b l e am ounts in th e l i v e r o f c e r t a i n b e a s ts o f p re y and
b ir d s a s w e ll a s i n th e h a r b iv o r a , b u t th e m a jo r ity o f th e
l i v e r s c o n ta in e d alm o st no c a ro te n o id p ig m en t. L arg e am ounts
o f e a ro te n e w ere fou n d in th e l i v e r s o f th e b a d g e r, fo x and
2 2 .
f i c h e t , th e v a lu e s b e in g o f th e same o rd e r a s th o s e fo u n d in
th e h o rs e and ro e d e e r.
E x c re tio n o f C aro ten e and V itam in A
A V a ria b le amount o f th e in g e s te d c a ro te n e i s e x c r e te d
in th e f e c e s ; De (19) r e p o r t s t h a t up t o 55 p e r c e n t o f th e
c a ro te n e f e d may be found in th e f e c e s .
No c a r o te n o id pigm ent o r v ita m in i s e x c r e te d by th e n o r
m al k id n e y . In mammals a c o n s id e ra b le amount i s e x c r e te d in
th e m ilk , and in fo w l in th e e g g s. The r e l a t i o n s h i p betw een
th e v ita m in A in th e d i e t and th e l e v e l o f c a r o te n o id s and
v ita m in A in th e m ilk h a s b een s tu d ie d by D euel (2 0 ) and in
c h ic k e n s by M a ttso n (6 8 ) and D euel ( 2 1 ) . H ere i t was fo u n d
t h a t th e fe e d in g o f m a ssiv e d o ses o f v ita m in A in c r e a s e d th e
am ount o f th e v ita m in in th e m ilk o r eggs and b lo o d and body
f a t w h ile th e c a ro te n o id l e v e l was lo w ered .
M echanism o f C o n v ersio n o f C a ro te n e to V itam in A
The m echanism o f c o n v e rs io n o f c a ro te n e to v ita m in A
i s s t i l l o b sc u re . The 1 :2 r a t i o f o r b i o l o g i c a l a c t i v i t y o f
°C- to ^ - c a r o t e n e (5 3 ) and th e s im i la r " h a lf a c t i v i t y " o f
s e m i- /^ c a r o te n o n e (6 3 ) and c ry p to x a n th in in d i c a te s t h a t t h i s
in v o lv e s f i s s i o n o f th e c e n t r a l double bond o f ^ - c a r o t e n e (4 4 ).
H u n ter (45) in a r e c e n t rev ie w a r t i c l e h a s d is c u s s e d
th e p ro b a b le ch em ica l r e a c t i o n s . He s t a t e s , f i t a p p e a rs
23.
p ro b a b le t h a t th e c o n v e rs io n w hich o c c u rs i n v iv o in v o lv e s o x i
d a tiv e a t t a c k o f th e c e n t r a l d o uble bond o f / 2 - c a r o te n e w ith
th e fo rm a tio n o f v ita m in A a ld eh y d e ( 3 9 ) , o r a p ro d u c t a f t e r
w ards c o n v e rte d to t h i s , w hich i s re d u c e d t o v ita m in A a lc o h o l
i n a su b se q u e n t s ta g e . A p u r e ly ch em ica l c o n v e rs io n o f c a ro
te n e to v ita m in A w hich s u g g e s ts i t s e l f i s th e r e f o r e d i r e c t
o x id a tio n o f ^ - c a r o t e n e t o v ita m in A ald e h y d e and r e d u c tio n
o f t h i s by th e P o n d o rff m ethod to v ita m in A a l c o h o l . ”
E u le r , K a rre r and S olm ssen (2 9 ) have r e p o r te d t h a t th e
o x id a tio n o f - c a r o te n e w ith a l k a l i n e p o ta ssiu m perm anganate
y ie ld e d f t -a p o -2 - and (2-a p o - 4 - o a r o te n o l w hich a re b i o l o g i c a l l y
a c t i v e .
H3C 0HS
c h s F s F s T O
H2C C ~ CH- CH “ C- CH~ CH-CH- C-CH“ CH -CH“ CH- C “ CH=CH~ CH-C ~ CH O
HgCs^ ^ C— CHg
P - a p o -2 - c a r o t e n o l
% C pHg
/C x 9 H 3 ch^ 9H3
HgC C-0H=CH- CiCH-CH=CH- C-CH~CH=CH~ CH-C" CIO
x C-OH3
c
H2
(2 -a g o - 4 - c a r o te n o l
By th e u se o f hydrogen p e ro x id e in g l a c i a l a c e t i c a c i d , H u n ter
and W illia m s (4 6 ) su cceed ed in fo rm in g v ita m in A a ld eh y d e from
24.
(.9 - c a r o te n e . R e d u c tio n o f th e ald e h y d e w ith alum inum is o p ro p -
o x id e r e s u l t e d in th e fo rm a tio n o f a compound p o s s e s s in g b io
l o g i c a l a c t i v i t y and a l l th e c h a r a c t i s t i c s o f v ita m in A.
T hese p a p e rs i n d i c a t e p o s s ib le c h e m ic a l r o u te s b y w hich
c a ro te n e may be c o n v e rte d t o v ita m in A. The f i n a l answ er a s
t o th e b i o l o g i c a l m ethod o f tr a n s f o r m a tio n w i l l n o t n e c e s s a r i ly
be th e sam e. H u n te r’ s arg u m en ts (4 5 ), b a se d on th e b i o l o g i c a l
p o te n cy o f p a r t i a l l y o x id iz e d c a r o te n e , may n o t f i t in t o th e
p h y s io lo g ic a l system when i t i s f i n a l l y e s ta b l is h e d .
The S i t e o f C o n v ersio n o f C aro ten e t o v ita m in A
I t was in 1930 t h a t Moore (7 7 ) f i r s t r e p o r te d t h a t c a ro
te n e i s c o n v e r tib le i n th e body to v ita m in A. In th e fo llo w in g
y e a r he (78) a t t r i b u t e d t h i s f u n c tio n to th e l i v e r . S in ce t h i s
p a p e r h a s been th e b a s i s o f many c o n c lu s io n s in th e f i e l d o f
c a ro te n e m e ta b o lism , i t w ould be w e ll to q u o te M oore’ s e x a c t
w o rd s. He s t a t e s , " I t i s deduced t h a t th e c o n v e rs io n o f c a ro
te n e to v ita m in A p ro b a b ly ta k e s p la c e in th e l i v e r . ” A uth o rs
s u b s e q u e n tly w r iti n g on th e s u b je c t u s u a ll y dropped th e word
•p ro b a b ly ’ from t h i s s ta te m e n t.
W ith in a few m onths a f t e r th e p u b li c a ti o n o f M oore’ s
w ork, th e f i r s t o f many p a p e rs r e p o r tin g th e in_ v i t r o c o n v e r
s io n o f c a ro te n e t o v ita m in A a p p e a re d . The p a p e r o f O lc o tt
and McCann (83) b a se d t h e i r c o n c lu s io n on a s l i g h t i n f l e c t i o n
in th e a b s o r p tio n sp e c tru m a t 328 fo llo w in g th e in c u b a tio n
25.
o f c a ro te n e w ith l i v e r . The r e s p o n s ib le enzyme was c la im e d
to be h e a t - l a b i l e and was g iv e n th e n a m e ? e a ro te n a s e ." ,A
s i m i l a r ty p e o f ex p erim en t by Bowden and Snow (10) was r e p o r te d
a t a b o u t th e same tim e , in w hieh com parable r e s u l t s w ere o b - -
* - • v
t a i n e d .
F o llo w in g th e a p p e a ra n c e o f th e s e p a p e rs , W oolf and
Moore (125) p u b lis h e d an e x c e lle n t a r t i c l e in w hich th e y d i s
c u ss th e c r i t e r i a n e c e s s a ry t o p ro v e th e c o n v e rs io n o f c a ro te n e
to v ita m in A. In r e p e a tin g th e in v i t r o w ork o f th e o th e r au
t h o r s , th e y o b se rv e d t h a t a c t u a l l y w hat h ad o c c u rre d was an
o x id a tio n o f th e c a ro te n e r a t h e r th a n a c o n v e rs io n o f i t to
v ita m in A.
T hese p a p e rs w ere th e f i r s t o f a s e r i e s c o n ta in in g
c la im s and c o u n te r-c la im s to s u c c e s s i n d e m o n stra tin g th e in
v i t r o c o n v e rs io n o f c a ro te n e to v ita m in A. Thus von E u le r (28)
r e p o r te d t h a t s h a rk l i v e r was in c a p a b le o f b r in g in g a b o u t th e
c o n v e rsio n u n d er in v i t r o c o n d itio n s . P a r ie n te and R a l l i (95)
r e p o r te d t h a t in one ex p e rim e n t o u t o f f o u r th e r e was an i n
c re a s e d re s p o n s e t o th e antim ony t r i c h l o r i d e r e a c tio n a f t e r
in c u b a tin g a c o l l o i d a l s o lu ti o n o f c a ro te n e w ith m inced dog
l i v e r . The r e s u l t s o f th e s e e x p e rim e n ts a r e q u e s tio n a b le in
t h a t th e c a ro te n e s o lu ti o n u se d d id n o t g iv e a p o s i t i v e C a rr-
P r ic e t e s t . Rea (96) was u n a b le to c o n firm th e in v i t r o con
v e r s io n o f c a ro te n e to v ita m in A e i t h e r w ith r a t l i v e r o r
cat; l i v e r . S im ila r u n s u c c e s s f u l r e s u l t s w ere o b ta in e d by-
Ahmad ( 3 ) . Drummond and Bea (25) w ere a ls o u n a b le t o o b ta in
a s u c c e s s f u l in v i t r o p r e p a r a tio n u s in g l i v e r . N e g a tiv e r e
s u l t s w ere a ls o o b ta in e d by Drummond and Mac W a lte r ( 2 4 ) , even
wiien tn e l i v e r c e l l s w ere a llo w e d to ta k e up c a ro te n e from
th e c i r c u l a t i n g blo o d b e fo re tb e m inced p r e p a r a tio n was in c u
b a te d . The l a t e r c la im o f f /ils o n , Ahmad and jsaajumdar (27)
w itn r e g a r d t o th e c o n v e rs io n by l i v e r a u t o ly s a te s i s n o t
s u p p o rte d by s p e c tr o s c o p ic e v id e n c e . In th e more r e c e n t
l i t e r a t u r e th e p a p e r o f K aplansky and B a la b a (5 0 ) r e p o r t s
t h a t io d in a te d c a s e in a c te d a s a c a t a l y s t i n th e in v i t r o
c o n v e rs io n o f c a ro te n e t o v ita m in A.
W i lls ta e d t (119) showed t h a t th e a d d i tio n o f c a ro te n e
t o v ita m in A -fre e l i v e r p a r t i c l e s and f i b r o b l a s t s in a v ita m in
A -fre e serum c u l tu r e r e s u l t e d in an in c re a s e in f i b r o b l a s t
grow th o v e r s im i l a r c u l tu r e s in w hich c a ro te n e was a b s e n t.
An e a r l i e r p a p e r by B aker (6 ) c laim e d to show t h a t v ita m in A
added to f i b r o b l a s t c u l tu r e s r e s u l t e d in an in c r e a s e in grow th.
W i lls ta e d t t h e r e f o r e co n clu d ed t h a t th e c a ro te n e h ad b een con
v e r te d t o v ita m in A in h i s e x p e rim e n ts . H owever, a c lo s e ex
a m in a tio n o f th e p a p e r o f B a rk e r in d i c a te s t h a t th e in c re a s e d
r a t e o f grow th o b ta in e d in h i s e x p e rim e n ts was p ro b a b ly due to
th e s o lv e n t u se d r a t h e r th a n th e v ita m in A i t s e l f .
G reav es and Schm idt (3 6 ) in a s tu d y on th e u t i l i z a t i o n
27
o f c a ro te n e by ja u n d ic e d and p h o s p h o ru s -tre a te d r a t s co n clu d ed
t h a t t h e i r e x p e rim e n ts in d i c a te d th e l i v e r i s th e s i t e o f con
v e r s io n . T h is was b a se d on th e i n a b i l i t y o f i c t e r i c o r phos
p h o ru s-p o iso n e d r a t s t o u t i l i z e c a ro te n e . However in t h e i r
p a p e r th e y r e p o r t t h a t r a t s t r e a t e d w ith such p o w e rfu l l i v e r
p o is o n s a s b e n z e n e , c h lo ro fo rm o r carb o n t e t r a c h l o r i d e w ere a b le
t o c o n v e rt c a ro te n e to v ita m in A. T h is in c o n s is te n c y i n t h e i r
r e s u l t s w ould a p p e a r to c a s t doubt on th e c o n c lu s io n s drawn in
t h e i r p a p e r.
B e n tle y and Morgan (8 ) r e c e n t l y r e p o r te d an i n t e r e s t i n g
e x p e rim e n t. By p a c k in g th e h e p a tic c e l l s w ith f a t by means
o f a c h o l i n e - d e f i c i e n t d i e t and th e K upfer c e l l s w ith tr y p a n
b lu e , an e f f o r t was made t o e s t a b l i s h one o f th e s e a s th e
s i t e o f c o n v e rs io n o f c a ro te n e to v ita m in A. In n e i t h e r c a se
was th e tr a n s f o r m a tio n b lo c k e d . A lth o u g h n o t d e f i n i t i v e , ....
th e s e e x p e rim e n ts make d o u b tfu l th e th e o ry t h a t th e l i v e r i s
th e r e s p o n s ib le o rg a n .
The work o f S ex to n , Mehl and D euel (10) f u r t h e r w eakens
th e h y p o th e s is t h a t th e l i v e r i s th e s i t e f o r th e tr a n s f o r m a tio n
o f c a ro te n e to v ita m in A. In t h e i r e x p e rim e n ts, v ita m in A-
d e f i c i e n t r a t s w ere a d m in is te re d c a ro te n e by v a r io u s p a r e n t e r a l
r o u t e s . Such a n im a ls grew f o r a s h o r t tim e , th e n th e w eig h t
d e c lin e d and th e an im a ls d ie d w ith a l l th e symptoms o f
v ita m in A -d e fic ie n c y ,
A n a ly s is o f th e l i v e r s showed them to c o n ta in la r g e am ounts o f
c a ro te n e * In o th e r w o rd s, th e an im a ls w ere d y in g from v ita m in
A -d e fic ie n c y when th e o rg a n su p p o se d ly r e s p o n s ib le f o r th e con
v e r s io n o f c a ro te n e to v ita m in A c o n ta in e d la r g e am ounts o f
p ro v ita m in A. The h y p o th e s is was t h e r e f o r e advanced t h a t some
o th e r o rg a n was r e s p o n s ib le f o r th e tr a n s f o r m a tio n and th e
i n t e s t i n e was s u g g e s te d a s th e m ost p ro b a b le on e.
In th e e x p e rim e n ta l s e c tio n o f t h i s p a p e r, e v id e n c e i s
p re s e n te d w hich in d i c a te s t h a t one s i t e o f c o n v e rs io n o f c a ro
te n e to v ita m in A i n th e r a t i s th e i n t e s t i n a l w a ll.
CHAPTER I I I
EXFSSIMSNTAt
1* M a te r ia ls
S o lv e n ts
Tween 80^ - T h is i s th e com m ercial name f o r a p o ly -
o x y a lk y le n e d e r iv a tiv e o f s o r b ita n ro o n o lea te.
P e tro le u m e th e r - B oth S k e lly s o lv e A2 ( e s s e n t i a l l y n -
p e n ta n e , B .P . ra n g e 3 2 -3 6 ° C .) and S k e lly s o lv e B (m ixed h ex
a n e s , B .P . ra n g e 63-69° q>) W e r e u s e d . T hese f r e q u e n tly con
t a i n s m a ll am ounts o f im p u r itie s w hich a b so rb l i g h t i n th e
u l t r a v i o l e t ra n g e . T hese im p u r itie s w ere rem oved by p a s s in g
th e s o lv e n t th ro u g h a 12'* colum n o f 80-8 0 mesh s i l i c a g e l ,
D ie th y l e t h e r - P r i o r to u s e , t h i s m a t e r i a l was r e d i s
t i l l e d t o rem ove im p u r itie s t h a t a b so rb l i g h t i n th e u l t r a
v i o l e t ra n g e .
C h lo ro fo rm - Any t r a c e s o f w a te r w ere rem oved by s to r in g
o v e r an h y d ro u s sodium s u l f a t e . The c h lo ro fo rm was th e n d i s
t i l l e d o f f and s to r e d o v e r anhydrous sodium s u l f a t e ,
B loxane - P u r i f i c a t i o n was c a r r i e d o u t by ad d in g sodium
m e ta l c h ip s , a llo w in g i t t o s ta n d f o r s i x h o u rs and th e n r e -
1 F u rn is h e d by th e A tla s Powder Company, W ilm in g to n , D el.
2 T rad e name o f S k e lly O il Company, Lyman, O kla.
30
c o v e rin g th e d io x an e b y d i s t i l l a t i o n ,
t
K re b s-R in g e r P h o sp h ate B u ffe r
T h is was p re p a re d o r i g i n a l l y a c c o rd in g t o th e d ir e c
t i o n s o f U m breit e t a l (1 1 0 ), The f i n a l ‘b u f f e r s o lu ti o n h a s a
pH o f 7 ,4 , At t h i s pH th e c a lc iu m p r e c i p i t a t e s a s th e p h o s
p h a te , At th e s u g g e s tio n o f D r, Jo h n Webb, th e c o n c e n tr a tio n
o f th e c alciu m s a l t was h a lv e d . W ith t h i s m o d if ic a tio n o f th e
b u f f e r no d i f f i c u l t i e s a s t o s o l u b i l i t y w ere e n c o u n te re d . The
r e s u l t i n g s o lu ti o n c l o s e l y ap p ro x im a te s th e c o n c e n tr a tio n o f
io n iz a b le calc iu m fo u n d in th e b lo o d . The f i n a l b u f f e r s o lu
t i o n had th e fo llo w in g com ponents:
1 ) NaCl
2) KP1
3) CaCl
&
4) KH2P04
5) MgS04 .?H 20
6 ) 0.1M p h o sp h a te b u f f e r , pH 7 ,4 (1 7 ,8 g . NagHPO^.,2HgO
p lu s 20 m l. 1 N HCl d i l u t e d t o 1 l i t e r )
To p re p a re th e K re b s-R in g e r s o lu ti o n th e fo llo w in g am ounts o r
th e .above a re j m ix e d :
100 p a r t s o f s o lu ti o n 1
4 it » » 2
3 " * * " 3
1 n w « 4
1 < * » « 3
1
M
0 .9 0 0*154
1 .1 5 0 ,1 5 4
0 ,6 1 0 .0 5 5
2 .1 1 0 .1 5 4
3 ,8 2 0*154
31.
To make up th e K re b s-R in g e r p h o sp h a te s o l u t i o n , 10 m l. o f s o lu
t i o n 6 a r e d i l u t e d to 100 m l. w ith th e K re b s-R in g e r s o l u t i o n .
C aro ten e S o lu tio n s
The so u re e p f c a ro te n e ' in th e fo llo w in g p r e p a r a tio n s
3
was e i t h e r a c r y s t a l l i n e p ro d u c t c o n s is tin g o f 90 p e r c e n t
/ ? - and 10 p e r c e n t - c a r o te n e o r a c o n c e n tra te d com m ercial
p r e p a r a tio n (V a lle y C a ro te n e )4 in c o tto n s e e d o i l . The l a t t e r
m a t e r i a l h a s a claim ed p o te n c y o f 2 2 ,4 0 0 ^ o f c a ro te n e p e r
gram . On th e b a s is o f th e c o lo r im e tr ic and b io lo g ic a s s a y
c a r r i e d o u t in t h i s l a b o r a to r y , th e pigm ent i s e s s e n t i a l l y
p u re /^ - c a r o t e n e .
C a ro te n e s o lu ti o n in c o tto n s e e d o i l - A p p ro x im ately
t h r e e - q u a r t e r s gram o f th e v a lle y C a ro te n e was d i l u t e d to
100 m l. w ith c o tto n s e e d o i l c o n ta in in g 0 .5 p e r c e n t added
o C -to c o p h e ro l. P h o to m e tric d e te rm in a tio n on th e K L e tt-
Summerson c o lo rim e te r showed th e c a ro te n e c o n c e n tr a tio n to
be 1 8 0 0 per ml.
3 O b ta in e d from G e n e ra l B io c h e m ic a ls, I n c , , C h ag rin
F a l l s , O hio.
4 T h is was o b ta in e d th ro u g h th e c o u r te s y o f D r. F r a n c is
P . G r i f f i t h s o f th e Y a lle y V ita m in s, I n c . , M cC allen, T ex as.
5 O b ta in e d from D i s t i l l a t i o n P r o d u c ts , R o c h e s te r, New
Y ork.
32.
The p r e p a r a tio n o f an o i l s o lu ti o n o f th e c r y s t a l l i n e
p ro d u c t i s more d i f f i c u l t b eca u se o f th e slow r a t e o f s o lu ti o n
o f th e c a r o te n e . T h is d i f f i c u l t y was circ u m v en ted by d is s o lv
in g th e c r y s t a l l i n e c a ro te n e in d ie th y l e t h e r . The r e s u l t i n g
s o lu tio n was m ixed w ith c o tto n s e e d o i l c o n ta in in g 0 .5 p e r c e n t
added «*C -tocopherol. The e t h e r was th e n rem oved u n d e r re d u c e d
p r e s s u r e in an atm o sp h ere o f n itr o g e n . The f i n a l c a ro te n e con
c e n t r a ti o n was d e te rm in e d p h o to m e tr ic a lly .
Aqueous s o l o f c a ro te n e - The c a r o te n e , d is s o lv e d in
a c e to n e , was r a p i d l y added w ith sh ak in g t o a warm s o lu ti o n o f
th e K re b s-R in g e r p h o sp h a te b u f f e r . The a c e to n e was rem oved
u n d e r re d u c e d p r e s s u r e , w ith th e s o lu tio n b e in g s t i r r e d by
a f i n e s tre a m o f n itr o g e n b u b b le s. T hese s o ls w ere p re p a re d
im m e d ia te ly b e fo re u s e .
C aro ten e in Tween - V a lle y c a ro te n e was found t o be
r e a d i l y m is c ib le w ith Tween. A w eighed sam ple o f c a ro te n e
was added to th e d e s ir e d amount o f Tween and re n d e re d homo
geneous by th o ro u g h s t i r r i n g .
V itam in A S o lu tio n s - The s o u rc e o f th e v ita m in A was
a h ig h -p o te n c y f i s h l i v e r o i l w hich by s p e c tro p h o to m e tric de
te r m in a tio n was shown t o have a p o te n c y o f 1 2 0 ,0 0 0 I.U . p e r m i.
t *
Aqueous C o llo id a l s o l o f v ita m in A - T h is s o lu ti o n was
p re p a re d by th e same m ethod a s d e s c rib e d f o r th e c a ro te n e s o l .
A ll c a ro te n e and v ita m in A s o lu ti o n s t h a t w ere s to r e d
33.
w ere k e p t u n d e r an atm o sp h ere o f n it r o g e n in th e r e f r i g e r a t o r .
Due p r e c a u tio n s w ere ta k e n t o p re v e n t p h o to -d e c o m p o sitio n .
A nim als
A nim als f o r in v iv o e x p e rim e n ts - The a n im a ls u sed
w ere from th e d e p a rtm e n t’ s s to c k c o lo n y . The d ie ta r y reg im e
was su ch a s to make th e a n im a ls s u i t a b l e f o r u se in th e b io
l o g i c a l a s s a y f o r v ita m in A, The p a r e n ts w ere m a in ta in e d f o r
a t l e a s t two m onths p re v io u s t o b re e d in g and f o r 14 days a f t e r
b i r t h o f th e l i t t e r on Sherman D ie t B (103) w ith o u t th e ad
d i t i o n o f su p p le m e n ta ry l e t t u c e o r m e a t. L i t t e r s w ere re d u c e d
t o sev en a t th r e e days o f a g e , and on th e 1 4 th day m o th ers
and l i t t e r s w ere p la c e d on a v ita m in A-low d ie t.® The a n i
m als w ere w eaned a t 21 days and p la c e d on USP X II v ita m in A-
d e f i e i e n t d i e t , on w hich th e y w ere m a in ta in e d th ro u g h o u t th e
d u r a tio n o f th e e x p e rim e n t. A f te r th r e e weeks on t h i s l a s t
d i e t th e a n im a ls w ere s u i t a b l e f o r u s e , in t h a t no v ita m in A
was fou n d on a n a ly s e s o f v a r io u s t i s s u e s . Any an im a ls t h a t
showed a m arked drop in w e ig h t o r x e ro p h th a lm ia w ere d is c a rd e d
a s u n f i t f o r u s e .
A nim als f o r in v i t r o e x p e rim e n ts - D'or th e s e e x p e rim e n ts
6 The same a s USP X II v ita m in A - d e f ic ie n t d i e t , e x c e p t
t h a t com m ercial c a s e in i s u se d in p la c e o f a lc o h o l- e x t r a c te d
c a s e in .
34.
i t was o n ly n e c e s s a ry t h a t th e i n t e s t i n a l w a ll he f r e e from
v ita m in A. S in c e th e i n t e s t i n a l w a ll c o n ta in s v ita m in A o n ly
w h ile th e v ita m in i s b e in g a b so rb e d , s to c k r a t s w ere s u i t a b l e
f o r u se a f t e r two w eeks on th e v ita m in A - d e f ic ie n t d i e t .
PROCEDURE FOR IN VIVO EXPERIMENTS
T reatm en t o f a n im a ls and t i s s u e s
C o tto n se e d o i l and c a ro te n e s o lu tio n s w ere a d m in is te re d
by sto m a c h -tu b e , th e v ita m in A was g iv e n p e r o s . A f te r th e
e x p i r a tio n o f th e d e s ir e d tim e , th e an im al was k i l l e d by a s
p h y x ia tio n in an atm o sp h ere o f ch lo ro fo rm .
F or th e a n a ly s is o f th e i n t e s t i n a l t r a c t , th e e n t i r e
t r a c t was rem oved and a p p ro x im a te ly th e u p p e r tw o - th ir d s o f
th e sm a ll i n t e s t i n e was s e p a r a te d . T h is p o r tio n was u s e d f o r
a n a ly s is a f t e r f lu s h in g o u t th e c o n te n ts w ith 100 m l. o f 0 .9
p e r c e n t aqueous sodium c h lo r id e . The e n t i r e l i v e r was r e
moved, c le a n s e d o f th e a d h e rin g b lo o d b y b l o t t i n g , and c u t
in t o s m a ll p ie c e s . The two k id n e y s w ere rem oved, any ad
h e rin g c o n n e c tiv e t i s s u e an d th e k id n e y c a p s u le w ere d is c a r d e d ,
and th e k id n e y s com bined in t o one sam ple a f t e r b e in g c u t in to
sm a ll p ie c e s . Each sam ple was p la c e d in a 150 m l. e x t r a c t i o n
f l a s k .
A n a ly s is o f t i s s u e s
To e a c h f l a s k was added 25 m l. o f 95 p e r c e n t e th a n o l
35.
and in a d d i tio n 1 m l. o f bO p e r c e n t aqueous KOH f o r eacn gram
o f t i s s u e . The f l a s k s w ere h e a te d on a h o t p l a t e u n d e r a r e r l u x
co n d en ser f o r one h a l f h o u r.
The s a p o n if ie d m ix tu re was c o o le d and s u f f i c i e n t w a te r
added to make th e f i n a l s o lu ti o n to an a lc o h o l c o n c e n tr a tio n o f
00 p e r c e n t. T h is was th e n e x t r a c te d f o r th r e e h o u rs w ith
S k e lly s o lv e A in a c o n tin u o u s e x t r a c t o r . The e x t r a c t was w ashed
w ith w a te r, d r ie d w ith anhydrous sodium s u l f a t e , and made t o
volum e. The E45 q v a lu e was d e te rm in e d in th e Coleman Model 6A
J u n io r S p e c tro p h o to m e te r. An a l iq u o t o f th e sam ple was th e n
e v a p o ra te d to d ry n e ss in a s tre a m o f n itr o g e n and th e r e s id u e
ta k e n up in . 1 m l. o f c h lo ro fo rm . V itam in A was d e te rm in e d by
th e C a r r - P r ic e r e a c t i o n u s in g h m l. o f a s a t u r a t e d s o lu tio n
o r antim ony t r i c h l o r i d e in c h lo ro fo rm . One drop o f a c e t i c
a n h y d rid e was f i r s t ad d ed , t o p re c lu d e th e developm ent o f a
clo u d y s o lu ti o n . H eadings w ere made a t 10 se c o n d s.
C a lc u la tio n s
A known s o lu ti o n o f p u re c a ro te n e was u se d f o r s ta n
d a r d iz a tio n . On th e b a s is o f t h i s i t was fou n d t h a t
4 x E4 gQ x d i l u t i o n = • Y c a ro te n e in sam ple (1 )
A s i m i l a r s ta n d a r d i z a t io n (T ab le I I ) u s in g p u r i f i e d
d i s t i l l e d v ita m in A e s t e r s 7 in th e C a r r - P r ic e r e a c t i o n
7 O b tain ed from D i s t i l l a t i o n P ro d u c ts , R o c h e s te r,
New Y ork.
TABLE I I
STANDARDIZATION OF CARR-PRICE REACTION
FOR VITAMIN A
E x p e rim e n ta l c o n d itio n s
V itam in A d is s o lv e d in S k e lly s o lv e A was added i n tn e
am ounts in d i c a te d to c o lo r im e te r tu b e s . S k e lly s o lv e rem oved
by a s tre a m o f n itr o g e n and r e s id u e d is s o lv e d in 1 m l. o f
c h lo ro fo rm . One drop o f a c e t i c a n h y d rid e and 0 m l. o f SbClg
w ere ad d ed . R ead in g s made 10 seco n d s l a t e r a t 620 m>(.
I . U ,
E620
I . U.
added
E620
7 .4 0 ;1 4 7 0.0199
1 4 .8 0 .2 9 5 0.0199
22.2 0 .4 4 2 0.0199
2 9 .6 0 .5 9 2 0,0200
A verage - 0.0199
37
gave tn e fo rm u la
0.0199 x l g20 x d i l u t i o n = I.U . v ita m in A in sam ple (2 )
S in c e c a ro te n e a ls o g iv e s a b lu i s h c o lo r w ith antim ony
t r i c h l o r i d e , i t i s n e c e s s a r y t o c o r r e c t th e v ita m in A v a lu e s
f o r t h i s e f f e e t when c a ro te n e and v ita m in A a r e p r e s e n t in th e
same s o lu ti o n . U sin g th e s ta n d a rd s o lu ti o n o f c a ro te n e (T ab le
I I I ) in th e C a r r - p r ic e r e a c tio n (b lu e v a lu e ) and com paring
th e v a lu e s w ith E ^ q ( y e llo w v a lu e ) , i t was found t h a t
y c a ro te n e in sam ple x 0 .0 0 4 9 3 = EggQ
Com bining fo rm u la s (2 ) and ( 3 ) , th e t r u e v a lu e f o r
v ita m in A in th e p re s e n c e o f c a ro te n e was c a l c u la te d by th e
fo rm u la
(®620^ ” '• { cd1* 0^ 81* ® i-1 1 sam ple x 0 .0 0 4 9 3 ) x d i l u t i o n
x 0.0199 =• I.U , v ita m in A in sam ple.
PROCEDURE P0R IN VITRO EXPERIMENTS
P r e p a r a tio n o f t i s s u e s
The a n im a ls w ere s a c r i f i c e d by d e c a p ita tio n , th e i n -
t e s t i n e rem oved im m ed iately and p la c e d in K re b s-R in g e r p h o sp h ate
b u f f e r . A ll f u r t h e r m a n ip u la tio n s o f th e i n t e s t i n a l t r a c t w ere
c a r r i e d o u t w ith th e t i s s u e im m ersed in th e l i q u i d .
3 8 .
TABLE I I I
STANDARDIZATION OF CARR-PRICE REACTION
FOR CAROTENE
E x p e rim e n ta l c o n d itio n s !
C r y s t a l l i n e 90 p e r c e n t f t - and 10 p e r c e n t * < --c a ro te n e
d is s o lv e d in S k e lly s o lv e A was added in th e am ounts in d i c a te d
t o c o lo rim e te r tu b e s . The S k e lly s o lv e was rem oved b y a s tre a m
o f n itr o g e n and th e r e s id u e d is s o lv e d in 1 m l. o f c h lo ro fo rm .
One drop o f a c e tic a n h y d rid e and 5 m l. o f SbClg ad d ed . R ead in g s
made 10 seco n d s l a t e r a t 620 mx f •
C aro te n e Added
y
e 620
E620
y c a ro te n e added
1 1 ,2 0 .0 6 1 0.00543
1 1 .2 0 .0 6 2 0.00553
2 2 .4 0 .1 1 0 0.00492
2 2 .4 0 .1 0 8 0.00482
3 3 ,6 0 .169 0,00503
3 3 .6 0 .1 6 6 0.00494
4 4 .8 0 .2 2 5 0.00502
4 4 ,8 0 .2 2 2 0.00494
5 6 .0 0 .2 7 5 0.00492
5 6 .0 0 .2 7 8 0.00497
5 6 .0 0 .2 7 1 0.00483
5 6 .0 0 .2 7 5 0.00492
A verage 0.00493
39.
I n a l l e x p e rim e n ts a p p ro x im a te ly tw o inch, segm ents o f
th e i n t e s t i n e w ere u s e d . I n some e x p e rim e n ts one end o f th e
i n t e s t i n a l s t r i p was t i e d o f f w ith s t r i n g and th e c a ro te n e
s o lu ti o n was in tro d u c e d in t o th e o th e r end from a s y r in g e .
As th e s y rin g e was w ithdraw n t h a t end o f th e i n t e s t i n a l s e c
t i o n was t i e d o f f . I n o th e r e x p e rim e n ts th e i n t e s t i n a l se g
m ent was s l i t le n g th w is e w ith th e b la d e o f a p a i r o f s c i s s o r s .
The i n t e s t i n a l hom ogenates w ere p re p a re d by g r in d in g
th e i n t e s t i n e , su sp en d ed in d i s t i l l e d w a te r in th e t i s s u e
hom ogenizer d e s c rib e d by P o t t e r (9 4 ).
The t i s s u e , p re p a re d by one o f th e above m e th o d s, was
th e n in tro d u c e d in t o a 50 m l. c o n s t r i c t e d to p c e n t r if u g e tu b e
c o n ta in in g th e a p p r o p r ia te s o lu ti o n s . I n to th e n eck o f th e
c e n tr if u g e tu b e was in s e r t e d a l rt le n g th o f l / 4 rt p l a s t i c
tu b in g . The a i r in th e u p p e r p a r t o f th e c e n tr if u g e tu b e was
r e p la c e d w ith n itr o g e n t o p re v e n t o x id a tio n o f th e c a r o te n e ,
and th e tu b e c lo s e d b y p la c in g a p in c h clamp on th e p l a s t i c
tu b in g . A d m itte d ly , i f th e sy stem i s one r e q u ir i n g oxygen,
t h i s p ro c e d u re w ould b lo c k i t s a c t io n . However, i f o x id a tio n
o f th e c a ro te n e w ere a llo w e d t o o c c u r, a s i t w ould i f oxygen
w ere p r e s e n t, th e p ro d u c ts w ould mask any v ita m in A fo rm ed .
The tu b e s w ere th e n p la c e d in a 37° w a te r b a th i n w hich th e y
w ere shaken f o r th e e x p e rim e n ta l p e r io d . At th e end o f t h i s
tim e th e tu b e s w ere a n a ly s e d f o r c a ro te n e and v ita m in A.
40.
A n a ly s is o f M edia and T isa n e s
To e a c h tu b e was added 1 gram o f s o l i d KQH and a volum e
o f 95 pel? c e n t e t h y l a lc o h o l e q u a l t o th e volum e o f th e m edia.
A 51 p ie c e o f 5 /1 6 " g la s s tu b in g was p la c e d w ith in th e p l a s t i c
tu b in g and th e c e n tr if u g e tu b e s h e a te d in a b o i l i n g w a te r b a th
f o r h a l f an h o u r. The tu b e s w ere th e n c o o le d , 10 m l. o f S k e lly
s o lv e B ad d ed and th e tu b e c lo s e d by in tr o d u c in g th e c o n ic a l
p a r t o f a 15 m l. c e n t r if u g e tu b e in t o th e p l a s t i c tu b in g .
The tu b e s were shaken th o ro u g h ly f o r two m in u tes* The p e tro le u m
e t h e r and a lc o h o l s o lu ti o n s s e p a r a te d on s ta n d in g f o r a few
m in u te s . An a l iq u o t o f th e p e tro le u m e t h e r was p ip e tte d o f f
and th e a n a l y s i s co m p lete d e i t h e r b y th e G a r r - P r ic e r e a c tio n
o r by s p e c tro p h o to m e tric d e te rm in a tio n a t 326 m ^ a s d e s c rib e d
above.
I n o rd e r t o check th e a c c u ra c y o f th e m ethod o f a n a l y s i s ,
p r e lim in a r y c o n t r o l e x p e rim e n ts w ere c a r r i e d o u t. Two r a t s t h a t
p r e v io u s ly h ad b een on a h ig h v ita m in A d i e t w ere s a c r i f i c e d .
T h e ir l i v e r s w ere rem oved, com bined in t o one sam ple and th o ro u g h ly
m inced t o o f f e r a homogeneous sam ple. F iv e a p p ro x im a te ly e q u a l
a l iq u o ts w ere ta k e n . T hree o f th e s e sam p les w ere p la c e d in
c e n tr if u g e tu b e s and to each was added 10 m l. o f K reb s-R in g er
p h o sp h ate b u f f e r , 10 m l. o f 95 p e r c e n t e t h y l a lc o h o l, and 1 g .
o f s o l i d KOH p e r gram o f t i s s u e . T hese w ere s a p o n if ie d , e x t r a c
te d an d th e v ita m in A d eterm in e d a s d e s c rib e d above.
The re m a in in g two sam ples w ere s a p o n if ie d and e x t r a c t
ed th r e e tim e s in a s e p a r a to r y f u n n e l. V itam in A was d e te r
m ined hy th e G a rr-P ric e r e a c t i o n .
S in g le E x t r a c ti o n T r ip le E x tr a c tio n
I.TJ. I.U .
1110 917
942 1110
1260
A verage 1104 A verage 1013
A lth o u g h th e r e s u l t s show c o n s id e ra b le v a r i a t i o n , th e
m ethod was b e lie v e d s u i t a b l e f o r th e p ro p o sed e x p e rim e n ts .
The m ethod has th e a d v a n ta g e o f th e g r e a t r a p i d i t y w ith Which
th e a n a ly s is can b e c a r r i e d o u t. S in c e th e i n v i t r o e x p e r i
m ents w ere e x p lo r a to r y in n a tu r e , th e ad v a n ta g e o f th e sp eed
o f th e m ethod outw eig h ed th e d isa d v a n ta g e o f th e in a c c u ra c y .
I t was a l s o n e c e s s a ry to a s c e r t a i n w h e th e r th e in c u b a tio n
c o n d itio n s em ployed w ould d e s tr o y v ita m in A, F o r t h i s p u rp o se
v ita m in A was s u b je c te d to th e e x p e rim e n ta l c o n d itio n s t o be
em ployed. T hese r e s u l t s a r e g iv e n in T ab le IV .
S in c e th e re c o v e ry o f v ita m in A was th e same w h eth er
th e sam ples w ere in e u b a te d o r n o t, th e e x p e rim e n ta l c o n d itio n s
a re s a t i s f a c t o r y . The lo w er re c o v e ry v a lu e s a f t e r in c u b a tio n
in th e ab sen ce o f th e i n t e s t i n e may p o s s ib ly be a t t r i b u t e d to
TABLE IV
VITAMIN A RECOVERY
I n t e s t i n e
p r e s e n t
Time o f
in c u b a tio n
E326
I . U.
re c o v e re d
( h rs )
Yes 3 0 .1 5 8 •31,9
Yes 3 0 .1 5 3 3 0 .9
Yes 3 0 .1 6 8 3 3.9
Yes 3 0 .1 7 2 3 4 .7
No 3 0 .1 3 2 2 6 .7
No 3 0 ,1 2 0 2 4 .2
No 0 0 .1 6 6 3 3 .4
No 0 0.159 3 2 .1
No 0 0 .1 7 2 3 4 .6
C o n te n ts o f r e a c t i o n f l a s k s ;
10 m l. o f K reb s-K in g er p h o sp h a te b u f f e r c o n ta in in g
200 mg. p e r c e n t g lu c o se and v ita m in A a s a s o l .
In c u b a tio n c o n d i tio n s :
Shaken a t 3?°c in th e d a rk f o r tim e in d ic a te d .
43.
tiie f a c t o r r e p o r te d toy V olk and P opper (114) to toe p r e s e n t in
t i s s u e s w hich p re v e n ts th e d e s tr u c t io n o f v ita m in A.
C a lc u la tio n s
The c o n v e rs io n o f c a ro te n e in to v ita m in A w ould toe i n -
d ic a te d toy an in c re a s e in th e a b s o r p tio n a t 328 o r by an
in c re a s e in th e b lu e to y e llo w r a tiO i.in th e C a r r - P r ic e r e a c t i o n .
I n th e fo llo w in g e x p e rim e n ts b o th m ethods have been u se d a s a
c r i t e r i o n . S in c e th e o x id a tio n o f c a ro te n e c a u se s th e same
e f f e c t , i t i s n e c e s s a ry to compare e a c h ex p erim en t w ith c o n tro ls
ru n a t th e same tim e . In t h i s way th e p re s e n c e o f v ita m in A
can toe d is tin g u is h e d from th e o x id a tio n p ro d u c ts o f c a r o te n e .
C e r ta in m a t e r i a l s , o th e r th a n v ita m in A, n o rm a lly p r e s e n t in
th e i n t e s t i n a l w a ll a l s o cau se an in c r e a s e in th e b lu e v a lu e
a s in d ic a te d in th e s e c ti o n on in vivg. m ethods. T h is e f f e c t
i s ev en more p ronounced when th e a b s o r p tio n a t 328 i s
u se d a s a c r i t e r i o n f o r th e p re s e n c e o f v ita m in A. Thus i t h a s
been fo u n d i n th e e x p e rim e n ta l p ro c e d u re em ployed f o r th e in
v i t r o work t h a t th e n o n - s a p o n if ia b le f r a c t i o n o f th e i n t e s t i n a l
w a ll w i l l o f i t s e l f g iv e an E v a lu e o f a p p ro x im a te ly 0 .0 3 .
T h e re fo re , in i n t e r p r e t i n g th e r e s u l t s o f th e in v i t r o e x p e r i
m ents i t i s n e c e s s a ry t o b e a r in m ind t h a t th e 620 m^ o r
328 m /t v a lu e w i l l be in c re a s e d toy c a r o te n e , th e o x id a tio n
p ro d u c ts o f c a ro te n e and th e n o n -s a p o n ifia to le f r a c t i o n o f th e
44.
i n t e s t i n a l w a ll. As th e in v i t r o e x p e rim e n ts w ere o f a q u a l i
t a t i v e n a tu re no a tte m p t was made to c a l c u la te th e amount o f
c a ro te n e o r v ita m in A p r e s e n t.
CHAPTER i y
RESULTS
I n Vivo E x p erim en ts
The E f f e c t o f th e A d m in is tra tio n o f C o tto n se e d O il
The v ita m in A -d e p le te d a n im a ls w ere sto m a c h -tu b e d w ith
0 .5 m l. o f c o tto n s e e d o i l c o n ta in in g 0 ,5 p e r c e n t to c o p h e ro l.
The an im a ls w ere k i l l e d a t v a ry in g i n t e r v a l s and a n a ly s e s o f
th e l i v e r s and i n t e s t i n a l t r a c t s c a r r i e d o u t. R e s u lts a re
g iv e n in T ab le V.
T hese a n im a ls , a lth o u g h th e y have n e v e r had a c c e s s to
any c a r o te n o id p ig m en t, have a chrom agen in th e l i v e r w hich
a b s o rb s l i g h t a t 450 • F o r p u rp o se s o f com parison tn e
v a lu e s a r e c a l c u la te d and r e p o r te d a s i f th e y w ere c a r o te n e .
S im i la r l y , a lth o u g h v a lu e s a re r e p o r te d f o r v ita m in A,
th e y u n d o u b te d ly a r e n o t a c t u a l l y th e v ita m in . I d e n t i c a l
r e s u l t s have been o b ta in e d on a n im a ls in th e l a s t s ta g e o f
v ita m in A d e f ic ie n c y . The a p p a re n t v ita m in v a lu e i s due
t o an amber c o lo r w hich d e v e lo p s im m ed iately on ad d in g th e
O a rr-P ric e r e a g e n t. T h is c o lo r re m a in s c o n s ta n t f o r 1 to 3
m in u te s and th e n g r a d u a lly in c r e a s e s in i n t e n s i t y . I t i s
p ro b a b le t h a t t h i s i s due t o th e r e a c t i o n o f th e antim ony
t r i c h l o r i d e w ith th e s t e r o l s . I t w ould be p o s s ib le t o c o r r e c t
46*
TABLE V
THE "CAROTENE" AND "VITAMIN A" CONTENT OF THE LIVER AND INTES
TINAL TRACTS OF YITAMIN A-DEPLETED RATS AT VARIOUS PERIODS AFTER
STOMACH-TUBING WITH COTTONSEED OIL
T iss u e
exam ined
Time e la p s e d i n h o u rs
A verage
1 2 4 . 5 6
T o ta l a p p a re n t c a ro te n e in Y
L iv e r 2 .6 0 2 ,0 8 1 .6 0 1 .0 0 2 .7 2 2 .0 0
I n t e s t i n a l
t r a c t
1 .8 0 1 .2 0 1 .6 0 1 .2 8 0 .7 2 1 .3 2
A p p aren t V itam in A i n I .U . / g ,
L iv e r 1 .5 3 1 ,7 5 1 .5 2 0 .8 0 2.6 9 1 .6 6
I n t e s t i n a l
t r a c t
1 .0 6 1 .1 9 2 .1 5 1 .1 7 0 .9 1 1 .3 0
47.
th e v ita m in A f a lu e in th e fo llo w in g e x p e rim e n ts f o r t h i s
r e a d in g . H ow ever, i t w ould seem to be p r e f e r a b l e t o r e p o r t
tn e v a lu e s o b ta in e d . I f , on ad d in g th e C a r r - P r ic e r e a g e n tj
a fa d in g b lu e c o lo r d id n o t d e v e lo p , t h i s f a c t i s in d ic a te d
by e n c lo s in g th e v a lu e s in p a r e n th e s e s . Such v a lu e s , a l
th ough r e p o r te d a s I n t e r n a t i o n a l U n its , a r e n o t b e lie v e d
a c t u a l l y r e p r e s e n t v ita m in A. S in ce t h i s n o n - s p e c if ic
c o lo r i s s n a re d a p p ro x im a te ly e q u a lly by b o th t h e l i v e r and
i n t e s t i n e , i t does n o t a l t e r th e co m p a ra tiv e r e s u l t s .
THe D isa p p e a ra n c e o f V itam in A from V a rio u s O rgans
In t h i s ex p erim en t a gro u p o f v ita m in A -d e p le te d
a n im a ls was su p p lem en ted p e r o s w ith a s in g le dose o f 0 .1 m l.
o f th e f i s h l i v e r o i l c o n c e n tra te c o n ta in in g a p p ro x im a te ly
1 2 ,0 0 0 l.U , o f v ita m in A. The a n im a ls w ere th e n c o n tin u e d
on th e v ita m in A - d e f ic ie n t d i e t . At v a r io u s i n t e r v a l s , a n i
m als o r t h i s group w ere s a c r i f i c e d and th e v ita m in A c o n te n t
o f th e v a rio u s t i s s u e s d e te rm in e d . T hese r e s u l t s a re g iv e n
i n T ab le V I.
H ere we see t h a t v ita m in A i s p r e s e n t in th e i n t e s t i n a l
w a ll in c o n s id e ra b le am ounts f o r a p p ro x im a te ly sev en days a f t e r
th e re e d in g o f v ita m in A. At th e end o f t h i s tim e , i t d is a p
p e a rs e n t i r e l y . The o n ly e x c e p tio n f o r t h i s was one an im al
w here a n a p p r e c ia b le q u a n t ity s t i l l rem a in e d in th e i n t e s t i n a l
48,
TABLE VI
THE VITAMIN A CONTENT OF VARIOUS TISSUES OF RATS SUPPLEMENTED
WITH A SINGLE DOSE OF HIGH POTENCY FISH-LIVER OIL CONCENTRATE
AND THEN MAINTAINED O N A VITAMIN A“ FREE DIET
E la p se d
p e r io d
L iv e r G, I , t r a c t K idneys
days
I.U , / g . I .U ./G . I .U , / g .
1 500 2 5 .2
3 907 1 0 ,3
7 985 1 9 .7 5S97:
7 832 1 4 .6 1 8 .0
9 674 ( 2 .4 )
11 880 2 .8
13 874 ( 2 .3 )
14 623
( 3 .1 )
79Wf
14 1030 7 .9 7 5 .3 7
17 677 (4 .4 2 ) 8 .5 0
17 842 (3 .4 3 ) 6 .9 0
20 738 (1 .0 7 ) 6 .2 7
20 603 7 .6 8
23 653 8 .3 2
23 952 9 .6 4
49.
w a ll on th e f o u r te e n th d ay . In th e c a s e o f th e k id n e y a d i f
f e r e n t s i t u a t i o n o b ta in s . H ere v ita m in A was p r e s e n t th ro u g h
o u t th e p e r io d s tu d ie d a t a r a t h e r c o n s ta n t l e v e l .
The R ate o f A ppearance o f V itam in A jLn th e I n t e s t i n a l T ra c t
and th e L iv e r A f te r F e e d in g C aro ten e
In t h i s s e r i e s v ita m in A -d e p le te d a n im a ls w ere stom ach-
tu b e d w ith 0 .5 m l. o f th e c a ro te n e s o lu ti o n c o n ta in in g a p p ro x i
m a te ly 900 Y o f c a ro te n e . A f te r v a ry in g p e r io d s , th e an im a ls
w ere s a c r i f i c e d and th e l i v e r and i n t e s t i n a l t r a c t a n a ly se d
f o r c a ro te n e and v ita m in A. T hese r e s u l t s a r e g iv e n in T ab le V II.
U nder th e e x p e rim e n ta l c o n d itio n s em ployed, v ita m in A
a p p e a re d in th e i n t e s t i n a l w a ll 1 .5 h o u rs a f t e r fe e d in g c a ro
te n e , w h ereas 2 .7 5 h o u rs e la p s e d b e fo re i t a p p e a re d in th e
l i v e r . U n t i l a p p ro x im a te ly 4 h o u rs a f t e r f e e d in g , th e v a lu e s
in th e i n t e s t i n e ex ceed ed th o s e in th e l i v e r .
The v a lu e s f o r th e y e llo w pigm ent in th e l i v e r showed
no s i g n i f i c a n t o r c o n s is te n t in c re a s e a t any tim e d u rin g th e
p e rio d s tu d ie d o r when com pared to th e v a lu e s fou n d i n th e
e a r l i e r e x p e rim e n ts a f t e r fe e d in g e o tto n s e e d o i l . However,
t h i s does n o t p re c lu d e e n t i r e l y th e p re s e n c e o f sm a ll am ounts
o f c a r o te n e .
TABLE V II
THE CAROTENE AND VITAMIN A CONTENT OF THE LIVERS AND INTESTINAL TRACTS
OF VITAMIN A-DEPLETED ANIMALS AT VARIOUS INTERVALS AFTER STOMACH-TUBING
WITH A SOLUTION OF CAROTENE IN OIL
T o ta l T o ta l i n t e s L iv e r I n t e s t i n a l t r a c t
E la p se d l i v e r t i n a l w a ll V itam in A V itam in A a v ita m in
p e rio d c a ro te n e c a ro te n e T o ta l P e r g. T o ta l P e r g , A
H rs. ■ z r
r I.U . i.u . I.U . I.U . I.U .
1 .5 v l . 28 3 .9 2 (6 .1 3 ) (1 .8 2 ) 6 .6 7 2 .3 8 0 ,5 6
2 . 0 1 . 0 0 1 . 0 0 (5 .8 2 ) (2 .1 6 ) 6 ,0 3 3 .3 7 1 , 2 1
2 .5 0 .7 2 3 ,6 0 (6 .1 3 ) (1 .9 2 ) 8 .5 7 3 .8 9 1 .9 7
2 .7 5 1 .8 0 1 0 .5 1 2 .7 2 .8 5 1 5 .3 5 ,1 4 2 ,2 9
5 .0 1 . 8 8 1 3 .1 1 5 .9 3 .6 2 2 6 .4 6 . 0 2 2 .4 0
3 .0 1 .6 0 1 3 .5 2 2 . 8 5 .4 8 4 0 .3 1 1 . 1 5 .6
3 .0 2 .3 2 1 4 ,7 2 7 .5 7 .2 3 3 6 .8 1 1 . 1 3 .9
3 .2 5 0 . 8 8 3 .5 0 8 .8 9 2 .7 0 9 .6 3 4 .1 3 1 .4 3
3 .5 0 1 , 8 8 6 .2 8 2 8 .6 6 .6 3 2 2 .5 5 .7 2 - 0 .9 1
3 .7 5 1 .9 2 3 2 .8 2 7 .8 6 .9 6 3 1 .5 8 .5 7 1 .6 1
4 .0 1 .5 2 7 .2 8 1 4 .6 3 ,8 1 1 6 . 2 5 .6 8 1 .8 7
4 .5 1 .8 0 • 6 .4 8 3 0 .7 ' 5 .6 7 2 7 .2 5 .3 2 - 0 .3 5
4 .7 5 1 .4 0 2 3 .8 33 .9 7 .6 8 3 0 .0 6 .3 7 -1 .3 1
5 .2 5 1 . 1 2 5 .5 6 3 0 .7 6 . 0 2 1 5 .9 2 .7 4 -3 .2 8
5 .7 5 2 , 1 0 13 .9 3 9 .1 7 ,9 7 3 0 .2 5 .4 8 -2 .4 9
*
( I .U . / g . i n t e s t i n a l t r a c t ) - I ,U . / g . l i v e r ) a? a v ita m in A.
51.
The N atu re o f th e L iv e r Pigm ent
T w e n ty -fiv e v ita m in A - d e f ic ie n t a n im a ls w ere stom ach-
tu b e d w ith 1 m l. o f th e e a ro te n e s o lu ti o n c o n ta in in g a p p ro x i
m a te ly 1800 Y o f t h i s c a r o te n o id . Seven h o u rs l a t e r th e a n i
m als w ere s a c r i f i c e d , th e l i v e r s p o o led and th e pigm ent ex
t r a c t e d a s p r e v io u s ly d e s c rib e d . C a lc u la tin g th e q u a n tity
o f pigm ent p r e s e n t on th e b a s is o f i t s re a d in g a t 450 m>(
a s i f i t w ere ^ - c a r o t e n e gave a v a lu e o f 6 3 ,7 Y • On a
w e ig h t b a s i s , t h i s am ounts to 0 ,4 ^ / g . T h is i s a p p ro x im a te ly
th e same v a lu e a s t h a t o b ta in e d e i t h e r a f t e r fe e d in g c o tto n
se e d o i l o r c a ro te n e in th e p re v io u s e x p e rim e n ts.
The a b s o r p tio n cu rv e (G raph 1) gave no i n d i c a ti o n o f
th e p re s e n c e o f c a r o te n e . W hereas c a ro te n e shows two maxima
a t 450 and 472 m ^ , th e maximum v a lu e o f th e l i v e r pigm ent
was in th e u l t r a v i o l e t ra n g e and th e r e a d in g s f e l l l i n e a r l y
from 320 to 520 i / ( w hich was th e re g io n o f th e sp ectru m
s tu d ie d . No i n f l e c t i o n in th e cu rv e was o b se rv e d a t 450 m ^ .
The pigm ent was fo u n d to be p r e c i p i t a t e d from a m e th y l a lc o
h o l s o lu ti o n when p la c e d in a d ry ic e b a th . I t was n o t p r e
c i p i t a t e d from 1 :1 a c e to n e -a lc o h o l by d ig i to n in . A lthough
th e p ro o f p r e s e n te d h e re i s n o t a b s o lu te , a t l e a s t th e ab
sen ce o f a c a r o te n o id pigm ent seems t o b e in d ic a te d .
5 2 .
TABLE V III
THE ABSORPTION VALUES O F ^-CAROTENE AND THE LIVER PIGMENT
-
L iv e r Pigm ent (2 - c a r o te n e
E
E E4.50
E
E E450
510 .0 5 7 .3 0 3
500 .0 7 2 .3 8 3 .035 .199
490 .088 .4 6 7
485 .1 3 7 .778
4 8 2 .1 5 4 .8 7 5
480 .1 0 6 .5 6 3 .1 6 3 ,9 2 6
477 .1 6 3 ,9 2 6
475 .1 6 2 .9 2 1
472 .161 .9 1 6
470 .1 3 3 .7 0 6 .1 5 6 .8 8 6
460 .1 5 5 .8 2 5 .149 .8 4 7
455 .1 5 1 .8 5 8
452 .1 7 4 .9 8 8
450 .1 8 8 1 . 0 i l ?6 1 . 0
445 .1 6 3 .9 2 7
440 .2 0 4 1 .0 8
450 .2 2 8 1 . 2 1
425 .1 2 1 .6 8 7
420 .258 1 . 2 1
410 .2 7 5 1 .4 6
400 .2 9 7 1 .5 8 ,0 6 3 ,3 5 8
5 3 .
GRAPH 1
THE ABSORPTION CURVE OP (? -CAROTENE AND THE LIVER PIGMENT
1 .4
1.2
1.0
®450
0 .8
0.6
G L iv e r pigm ent
-A - c a r o te n e
0 .4 /
0.2
500
460 480 440 480 400
54.
Q u a li ta ti v e I d e n t i f i c a t i o n o f V itam in A in th e I n t e s t i n a l
T ra c t a f t e r d e e d in g C aro te n e
A f u r t h e r group o f 12 v ita m in A - d e f ic ie n t an im a ls was
sto m a c h -tu b e d w ith 0 .5 m l. o f th e c a ro te n e s o lu tio n c o n ta in in g
900 Y . A fte r fo u r h o u rs th e a n im a ls w ere k i l l e d . The in
t e s t i n e s w ere rem oved, com bined in t o a s in g le sam ple and tn e
n o n - s a p o n if ia b le f r a c t i o n s e p a r a te d . The p e tro le u m e t h e r
s o lu ti o n was e x t r a c te d th r e e tim e s w ith 90 p e r c e n t m e th y l
a lc o h o l. The c a ro te n e rem a in ed in th e p e tro le u m e t h e r s o lu
t i o n w h ile any v ita m in A p r e s e n t w ould be t r a n s f e r r e d to
th e m e th y l a l c o h o l. S u f f i c i e n t w a te r was added to re d u c e
th e m e th y l a lc o h o l to a. c o n c e n tr a tio n o f 50 p e r c e n t and
t h i s s o lu ti o n was th e n e x t r a c te d w ith S k e lly s o lv e A. The
p e tro le u m e t h e r was w ashed w ith w a te r and d r ie d w ith anhy
d ro u s sodium s u l f a t e .
An a l iq u o t was chro m ato g rap h ed on a column o f 1 :1
m agnesium o x id e -h y f lo . U nder u l t r a v i o l e t l i g h t th e f l u o r e s -
e in g band was s e p a r a te d m e c h a n ic a lly . T h is was e l u te d by
th e a d d itio n o f a sm a ll amount o f e t h y l a lc o h o l. The e l u a te
was d i l u t e d w ith S k e lly s o lv e A and th e a b s o r p tio n curVe
d e te rm in e d on th e Beckman S p e c tro p h o to m e te r. T hese v a lu e s
a r e shown in G raph I I and com pared w ith th e cu rv e o b ta in e d
from a known sam ple o f v ita m in A a lc o h o l.
A n o th er a l iq u o t o f th e i n t e s t i n a l t r a c t e x t r a c t was
55
TABLE IX
THE ABSORPTION VALUES OF VITAMIN A ALCOHOL
AND THE FLUORESCENT MATERIAL OF THE INTESTINAL TRACT
V itam in A a lc o h o l F lu o re s c e n t M a te r ia l
E
E . E326
E
I Eggg
300 ,4 0 5 ,5 8 8 .4 0 2 ,8 8 7
310 .5 6 4 .8 2 1 .4 2 8 .9 5 0
320 .6 4 3 .9 3 5 .4 4 6 .9 9 0
326 .6 8 8 1 . 0 .4 5 2 1 . 0
330 .6 6 2 .9 6 2 .4 1 3 .9 1 5
340 .5 4 3 .7 9 0 .3 0 2 ,6 6 8
350 .3 4 1 .4 9 6 .1 8 3 .4 0 4
66
GRAPH 3 .
THE ABSORPTION CURVES OF VITAMIN A ALCOHOL
AND THE FLUORESCENT MATERIAL OF THE INTESTINAL TRACT
1.0
0.8
O F lu o re s c e n t m a t e r i a l o f i n t e s t i n e s
0.2
A V itam in A a lc o h o l
310 300 350
57.
m ixed w ith a sam ple o f known v ita m in A a lc o h o l. On a column
o f 1 : 1 m agnesium o x id e -h y flo o n ly one f lu o r e s c e n t hand d e v e l
oped.
I n V itr o E x p erim en ts
The fo re g o in g jLn v iv o e x p e rim e n ts w ould be g r e a t l y
s tre n g th e n e d i f t h i s c o n v e rs io n o f c a ro te n e t o v ita m in A
c o u ld a l s o be d e m o n stra te d in v i t r o . The fo llo w in g e x p e r i
m ents w ere e x p lo r a tio n s in t h a t d i r e c t i o n in an a tte m p t to
e l u c id a t e th e c o n d itio n s n e c e s s a ry f o r th e d e m o n stra tio n
in v i t r o o f th e c o n v e rsio n o f c a ro te n e t o v ita m in A.
K reb s-H in g e r p h o sp h a te b u f f e r was u se d a s a medium.
An in c u b a tio n te m p e ra tu re o f 37°C was s e le c te d . The n e x t
q u e s tio n was th e p ro p e r s o lv e n t f o r th e c a ro te n e . The
number o f p o s s ib le s o lv e n ts i s li m it e d in t h a t i t n o t o n ly
h a s t o be c a p a b le o f d is s o lv in g th e c a ro te n e b u t m ust a ls o
be m is c ib le w ith th e aqueous m edia.
Tween a s th e C aro ten e S o lv e n t
Tween was s e le c te d a s a s o lv e n t f o r th e c a ro te n e
b e c a u se i t p o s s e s s e s th e s e p r o p e r t i e s . The m arked s u c c e s s
T o m e ra lli (108) had w ith Tween a s a s o lv e n t i n h is e x p e r i
m ents was a f u r t h e r recom m endation. The r e s u l t s o f t h i s
s e r i e s o f e x p e rim e n ts i s g iv e n i n T ab le X.
T here was no m arked o r c o n s is te n t in c re a s e in th e
58
TABLE X
WEEN AS THE CAROTENE SOLVENT
C o n te n ts o f r e a c t i o n f l a s k a :
As in d i c a te d in t a b l e , p lu s 10 m l. o f K re b s-R in g e r
p h o sp h a te b u f f e r .'
I n c u b a tio n c o n d itio n s :
Shaken a t 37°C
sp h e re o f n itr o g e n .
in th e d a rk f o r 3 h o u rs in an atm o-
F la sk
No.
I n t e s t i n e
P re s e n t
T w een-C arotene
s o lu ti o n u s e d ,
m l.
®450
(B)
^620
m
% 2 0
e 450
(B:Y)
F i r s t S e r ie s
L Yes 0 . 2 ,287 .332 1 . 1 6
2 Yes 0 . 2 .306 .378 1 .2 3
3 No 0 . 2 .3 2 6 .410 1 .2 6
4 No 0 . 2 .3 7 6 .468 1 . 24
5 Yes 0 . 0 .0 0 0 .032
-
6 Yes 0 . 0 . 0 0 0 .038
Second S e r ie s
1 Yes 0 . 1 .1 5 0 .183 1 . 2 2
2 Yes 0 . 1 .1 5 5 ,206 1 .3 3
3 Yes 0 . 1 .1 8 7 .2 2 2 1 .1 9
4 Yes 0 . 1 .1 8 2 .2 4 2 1 .3 3
5 No 0 . 1 .1 9 3 .2 7 5 1 .4 2
6 No 0 . 1 .1 5 7 ,215 1 .3 7
59.
TABLE X
(c o n tin u e d )
TW EEN AS THE CAROTENE SOLVENT
F la s k I n t e s t i n e
T w een-C arotene
S o lu tio n u se d
E326 E450 E620 E326
E620
No. P re s e n t m l.
E450
E450
(Y) (B) (B:Y)
T h ird S e r ie s
1 Yes 0 , 1 .0 2 8 .1 5 0 .1 9 3 .1 8 6 1 .2 9
2 Yes 0 . 1 .0 2 8 .1 5 4 .1 9 5 ,1 8 2 1 .2 7
3 Yes 0 . 1 .0 3 0 .1 4 4 .2 0 0 .2 0 8 1.3 9
4 Yes 0 . 1 .0 3 2 .1 6 2 .2 2 8 .197 1 .4 1
5 No 0 . 1 .0 2 5 .1 7 4 .2 2 3 .1 4 4 1 .2 8
6
No 0 . 1 .0 2 7 ,179 .2 4 1 ,1 5 1 1 .3 5
F o u rth S e r ie s
1 Yes 0 . 1 .0 2 6 .1 9 4 ,2 4 6 .1 3 4 1 .2 7
2 Yes 0 . 1 .0 2 7 .198 .2 5 0 .1 3 6 1 .2 6
3 Yes 0 . 1 .0 2 3 .1 8 1 .2 3 0 .1 2 7 1 .2 7
4 Yes 0 . 1 .0 2 3 .1 7 4 #216 .1 3 2 1 .2 4
5 Yes 0 . 1 .0 2 2 .178 .237 .1 2 4 1 .3 3
60
b lu e t o y ello w r a t i o in th e f l a s k s w here th e i n t e s t i n e was
ad d ed , a s com pared t o th o s e in w hich o n ly th e m edia and c a ro
te n e w ere p r e s e n t. S in c e th e a p p e a ra n e e o f v ita m in A would
in c r e a s e th e r a t i o , i t was c o n clu d ed t h a t th e r e was no v i t a
m in A b e in g form ed from th e c a r o te n e . I t was f e l t t h a t a
p o s s ib le re a s o n f o r t h i s f a i l u r e c o u ld be a b lo c k in g by th e
Tween o f th e sy ste m r e s p o n s ib le f o r th e c o n v e rs io n i n v iv o .
A s im i l a r s e r i e s o f e x p e rim e n ts u s in g v a rio u s s o lv e n ts was
th e n d e c id e d upon.
D ioxane a s th e C a ro te n e S o lv e n t
D ioxane ap p e a re d t o be a s u ita b le c a r r i e r in t h a t i t
p o s s e s s e s th e d e s ir e d p r o p e r t i e s . The c a ro te n e was r e a d i l y
s o lu b le in a p p r e c ia b le am ounts in t h i s s o lv e n t. H ow ever,
i t was fou n d t h a t a r a t h e r c o n c e n tra te d s o l u t i o n , even when
s to r e d in th e r e f r i g e r a t o r f o r a few d a y s, became d e c o lo r iz e d
a s th e r e s u l t o f o x id a tio n . P u r i f i c a t i o n o f th e d io x an e by
tre a tm e n t w ith sodium d id n o t p re v e n t t h i s change from
o c c u rrin g . Such a sam ple s to r e d o v e rn ig h t in th e r e f r i g e r a t o r
showed a 326/450 r a t i o o f 0 .2 5 6 a s com pared to a norm al
r a tio i- o f a p p ro x im a te ly 0.06* I t was a p p a re n t t h a t d io x an e
was n o t a s u i t a b l e s o lv e n t.
61.
A cet one a s th e C aro ten e S o lv e n t
S in c e a c e to n e p o s s e s s e s th e r e q u ir e d p r o p e r t i e s , a
s e r i e s o f e x p e rim e n ts was p erfo rm e d u s in g i t a s th e s o lv e n t.
The r e s u l t s a re g iv e n in T ab le X I.
A lth o u g h f l a s k s 2 , 3 , and 4 showed an in c re a s e i n th e
b lu e to y ello w r a t i o n , t h e d if f e r e n c e s w ere v e ry s m a ll. Such
an A lte r a tio n co u ld be e x p la in e d by s l i g h t v a r i a t i o n s in th e
am ount o f o x id a tio n o f th e c a ro te n e o r by th e p re s e n c e o f
i n t e s t i n e . B ecause o f t h i s g r e a t l a b i l i t y , c a u tio n m ust be
e x e r c is e d in i n t e r p r e t i n g a l l r e s u l t s . On t h i s b a s i s , i t
was co n clu d ed t h a t t h i s ex p erim en t h ad n o t d e m o n stra te d th e
c o n v e rs io n o f c a ro te n e to v ita m in A.
A queous C aro ten e S o l
I n th e p re c e d in g e x p e rim e n ts, th e m a te r ia l u s e d a s a
s o lv e n t f o r th e c a ro te n e was p r e s e n t d u rin g th e p e rio d o f
in c u b a tio n . Under th e s e c irc u m s ta n c e s , i t was p o s s ib le t h a t
th e la c k o f s u c c e s s was due to an i n a c t i v a t i o n o f th e sy stem
by th e s o lv e n t.
The n e x t s e r i e s was s e t up t o a v o id t h i s d i f f i c u l t y
by u s in g an aqueous c a ro te n e so l* The r e s u l t s a re g iv e n in
T a b le s X II and X I I I.
The r e s u l t s i n T ab le X II i l l u s t r a t e th e d i f f i c u l t i e s
in v o lv e d in i n t e r p r e t i n g th e s e e x p e rim e n ts . The E326/E450
62.
TABLE XI
ACETONE AS: THE CAROTENE SOLVENT
C o n te n ts o f r e a c t i o n f l a s k s ;
9 m l. K re b s-R in g e r p h o sp h ate b u f f e r
1 m l. a c e to n e s a tu r a t e d w ith c r y s t a l l i n e c a r o te n e .
In c u b a tio n c o n d i tio n s :
Shaken a t 37°C i n th e d a rk f o r two h o u rs in an atm osphere
o f n itr o g e n .
F la s k
No.
I n t e s t i n e
P r e s e n t
E386 E450 E620 E326
> E450
E620
E450
(Y) (B) (B:Y)
1 Yes .0 1 6 .1 5 5 .167 ,1 0 3 1 .0 8
2 Yes . 0 2 0 .1 5 6 .1 7 5 .1 2 8 1 . 1 2
3 Yes ,019 .1 5 1 ,1 7 6 ,1 2 6 1 .1 6
4 Yes ,0 8 0 ,1 5 0 ,1 7 6 .1 3 3 1 .1 7
5 No ,0 1 8 ,1 7 4 ,1 7 8 ,1 0 2 1 . 0 2
6 No .0 1 8 ,1 6 4 .1 7 0 ,1 1 0 1 .0 4
6 3 .
TABLE X II
AQUEOUS CAROTENE SOL
C o n te n ts o f r e a c tio n f l a s k s
10 m l. o f K re b s-R in g e r p h o sp h a te b u f f e r c o n ta in in g th e
c a ro te n e a s a s o l .
In c u b a tio n c o n d itio n s ;
Shaken a t 37°c in th e d a rk f o r 2 h o u rs in an atm o sp h ere
o f n itr o g e n .
I n t e s t i n e
P re s e n t
E326 E45Q E6 2 0 E526
e 450
E620
^450
(* )
(B) (B:Y)
Yes . 0 2 0 .1 5 0 .187 .1 3 3 1 .2 5 '
Yes .0 1 7 ,1 5 5 .1 8 2 , 1 1 0 1 .1 7
Yes .019 ,1 4 6 ,1 7 8 .1 3 0 1 . 2 2
Yes ,0 1 6 .149 .178 .117 1.1 9
No .0 1 3 .1 8 3 .1 9 8 .0 7 1 1 .0 8
No .0 1 4 .1 8 3 .205 .0 7 7 1 . 1 2
64
TABLE X III
AQUEOUS CAROTENE SOL
C o n te n ts o f r e a c tio n f l a s k s
10 m l. K re b s-R in g e r p h o sp h a te b u f f e r c o n ta in in g
2 0 0 mg. p e r c e n t g lu c o s e and th e c a ro te n e
a s a s o l .
In c u b a tio n c o n d itio n s
S haken a t 37°C in th e d ark f o r 2 h o u rs , in an atm o
sp h ere o f n itr o g e n .
I n t e s t i n e p r e s e n t
326
E450
S326
e 450
Y es; 2 m l. o f c a ro te n e s o l
in tro d u c e d in to lum en
. 0 2 2 ,182 .1 2 1
Y es; 3 m l. o f c a ro te n e s o l
in tro d u c e d in t o lumen
.0 2 4 .168 .1 4 3
Y es; i n t e s t i n e s p l i t .0 2 5 .1 5 1 .1 6 5
Y es; i n t e s t i n e s p l i t , 0 2 0 .1 6 5 . 1 2 1
N O . 0 1 2 ,168 ,0 7 1
No .0 1 1 .169 .0 6 5
65*
r a t i o w ould a p p e a r t o i n d i c a te th e c o n v e rs io n o f c a ro te n e in to
v ita m in A. H ow ever, th e in c r e a s e i n th e r a t i o in th e p re se n c e
o f i n t e s t i n e i s due to a d e c re a se in th e v a lu e s r a t h e r
450
th a n an in c r e a s e in th e Eggg r e a d in g . T h is c o u ld w e ll he due
to th e o x id a tio n o f some o f th e c a ro te n e t o a p ro d u c t w hich
does n o t a b s o rb l i g h t a t 326 The ab sen ce o f v ita m in A
i s f u r t h e r in d i c a te d b y th e f a c t t h a t th e b lu e t o y e llo w r a t i o
in c re a s e d o n ly 10 p e r c e n t, w hich i s o f d o u b tfu l s i g n i f i c a n c e .
T a b le X III shows a s i m i l a r in c r e a s e in th e E g g g /l^ Q
r a t i o . H owever, s in e e th e n o n - s a p o n if ia b le f r a c t i o n o f th e
i n t e s t i n a l w a ll w i l l o f i t s e l f r a i s e th e 326 m ^ v a lu e by a t
l e a s t t h i s am ount, th e in c r e a s e o v er th e c o n tr o l v a lu e s c an n o t
be c o n s id e re d s i g n i f i c a n t .
Use o f I n t e s t i n a l Hcanogenate and Added A denosine T rip h o sp h a te
L e h n in g e r i n h i s work on th e in v i t r o o x id a tio n o f
f a t t y a c id s h a s d e m o n stra te d th e n e c e s s i t y o f u s in g hom ogenates
and added ATP. I t i s c o n c e iv a b le t h a t th e r e i s a s im i l a r r e
q u irem en t f o r th e c o n v e rs io n o f c a ro te n e to v ita m in A. A
s e r i e s o f tu b e s was s e t up u s in g th e s e c o n d itio n s . The r e s u l t s
a r e g iv e n in T ab le XIV.
A lthough no c o n t r o l s w ere u se d in t h i s s e r i e s , th e
Eggg/E^gQ ap p ro x im a te th o s e o f th e p re v io u s e x p e rim e n ts .
I t was co n clu d ed t h a t t h i s ex p erim en t was a ls o u n s u c c e s s f u l.
6 6 ,
TABLE XIV
INTESTINAL HO M OGENATE AND ADENOSINE TRIPHOSPHATE
C o n te n ts o f r e a c t i o n f l a s k s
4 m l, o f 11$ i n t e s t i n a l hom ogenate in d i s t i l l e d w a te r
4 m l, o f K re b s-R in g e r p h o sp h a te b u f f e r c o n ta in in g
c a ro te n e a s a s o l
2 m l. 0 ,0 0 1 M Na ATP.
In c u b a tio n c o n d itio n s
Shaken a t 37°C in th e d a rk f o r t h e tim e in d ic a te d ,
in an atm o sp h ere o f n itr o g e n .
Time o f
in c u b a tio n
h r s .
E326
E450
E3 2 6 /E 450
2 .0 7 0 .4 9 4 .142
2 .0 8 0 .4 9 0 .1 6 3
1 .0 7 1 .4 8 2 .147
3 /4 .0 6 7 ,4 5 9 .1 4 6
3 /4 .0 7 8 .4 5 5 .1 7 1
CHAPTER ?
DISCUSSION
The la c k o f s u c c e s s in d e m o n stra tin g th e in v i t r o con
v e r s io n o f c a ro te n e to v ita m in A l i m i t s th e d is c u s s io n o f th e s e
e x p e rim e n ts to th e d i f f i c u l t i e s in v o lv e d . T hese same d i f f i
c u l t i e s a re p ro b a b ly th e b a s i s o f th e num erous c la im s and
c o u n te r-c la im s found in th e l i t e r a t u r e on t h i s p ro b lem . The
n o n - s p e c if ic a b s o r p tio n o f l i g h t by th e n o n - s a p o n if ia b le f r a c t i o n
o f th e i n t e s t i n a l w a ll and by th e o x id a tio n p ro d u c ts o f c a ro te n e
a r e two im p o rta n t l i m i t i n g f a c t o r s in t h i s ty p e o f w ork.
S u c c e s s fu l e x p e rim e n ts w ould depend e i t h e r on overcom ing t h i s
w eakness in th e m ethod o f a n a l y s i s o r on th e v ita m in A p ro d u ced
from th e c a ro te n e b e in g in such q u a n tity t h a t i t s i d e n t i f i c a t i o n
w ould be u n m is ta k a b le .
The c o n d itio n s em ployed in th e s e e x p e rim e n ts w ere s im i la r
t o , b u t n o t i d e n t i c a l w ith , th o s e u se d by W iese e t a l . (117)
who w ere s u c c e s s f u l in d e m o n stra tin g th e in v i t r o c o n v e rs io n
o f c a ro te n e to v ita m in A, The p a p e r o f G lo v e r, Goodwin and
K o rto n (33) a ls o r e p o r t s p o s i t i v e r e s u l t s u s in g r a t i n t e s t i n e .
The e x p la n a tio n f o r th e la c k o f s u c c e s s in th e p r e s e n t work
i s n o t a p p a re n t a t th e p r e s e n t tim e . I t w ould a p p e a r t h a t
c e r t a i n e x p e rim e n ta l c o n d itio n s , h e re o v e rlo o k e d , a re e s s e n t i a l
f o r th e p ro p e r f u n c tio n in g o f th e sy stem .
6 8 .
The in v iv o e x p e rim e n ts, on th e o th e r h an d , p r e s e n t
r e s u l t s w hich w a rra n t f u r t h e r d is c u s s io n .
The e x p e rim e n ts in w hich th e v ita m in A l e v e l was
fo llo w e d a f t e r fe e d in g c a ro te n e d e m o n stra te s t h a t v ita m in A
a p p e a rs f i r s t in th e i n t e s t i n a l w a ll and o n ly l a t e r in th e
l i v e r . T h is e x c e ss o f v ita m in A in th e i n t e s t i n a l w a ll o v er
t h a t in th e l i v e r c o n tin u e d f o r a p p ro x im a te ly 4 h o u rs . I f
one i s to a c c e p t th e l i v e r a s th e s i t e o f c o n v e rs io n , i t i s
d i f f i c u l t t o e x p la in how t h i s s i t u a t i o n co u ld be b ro u g h t
a b o u t.
Even th e q u a l i t a t i v e p re se n c e o f v ita m in A u n d er th e s e
c irc u m s ta n c e s ta k e s on added s ig n if ic a n c e in view o f th e f a c t
t h a t i t was shown t h a t , fo llo w in g v ita m in A s u p p le m e n ta tio n ,
th e v ita m in i s a b s e n t from th e i n t e s t i n a l w a ll even when la r g e
am ounts a r e p r e s e n t in th e l i v e r . T h is w ould in d i c a te t h a t
th e so u rc e o f th e v ita m in A in th e i n t e s t i n a l w a ll i s n o t th e
l i v e r .
T h at th e l i v e r i s n o t th e s i t e o f c o n v e rs io n i s sug
g e s te d f u r t h e r by th e la c k o f any in c r e a s e in i t s c o n te n t o f
c a r o te n e , w h ereas t h a t o f th e i n t e s t i n a l w a ll does in c re a s e
fo llo w in g th e f e e d in g o f c a r o te n e .
The f a i l u r e o f th e l i v e r t o show an in c re a s e in y ello w
pigm ent i s in d isa g re e m e n t w ith th e work o f Moore ( 7 8 ) , b u t
in agreem ent w ith S ex to n e t a l . (1 0 1 ). However, th e c o n d itio n s
6 9 .
o r M oore’ s ex p erim en t w4re d i f f e r e n t from th e s e , in ti i a t n i s
r a t s w ere fe d la r g e am ounts o f c a r r o t s o r re d palm o i l f o r
s e v e r a l w eeks. U nder su cn c irc u m s ta n c e s Moore showed an i n
c re a s e in th e "y ello w u n i t s ” o f th e l i v e r s , b u t th e pigm ent
r e s p o n s ib le was n o t i d e n t i f i e d . S in c e th e supplem ent he
u se d c o n ta in s a m ix tu re o f c a r o te n o id s , i t i s p o s s ib le t h a t
th e pigm ent was n o t one o f th e p ro v ita m in s A.
The c o n c lu sio n t h a t th e pigm ent found in th e l i v e r
u n d e r th e e x p e rim e n ta l c o n d itio n s em ployed i s n o t caro g en e
i s in agreem ent w ith th e work o f Jo h n so n and Baumann (4 9 ).
They r e p o r t t h a t th e " c a ro te n e " m easured in th e l i v e r s and
k id n e y s o f t h e i r a n im a ls was e x tra n e o u s y ello w pigm ent u n re
l a t e d to th e c a ro te n e o f th e d i e t s in c e th e am ounts w ere th e
same w h eth er c a ro te n e was f e d o r n o t. F u rth e rm o re , th e p ig
m ent co u ld be s e p a r a te d q u a n t i t a t i v e l y from added c a ro te n e
on a chrom atogram .
S in c e th e i n t e s t i n e i s th e o n ly o rg an o f th e r a t in
w hich an in c re a s e in c a ro te n e can be fo u n d fo llo w in g i t s
a d m in is tr a t io n , and s in c e v ita m in A i s found in th e i n t e s t i n a l
w a ll o n ly in a p e r io d fo llo w in g th e fe e d in g o f c a ro te n e o r
v ita m in A, i f w ould ap p e a r t h a t th e s i t e o f c o n v e rsio n o f
c a ro te n e to v ita m in A in th e r a t i s th e i n t e s t i n a l w a ll. A fte r
th e v ita m in h as b een form ed by th e i n t e s t i n e fo llo w in g th e
fe e d in g o f c a ro te n e , th e l i v e r a c t s a s a s to ra g e d e p o t.
70.
C e r ta in p a p e rs in th e l i t e r a t u r e w ould le a d one d i r e c t l y
o r i n d i r e c t l y to th e same c o n c lu s io n . Popper (92) s tu d ie d th e
r a t e o f a p p e a ra n c e o f v ita m in A in v a r io u s t i s s u e s o f v ita m in A-
d e f i c i e n t r a t s fo llo w in g th e fe e d in g o f c a r o te n e . As th e organ
in w hich th e v ita m in A f i r s t a p p e a re d was fo u n d t o v a r y , he
a s c r ib e d th e s i t e o f c o n v e rs io n to e i t h e r th e i n t e s t i n e , l i v e r ,
k id n e y , lu n g , r e n a l c o r te x , o r a d r e n a l c o r te x , o r t o a l l o f
th e s e . In view o f th e many f a i l u r e s in a tte m p tin g t o d em o n strate
c a ro te n e in any r a t t i s s u e e x c e p t th e i n t e s t i n e h i s c o n c lu sio n
i s re n d e re d d o u b tfu l a s i t p e r t a i n s to o th e r t i s s u e s , The low
s e n s i t i v i t y o f th e p ro c e d u re he em ployed may e x p la in h i s r e s u l t s .
A p r e lim in a r y r e p o r t o f th e p a p e rs p re s e n te d a t th e
A p r il, 1947, m e e tin g o f th e B io c h em ical S o c ie ty in c lu d e d one
w hich s u p p o rts th e c la im t h a t , in th e r a t , c a ro te n e i s con
v e r te d to v ita m in A in th e i n t e s t i n a l w a ll ( 3 2 ) . The p a u c ity
o f in fo rm a tio n w hich can bfe in c lu d e d in su ch a r e p o r t makes
e v a lu a tio n o f th e r e s u l t s d i f f i c u l t , a lth o u g h i t w ould seem
t h a t th e p r e s e n t ap p ro ac h t o th e p roblem h a s b e e n somewhat
d i f f e r e n t .
An a tte m p te d c o r r e l a t i o n o f th e p a p e r o f Goodwin (34)
on th e a b s o rp tio n o f c a ro te n e in th e g o a t and t h a t o f iJrummond
(23) on a human s u b je c t i s d i f f i c u l t i f one a s c r ib e s th e s i t e
o f c o n v e rsio n t o th e l i v e r . In th e ex p erim en t on th e g o a t,
a d m in is te re d c a ro te n e co u ld n o t be re c o v e re d in e i t h e r th e
p o r t a l v e in o r ju g u la r v e in . T here was a s im i la r la c k o f s u c c e s s
in r e c o v e rin g c a ro te n e from th e c h y le o f a human s u b je c t fe d
c a r o te n e , a lth o u g h v ita m in A c o u ld be re c o v e re d alm o st q u a n ti
t a t i v e l y when i t was a d m in is te re d . U n f o r tu n a te ly , in n e i t h e r
o f th e s e e x p e rim e n ts was an a n a ly s is c a r r i e d o u t f o r v ita m in A
fo llo w in g th e fe e d in g o f c a r o te n e . I f one assum es t h a t in
human b e in g s and g o a ts , th e i n t e s t i n e i s a l s o th e s i t e o f
c o n v e rs io n o f c a ro te n e t o v ita m in A, th e n th e r e s u l t s o b ta in e d
a r e e a s i l y e x p la in e d . A lthough th e s e e x p e rim e n ts do n o t o f f e r
d i r e c t e v id e n c e t h a t c a ro te n e i s c o n v e rte d to v ita m in A in
th e i n t e s t i n e o f th e g o a t and th e human s u b je c t , i t c e r t a i n l y
i s in d i c a te d t h a t such a m echanism may e x i s t in th e s e s p e c ie s .
I f th e i n t e s t i n a l w a ll i s th e s i t e o f c o n v e rs io n in th e
r a t , i t i s n e c e s s a ry to e x p la in how c a ro te n e can be u t i l i z e d
a s v ita m in A fo llo w in g p a r e n t e r a l a d m in is tr a tio n . Very li m it e d
u t i l i z a t i o n i s in d ic a te d by th e work o f S ex to n e t a l . (101) and
m ore e f f e c t i v e u t i l i z a t i o n by T o m a re lli e t a l . (1 0 8 ). U nder
su ch c irc u m s ta n c e s i t c o u ld w e ll be t h a t th e c a ro te n e i s ex
c r e t e d i n t o th e lum en o f th e i n t e s t i n e , c a r r i e d th e r e by e i t h e r
th e b lo o d o r i n th e b i l e . T hat su ch a m echanism i s p o s s ib le
i s s u g g e s te d by th e work o f S ex to n , in w hich he showed t h a t ,
fo llo w in g th e i n t r a p e r i t o n e a l a d m in is tr a tio n o f c a r o te n e ,
5 . 5 p e r c e n t c o u ld be re c o v e re d in th e g a s t r o i n t e s t i n a l t r a c t
and f e c e s . The f i n a l s te p in t h i s scheme w ould th e n be th e
r e a h s o r p tio n o f th e c a r o te n e , a t w hich tim e i t w ould he
c o n v e rte d t o v ita m in A,
CHAPTER V I
SUMMARY
1 . A nim als f e d a s in g le dose o f v ita m in A and tn e n
m a in ta in e d on a v ita m in A - d e f ic ie n t d ie t f o r a p p ro x im a te ly
two weeks show no v ita m in A in th e i n t e s t i n a l w a ll, even
though la r g e am ounts a re p r e s e n t in th e l i v e r and a p p r e c ia b le
am ounts in th e k id n e y .
2 . V itam in A was d em o n stra te d q u a l i t a t i v e l y in th e
i n t e s t i n a l w a ll 4 h o u rs a f t e r fe e d in g c a ro te n e t o v ita m in A-
d e f i c i e n t r a t s .
5 . F o llo w in g th e a d m in is tr a tio n o f c a ro te n e t o v i t a
m in A - d e f ic ie n t r a t s , th e v ita m in a p p e a rs f i r s t in th e i n t e s
t i n a l w a ll. F or a p e r io d o f a p p ro x im a te ly 4 h o u rs th e q u a n tity
in th e i n t e s t i n a l w a ll ex c e e d s t h a t in th e l i v e r .
4 . F o llo w in g th e a d m in is tr a tio n o f c a ro te n e u n d e r th e
e x p e rim e n ta l c o n d itio n s em ployed, th e r e i s no in c r e a s e in th e
y e llo w pigm ent o f th e l i v e r , w h ereas th e r e i s an a u g m e n ta tio n
o f i t in th e i n t e s t i n a l w a ll. The pigm ent o f th e l i v e r ap
p a r e n t ly i s n o t c a r o te n e .
1 . I t i s , t h e r e f o r e , co n clu d ed t h a t one s i t e o f con
v e r s io n o f c a ro te n e t o v ita m in A in th e r a t i s th e i n t e s t i n a l
w a ll.
6 . A tte m p ts to d e m o n stra te t h i s c o n v e rsio n in v i t r o
w ere u n s u c c e s s f u l.
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\iniv3Jsity of Southern California L .ib f & 'r s )

Asset Metadata

Creator Mattson, Fred Hugh (author)

Core Title The site of conversion of carotene to vitamin A.

Contributor Digitized by ProQuest (provenance)

Degree Doctor of Philosophy

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

Tag health sciences, nutrition,OAI-PMH Harvest

Language English

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-606748

Unique identifier UC11354931

Identifier DP21538.pdf (filename),usctheses-c17-606748 (legacy record id)

Legacy Identifier DP21538.pdf

Dmrecord 606748

Document Type Dissertation

Rights Mattson, Fred Hugh

Type texts

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

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

Repository Name University of Southern California Digital Library

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