Significance of essential fatty acids in the utilization of carotene by the rat

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SIGNIFICANCE OF ESSENTIAL FATTY ACIDS IN THE UTILIZATION OF CAROTENE BY THE RAT

BY JOHN S* McANALLY

S ubm itted to th e F a c u lty o f th e G raduate School in p a r t i a l f u l f i l l m e n t of th e req u irem en ts f o r th e d eg ree, D octor o f P h ilo so p h y , in th e Departm ent o f Chem istry In d ia n a U n iv e rs ity May, 1950 i, W'A

ProQuest Number: 10295207

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ACKNOWLEDGEMENTS

The a u th o r w ishes to ex p re ss h is thanfcs to th e N u tr itio n F oundation f o r a fe llo w s h ip g ra n t from F eb ru ary , 19^9

A p ril, 195°* an but s c a r c e ly a n y th in g i s known about th e n a tu re o f th e p ro c e s s .

H unter (69) p o s tu la te d th a t th e p resen ce o f u n s a tu r­

a te d f a t t y a c id s a re im p o rta n t, in c o n ju n c tio n w ith an enzyme, in re n d e rin g th e c e n tr a l double bond o f c a ro te n e prone to o x id a tio n .

O bviously o x id a tio n i s a p ro c e ss in th e c o n v e rsio n .

On t h e o r e t i c a l grounds th e c e n t r a l double bond i s th e most un­ s ta b le .

C onsequently a p ro c e ss o f sym m etrical f i s s i o n m ight

be ex p ected to c h a r a c te r iz e th e fo rm a tio n o f v itam in A from c a ro te n e . The su g g e stio n t h a t e s s e n t i a l f a t t y a c id s m ight be Im p o rtan t in th e form ation o f v itam in A In v o lv es some c o n sid e ra ­

2

t i o n o f th e f a o t th a t c a ro te n e d e s tr u c tio n I s c a ta ly z e d by th e p re se n ce o f th e n u t r i t i o n a l l y e s s e n t i a l f a t t y a c id s and lip o x id a s e * w hich i s p re s e n t in c e r t a i n t i s s u e s (7&)«

The

lip o x id a s e c a ta ly z e s th e p e ro x id a tio n o f l i n o l e i c acid* l i n o l e n i c acid* t h e i r e s te rs * and m ethyl arach id o n ate* th e n u t r i t i o n a l l y e s s e n t i a l f a t t y a c id s .

These a re

O ther f a t t y a c id s do

n o t se rv e a s s u b s tr a te s o f th e enzyme system .

The p e ro x ld iz e d

f a t t y a c id s d e c o lo r iz e c a ro te n e , b u t th e n a tu re o f th e decompo­ s i t i o n p ro d u c ts have n o t been c h a ra c te riz e d *

I t i s c o n sid e re d

h e re a s a t l e a s t rem otely p o s s ib le t h a t th e e s s e n t i a l f a t t y a c ld s —lip o x ld a s e system m ight be a f a c t o r in th e co n v e rsio n o f c a ro te n e to v ita m in A.

At l e a s t th e p o s s i b i l i t y m e rits an

e x p lo ra to ry in v e s tig a tio n . C e rta in ly i t i s unknown what function* i f any* th e e ssen ­ t i a l f a t t y a c id s have in th e u t i l i z a t i o n o f c a ro te n e .

Moreover*

l i t t l e i s known about th e e f f e c t s o f e s s e n t i a l f a t t y a c id d e f ic ie n c y on th e com position o f th e t i s s u e s .

T h erefo re th e

pu rpose o f t h i s in v e s tig a tio n was to e x p lo re th e q u e s tio n o f th e s ig n if ic a n c e o f e s s e n t i a l f a t t y a c id s and lip o x id a s e in th e u t i l i z a t i o n o f c a ro te n e .

In ad d itio n * evidence was sought

co n cern in g th e e f f e c t o f e s s e n t i a l f a t t y a c id d e f ic ie n c y on th e com position o f th e p h o sp h o lip id s in th e t i s s u e s s in c e no rm ally th e e s s e n t i a l f a t t y a c id s a re c o n c e n tra te d in th e s e compounds.

Owing to th e a b i l i t y o f l i n o l e i c a c id , as th e

m ethyl e s t e r , to s a t i s f y th e req u irem en ts f o r a l l th e f a t t y a c id s on ly m ethyl l i n o l e a t e was used in th e in v e s tig a tio n .

HISTORICAL

THE CONVERSION OF CAROTENE TO VITAMIN A E a rly s p e c u la tio n th a t c a ro te n e p o sse s se s v ita m in A a c t i v i t y r e s u l t e d in c o n tr a d ic to r y fin d in g s*

S teenbock, e t

al* ( 1 ) found t h a t c r y s t a l l i n e c a ro te n e had v itam in A a c t i v i t y , w h ile Drummond (2 ) and Stephenson (3) came to th e o p p o site co n clu sio n *

U nequivocal p ro o f th a t c a ro te n e would su p p o rt

grow th on a d i e t d e f i c i e n t in v itam in A was n o t o b ta in e d u n t i l a f t e r th e d is c o v e ry o f v itam in D ( ^ , 5 )* Both Moore (6) and Drummond, Ahmad and Morton (7 ) found th a t th e in g e s tio n o f c a ro te n e gave r i s e to a su b stan ce sim i­ l a r to v itam in A in th e l i v e r s o f r a t s .

Very s h o r tly t h e r e a f t e r

Moore (£>) d em onstrated r a t h e r c o n c lu siv e ly t h a t th e r a t co n v e rts d i e t a r y ca ro te n e to v itam in A and s to re d th e v itam in A in th e liv e r.

F u rth e r experim ents employing a b s o rp tio n s p e c tr a d a ta

and th e antim ony t r i c h l o r i d e c o lo r r e a c tio n v e r if ie d th e se fin d in g s f o r th e r a t , th e chicken and th e pigeon (9-12)* E arly a tte m p ts to e lu c id a te th e s i t e of th e co n v ersio n o f c a ro te n e to v itam in A f e l l in to two c a te g o r ie s .

F irs tly

th e re were th o se o b s e rv a tio n s made on la b o ra to r y a n im a ls, and seco n d ly th o se c l i n i c a l o b s e rv a tio n s which could be made on human p a t i e n t s in whom some p a th o lo g ic a l c o n d itio n had brought ab o u t a secondary derangem ent in ca ro te n e m etabolism .

When

Moore (13) found la r g e q u a n t i tie s o f v itam in A and sm all q u a n t i t i e s o f a y ellow pigm ent in th e l i v e r s o f r a t s fe d c a ro te n e , he assumed t h a t co n v ersio n took p la c e in th e liv e r *

4

However, Drummond and M acw alter (lM*) I n je c te d c a ro te n e su sp e n sio n s in to th e p o r t a l c i r c u l a ti o n o f th e r a t and found t h a t , alth o u g h th e c a ro te n e was tak en up by th e l i v e r , th e r e was no evidence t h a t any o f i t was co n v erted to v ita m in A. These r e s u l t s were confirm ed f o r th e r a t and th e dog, b u t th e r a b b it ap p eared to be a b le to u t i l i z e in tra v e n o u s ly i n ­ je c t e d c a ro te n e ( 15 ).

This was th e f i r s t in d ic a tio n th a t

c a ro te n e m etabolism m ight v ary in d i f f e r e n t h ig h e r s p e c ie s , a q u e s tio n which w i l l be d is c u s se d l a t e r in more d e ta il*

D esp ite

th e fo re g o in g , v a rio u s ex p erim en ters co n tin u ed to su g g e st th a t th e l i v e r i s th e s i t e o f co n v e rsio n .

Greaves and Schmidt (1 6 )

found t h a t th e i c t e r i c r a t does n o t c o n v e rt c a ro te n e to v i t a ­ min A, and t h a t phosphorus p o iso n in g g r e a tly reduces the a b i l i t y o f th e r a t to c a rry o u t th e tra n s fo rm a tio n .

O ther

ex p erim en ts In d ic a te d th a t in man th e l i v e r i s th e s i t e o f c o n v e rsio n ( 17, 1#)*

The s t a t u s o f th e problem i s t y p i f i e d by

th e o p p o site r e s u l t s o b ta in e d by R okhlina, e t a l . (19) and T o m a re lli, e t a l . (20) .

The form er found th a t th e v ita m in

e f f e c t o f c a ro te n e was com pletely la c k in g i f i t was giv en p a r e n t e r a l l y , w hile th e l a t t e r found th a t p ro v id ed th e c a ro te n e was d is p e rs e d to a s u f f i c i e n t l y g re a t degree i t was r e a d ily u tiliz e d a f te r in je c tio n . The f i r s t Im p ressiv e s u g g e stio n th a t c a ro te n e co n v e rsio n i s accom plished in t i s s u e s o th e r than th e l i v e r was made by S ex to n , Mehl and Deuel (2 1 ) .

They dem onstrated th a t ca ro te n e

i n t r a p e r l t o n e a l l y in je c te d i n th e r a t i s n o t co n v erted to

v ita m in A, b u t i s s to re d as such by th e l i v e r .

This s to re d

e a ro te n e d id n o t I n t e r f e r e w ith th e u t i l i z a t i o n o f o r a ll y a d m in is te re d v ita m in A o r c a ro te n e , b u t th e r a t s could and d id d ie o f v itam in A d e f ic ie n c y even though th ey had s to re d i n t h e i r l i v e r s q u ite la rg e amounts o f c a ro te n e .

Almost

sim u lta n e o u sly in 19^7 th re e s e r i e s o f in v e s tig a tio n s showed c o n c lu s iv e ly t h a t in th e r a t and in th e p ig th e conversion o f c a ro te n e to v ita m in A o ccu rs in th e i n t e s t i n a l w a ll (22- 210. Subsequent in v e s tig a tio n s have fu rth e re d t h i s view f o r th e r a t (25)> and have extended i t to th e r a b b it , g o at and sheep (2 6 ). P a r t i c u l a r l y co n v in cin g evidence f o r the i n t e s t i n a l w a ll as th e s i t e o f co n v ersio n was p re se n te d by M attson ( 27 )» who s e p a ra te d v itam in A from th e o th e r n o n -s a p o n ifia b le components o f th e i n t e s t i n a l w a ll.

He found th a t th e u l t r a v i o l e t ab so rp ­

ti o n curve o f th e I s o la te d m a te r ia l was alm ost i d e n t i c a l w ith t h a t o f pure v itam in A. The co n v ersio n o f c a ro te n e to v itam in A i s a f f e c te d by s e v e r a l f a c t o r s , both p h y s io lo g ic a l and n u t r i t i o n a l in o r i g i n . The im portance o f v itam in E i n th e d i e t f o r th e e f f i c i e n t u t i l i z a t i o n o f c a ro te n e has been dem onstrated by s e v e r a l in ­ v e s tig a to rs . ta n t.

The fin d in g s o f Koehn (2£) a re e s p e c ia lly impor­

By in c lu d in g ad eq u ate q u a n titie s o f to c o p h e ro l in th e

d i e t Koehn was a b le to o b ta in eq u a l growth resp o n ses w ith e q u a l w eights of ^ - c a r o te n e and v itam in A.

P re v io u sly th e

b i o l o g ic a l potency o f p -c a ro te n e had been e s ta b lis h e d a t about o n e - h a lf th a t o f v itam in A on a w eight b a s i s .

I t has been

8

assum ed th a t t h i s e f f e c t I s dependent upon th e a n tio x id a n t p r o p e r tie s o f th e to c o p h e ro l, hut i t has a ls o been d em onstrated t h a t excess amounts o f to c o p h e ro l i n t e r f e r e d w ith th e a s s im ila ­ t i o n of c a ro te n e (29)* C e rta in c a ro te n o id s may a ls o a f f e c t the u t i l i z a t i o n o f c a ro te n e .

The e f f e c t seems to vary w ith th e r e l a t i v e q u a n ti­

t i e s o f ca ro ten e and x an th o p h y ll fe d .

Sherman (30) found th a t

th e a d d itio n o f 5 /* £ of x an th o p h y ll ( l u t e i n ) to a d a ily su p p le­ ment o f

o f ca ro ten e improved th e grow th response o f v itam in

A - d e f ic ie n t r a t s on a d i e t f r e e o f v itam in E d u rin g th e p e rio d o f su p p lem en tatio n . 6

0

Kemmerer, P raps and D eM ottier (31) fed

of ea ro ten e p er day, p lu s th e q u a n tity o f x an th o p h y ll

which would o ccu r w ith th a t amount o f ca ro te n e in f r e s h sp in ach , and found an im pairm ent in th e u t i l i z a t i o n of c a ro te n e -a s m easured by v itam in A s to re d in th e l i v e r .

K elley and Day (32)

and High (J2 a.) o b ta in e d r e s u l t s which su p p o rted and extended th e o b se rv a tio n o f Kemmerer, e t a l . The c h a r a c te r o f th e d ie ta r y f a t a f f e c t s th e u t i l i z a t i o n o f c a ro te n e to some d e g re e .

W ilson, das Gupta and Ahmad (33)

found th a t in humans a low c o n c e n tra tio n o f f a t in th e d i e t red u ced ca ro ten e a b s o rp tio n from v e g e ta b le s by about o n e - h a lf. F u rth e r in v e s tig a tio n showed th a t th e in c lu s io n o f b i l e w ith a f a t - f r e e d i e t improved caro ten e a b s o rp tio n ( 3*0 .

One o f th e

p rim ary f a c to r s in ca ro te n e a b s o rp tio n on a f a t - f r e e d i e t i s th e d eg ree o f d is p e rs io n o f th e ca ro te n e su p p lie d .

Majumdar

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