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Interrelations between vitamins A and E
Exp. Eye Res. (1964) 3, 388-391
Interrelations b e t w e e n V i t a m i n s A and E 5. GF, E E N
Walton Oaks ExTerimental ,Station, Vitamins Ltd., Tadworth Su~'zey, England Thel~ arc important interrelations between vitamins A and E which manifest t h e m s e l v e s i n v a r i o u s w a y s . I n t h e die~, t h e p r v s c n c c o f ~:itamin ]~ is of mrrjor i m p o r t a n t e i a p r o t ~ c t l n g small q u a n t i t i e s o f v i t a m i n A f r o m o x i d a t i o n . U n d e r p h y s i o l o g i c a l c o n d i t i o n s , v i t a m i n I~ h a s a s p a r i n g e f f e c t o n vitatLain A a n d o n t h e earot, e n o i d s . i n e x p e r i m e n t a l arfimals t h a t iLt~ve b e e n gig-on d i e t s o n l y m a r g i n a l l y s u f f i c i e n t in v i t a m i n s A a n d ]E, c a r e m u s t be t a k e n t h a t d e f i c i e n c y s y m p L o m s d u e t o t h e abse.nee o f o n e v i t a m i n ~re n o t c o m p l i c a t e d b y s y m t . o m s p r o d u c e d b y a d e f i c i e n c y o f t h e o t h e r .
V i t a m i n s A a n d E are both m e m b e r s of the fat-soluble group of v i t a m i n s a n d , f r o m t h e n u t r i t i o n i s t ' s p o i n t of view, t h e r e are several in tcrcstlng a n d often i m p o r t a n t interrelations b e t w e e n them. These m a y be considered as arising, a t least p a r t l y , from differences in the c h e m i s t r y of the two substances. V i t a m i n A is a h i g h l y u n s a t u r a t e d alcohol and, as such, is sensitive to d e s t r u c t i o n by o x i d a t i o n , espccially in the presence of pcroxidizing lipids, as m a y occur u n d e r physiological conditions. S u b s t a n t i a l a m o u n t s of v i t a m i ~ A, therefore, t h a t m a y be originally preserlt in foodstuffs can be lost a t various stages in the process by which the v i t a m i n is t r a n s f e r r e d from the diet of an a n i m a l to its final d e s t i n a t i o n , t h a t is, the u l t i m a t e site of a c t i o n in t h e tissues of the a n i m a l itself. I n a s m u c h as a large p r o p o r t i o n of the loss of v i t a m i n A during this process is b y o x i d a t i o n , it c a n be p r e v e n t e d or delaycd by the presence of f a t a n t i oxidants. Of these, t h e m o s t i m p o r t a n t n a t u r a l l y - o c c u r r i n g s u b s t a n c e is v i t a m i n E. V i t a m i n E, in fact, is n o t a single s u b s t a n c e b u t a complex of several c o m p o u n d s , t h e tocopherols, which are s t r u c t u r a l l y closely related. These substances are w i d e l y dist r i b u t e d in h a t , ire in v a r y i n g a m o u n t s a n d h a v e s o m e w h a t differing a n t i o x i d a n t properties. However, one of ~hese, ~-tocopherot, is the m o s t widespread, a n d is u s u a l l y the one w i t h which we are concerned in a n i m a l n u t r i t i o n , Since it is the one m o s t r e a d i l y absorbed b y animals. a-Tocopherol is f o u n d in t h e ]ipids of all green pIants, from the unicellular organisms such as E~glena gracilis t o t h e higher flowering species. Chemically, it is a h i g h l y s u b s t i t u t e d phenol, r e n d e r e d far-soluble b y v i r t u e of its i s o p r e n o i d - t y p e side chain. I n c o n t r a s t to vitazrdn A, i t is a powerful reducing s u b s t a n c e a n d loses its p h e n o l i c h y d r o g e n a t o m , by a one-electron o x i d a t i o n reaction, to give a t o e o p h e r y l radical, which is stable enough to a c t as c h a i n - b r e a k e r in radical chain-reactions. The processes b y which u n s a t u r a t e d lipids become oxidized arc o u t l i n e d below: [ . . . . * R " + O2 [ lZH + RO0"
-->ROO" --> ~ O 0 ~ . t
+ R" I
I n t h e presence of small a m o u n t s o£ a n a n t i o x i d a n t such as t o c o p h e r o l (TH), i n h i b i t i o n of ttle process t a k e s place, for t h e radicaI 1-¢00" a b s t r a c t s a h y d r o g e n a t o m m o r e readily f r o m a t o c o p h e r o l molecule t h a n fi'om a n o t h e r molecule of R H . RO0" + TH-* l%00H + T'. 388
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VITAMI:NS
A AND
]~
389
F r o m the Iipids ol plants, ~-tocopherol finds its w a y into the tissues of animals, a n d s u b s t a n t i a l a m o u n t s occur in fish liver oils, which supply us a n d m a n y of our domestic animals with m u c h of our vit~min A. These oils are, therefore, ~lready stabilized to some degree against~ t h e processes of oxidation. Moore (1939) was t h e first worker to report a connection between the v i t a m i n A. reserves of an a n i m a l a n d the v i t a m i n l~ content of its diet a n d showed t~hat r a t s given v i t a m i n E in their food stored up to 10 times a s much v i t a m i n A in their livers as those w i t h o u t v i t a m i n E. D a v i e s a n d Moore (1941) later d e m o n s t r a t e d t h a t a t least p a r t of the effect was due to proteetipn of v i t a m i n A t h a t h a d a l r e a d y been stored before the e x p e r i m e n t wag. Started. S t a r t i n g with rat~ containing a b o u t 20,000 I U of vitamin A in their livers, t h e y found t h a t 7 weeks of diet deficient in b o t h vitamins was sut Iieient to reduce t h e r e s e r v e s a l m o s t t o zero, whereas r a t s ~ v c n v i t a m i n E still contained a b o u t 5,000 I U of v i t a m i n A a f t e r this time. H i c k m a n , K a l e y and H a r r i s (1944) carried out. several refined e x p e r i m e n t s in order to disclose more a b o u t the precise n a t u r e of t h e protective effect. T h e y showed t h a t if v i t a m i n A, in a m o u n t s j u s t sufficient to l~eep t h e m alive a n d growing, was given to rata, smalI amotlnts of tocopherol, a d m i n i s t e r e d a t t h e same time, significantly increased their growth r a t e s a n d survival times. T h e y concluded t h a t t h e role of vit,umln E was mainly due to it,s action in p r e v e n t i n g v i t a m i n A becoming oxidized in the gut, although, t h e y did n o t r~lle o u t the possibility t h a t protection would tal~e place a t s e c o n d a r y sites in the tissue.s. I t was suggested b y Clausen, McCoord, Golf and L a v e n d e r (1946) t h a t the effect of v i t a m i n E was g r e a t e r in m a l e t h a n in female rats. L a t e r studies, however, b y several groups of workers, has t e n d e d to suggest t h a t t h e v i t a m i n A - v i t a m i n E relationship m a y be m o r e c o m p l e x t h a n at first a p p e a r e d . Thus, if v i t a m i n A p a l m : t a t s and ~-tocophcroI are adminisLered, in Tween-based a q u e o u s ~iispersions, to weanling, v i t a m i n A-deficient ruts, no s p a r i n g effect on v i t a m i n A could be observed, a l t h o u g h this was clearly observed w h e n b o t h v i t a m i n s were given in oily solutions (Esh a n d B h a t t a c h a r y ~ , 1953). P o p p e r a n d Volk (:[944) m a d e a carei'ul exambaation of r a t L~testine b y fluorescent microscopy a n d r e p o r t e d t h a t t h e y could, in fact, find no izmrease in the a b s o r p t i o n of v i t a m i n A a f t e r tocophcrol a d m i n i s t r a t i o n . Lemley, :Brown, :Birch. and E m m e t t (1947) found t h a t soltibilJzed toeopherol given pa.renterally to ratz h a d the same effect in s u p p l e m e n t i n g v i t a m i n A a c t i v i t y as when g:-ven orally. This suggested to t h e m t h a t t h e action of tocophcrol could n o t be limited to the gut. H o w c v c r , these result'~ m u s t be viewed in t h e light of r e c e n t evidence t h a t ~-tocopherol is itself excreted a n d absorbed ~da the intestine in e eylical mam~er (Sternberg a n d P a s c o z - D a w s o n , 1959). h[ore reccr~ly, Dicks et el. (1959) h a v e described some e x p e r i m e n t s in d o u b l y deficient male calves; t h e y found a s ~ e r g i s t i c relationship between vitamins A a n d E only when either v e r y low or v e r y high levels of v i t a m i n A were given, i n t e r m e d i a t e levels showing no effect: F u r t h e r , ][)range, S S n d e r g a a r d a n d D a m (1952) have s h o w n tha~ a third, p a r a m e t e r in the relationship is the n a t u r e of the f~t in t h e to~al diet. T h u s , i n rats, the sparing .effec~ of v i t a m i n E eouid only be observed if there was ConsiderabIe u n s a t u r a t e d fat present: w h e n a s a t u r a t e d f a t was used, no s y n e r g i s m was o b s e r v e d . I t seems, therefore, t h a t m a n y factors can influence the v i t a m i n A - v i t a m i n :E relationship a n d these h a v e n o t so f a r been precisely defm.ed: The o b s e r v a t i o n of Cox, Deuel e n d :Ershoff (1957) is a case in point. T h e y f e d r a t s a diet containing 3 × 10 ~ I U v i t a m i n A / k g t6 p r o d u c e hypervit~minosis A a n d w e r e a b l e to de2~ress t h e s y m p t o m s b y giving s u p p l e m e n t a r y tocopherol. Several reports ha.re a p p e a r e d t h a t pose t h e questio n as t o : w h e t h e r there is in f a c t ~. synergism in t h e action of the two v i t a m i n s a t a more f u n d a m e n t a l biochemical
390
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level. It, is k n o w n , for example, t h a t v i t a m i n E deficiency a n d v i t a m i n A deficiency m a y induce c r e a t i n u r i a in ra~s. B u t t u r i n i (1943) claimed t h a t although 1-10 m g of a-tocopherol m u s t be given e v e r y other d a y for 2 wee]~s t o n o r m a l r a t s to reduce the excretion of creathae to zero, as little as 0.1 m g is effective if 12,000 I U of v i t a m i n A is given a t the s a m e time. Similar results were f o u n d with v i t a m i n A-deficient r a t s , a l t h o u g h t h e c r e a t i n u r i a of v i t a m i n E deficiency was m o r e difficult to p r e v e n t . A n o t h e r possibility of s y n e r g i s m a t t h e cellular level spr!ngs f r o m the f a c t t h a t both v i t a m i n A a n d a-tocopherot h a v e high surface tensior~ a c t i v i t y a n d c a n f o r m m o n o l a y c r s on hydrophilic surfaces in vitro. B o t h v i t a m i n s can p e n e t r a t e and stabilize m e m b r a n e s of various kinds, especially the e r y t h r o c y t e m e m b r a n e , Ttnm, b o t h vitamins can, u n d e r differing conditions, protect r a b b i t e r y t h r o c y t e s f r o m haemolysis, a n d a synergistic action between t h e two s u b s t a n c e s has been d e m o n s t r a t e d in this s y s t e m (Dingle, ]964). Effects a t o t h e r m e m b r a n e s , especially vasctflar ones, m a y occur; Weitzel, Sch0n a n d Gey (1955), for example, h a v e shown t h a t a combination of v i t a m i n A a n d v i t a m i n E w a s exceptionally active in reduc~ug plaque f o r m a t i o n a n d t h e f a t a n d cholesterol c o n t e n t s of the a o r t a of hens on a n atherosclerotogenic diet. As m i g h t be expected, a f t e r the n u t r i t i o n a l relationship between v i t a m i n A a n d E had been d e m o n s t r a t e d , it was soon shown t h a t a similar connection could be obesrved b e t w e e n vif~amin ]~J a n d carotene utilization, I-Iowever, certain differences, still of a r a t h e r puzzling kind, h a v e emerged. Z'Ioore (1940) first r e p o r t e d t h a t v i t a m i n E h a d less effect on t h e storage of v i t a m i n A derived f r o m carotene ~han on the s t o r a g e of t h e p r e f o r m e d v i t a m i n . Harris, K a l e y a n d t ~ c k m a n (1944) confirmed t h a t small daily doses of 0"5 m g of mLxed tocopherols increased t h e g-rowth response of r a t s t o m a r g i n a l doses of fl-carotene, b u t larger doses of tocopherols reversed t h e eifec~, which could also be ir~fluenced b y a complex i n t e r p l a y of o t h e r d i e t a r y factors. I t seems, therefore, that,, in the case of carotenc, t h e a b s o r p t i o n process itself m a y be affected b y t h e presence of tocopherol in t h e gut. This seern~ to be s u p p o r t e d b y the work of Bieri (1955), who foo_nii t h a t the utilization of single, small (13/zg) doses of fl-carotene b y d o u b l y deficient r a t s was unaffected b y tocopherol, if t h e f o r m e r was given in solubilized form. I-Iowevcr, o t h e r workers h a v e s u g g e s t e d t h a t tocopheroI m i g h t affect a step in the actual conversion of fl-carotene to vitamin A. Thus, Johnson and B a u m a n n (1948) f o u n d t h a t if 40 t~g of carotene a d a y w a s given to v i t a m i n A-deficient rats, increased a m o u n t s of tocopherol could lead to m a r k e d l y diminished storage of v i t a m i n A itself in t h e liver a n d the effect persisted, a l t h o u g h it was less if the tocopherol was g i v e n i n ~ a p e r i t o n e a l l y . I t seems almost c e r t a i n t h a t there are in f a c t two opposing effects of v i t a m i n E on c a r o t e n e metabolism. F r o m t h e p r a c t i c a l point of view, therefore, it is possible to s u m m a r i z e as foUows. VChile the full natltre of the t o e o p h c r o l - - v i t a m i n A - - c a r o t e n e interreh-.ti~nships is p r o b a b l y n o t y e t understood, it is clear t h a t e x p e r i m e n t s w i t h v i t a m i n A, especially those involving m a r g i n a l deficiency o r sufficiency, c a u be m a r k e d l y influenced b y t h e v i t a m i n E s t a t u s of t h e animals concerned. I t is even possible t h a t t h e signs of one deficiency m a y be confused w i t h t h e signs of t h e o t h e r if botda v i t a m i n supplies are marginal. M a n y reports in the l i t e r a t u r e indicate t h a t v i t a m i n A deficiency signs, p a r t i c u l a r l y x e r o p h t h a l m i a , are m a r k e d l y accelerated in animals low in v i t a m i n ~E reserves, especially on low protein diets (McLaren, 1959). Conversely, several descriptions of ocular lesions ascribed to ~dtamin E deficiency h a v e been reported. Th.u s, Corrado (1953) f o u n d interstitial keratitis of t h e u r e a a n d alterations in t h e lens in x~itamin E-deficient r a b b i t s , a n d effects of v i t a m i n E deficiency on t h e e m b r y o n i c d e v e l o p m e n t of t h e t u r k e y eye h a v e been described (I~erguson, Y a u g h t , Cross,/-tander
[ N T E R R E L A T I O N S BETWI~EN VITAMINS A AND ]:]
391
and 0ouch, 1955; Ferguson, Rigdo a and Couch, 1956a, b). It is necessary for the vitamin A stratus of the ahimals to be ]~nown before certain of these effects can be positively ascribed to the vitamin1 E-deficient state. Knowledge about the cellular levels of both vitamins is often, lmfortunatcly, not to be obtained from the literature. Practical experience has shown that urdess care is taken with the composition and storage o f laboratory diets, or un]ess positive steps are taken to stabilize vitamin A or to administer this vitamin separately from the diet, a deficiency of vitamin E may be induced in the ration and this in turn o,~n precipitate vitamin A deficiency symptoms. F, E F E R E N C E S ]3ierl, J. G. (1955). Prec, Soc. exl±. Biol., N.Y. 88, 482, .Bufturini, H. (1943). G. Cli~,. reed. 24, (16), 30. Ctausen, S. W., MeCoord, A. B., Goff, B. L. and Lavender. C. F. (1946). Abstr. Amer. Chem. Soc. 110th Meeting. 2913. Chicago, September 194G. Corrrtdo, M. (1953). Ann. Ottal. 79, 211. Cox, R. P., Dcuel, A. J. and Ershoff, B. A. (1957). Exp. reed. Surg. 35, 328. Davies, A. W. and Moore, T. (1941). Nature, Lend. 147, 794. Dicks, ]~1.W., l~0ussoau, J. E., Eaton, H. D., TMchamn, R., Grlfo, A. P. ~nd Kommerer, I-I, A. (1959). J. Da~rySci. 42, 501. Dingle, J. T. (1964). Biochem. J. 90, 36. Esh, G. C. and Bhattacharya, S. (1953). Indian J. Pt~ysiol. 7, 153. Ferguson, T. M., Rigdon, R. El. and Couch, J. P~. (1956a). Arch.. Ophthal., Chicago, 55, 346. Fcrguson, T. M., Rigdon, R. H. and Couch, J. R. (1956b)..Fed. Prec. 15, 550. Ferguson, T. M., Vaught, I-I..P., Cross, M. S., Hander, O. if. and Couch, J. R. (1955). Poultriji~ci 34, 1193. Harris, P. L., KMev, bi,-W, and Hickm~n, K. C. D. (1944). J. bloI Chem. 152, 313. Hickman, K: C. D., Kaley, M. W. and Harris, P. L. (1944). J. biol. GheVi.:~S2, 303. Johnson, ~. M. and ]3aumann. C. A. (1948). J. blol. Chem, 175, 881. Lemley, J. M., Brow~, R. A., Bird, O. D. and Emmctt, A. D. (1947). J. NuSr. 34, 205. McLarcn, D. 8. ([959). Brit. J. Ophthal. 43, 234. Moore, T, (1939). Chem. & Ind. (Roy.) 58, 65. ~[ooro, T. (19~t0). Biochem. J. 34, 1321. Popper, H. and Volk, B. W. (1944), Arch. Path., Chicago. 38, 71. Pran ge, t., SSndergaard, E. and ]')am, H. (1952). zI bstr. Communs. 9rid I~¢. Coheir. 13iochem.(Paris) 1952, 330. 8ternberg, J. and Pascoe-Dawson, E. (1959). Canad. meg. Ass. J. 80, 266. Weitzel, G., Sch6n, R. and Gcy, ~.~.(1955). Kl¢n. Wschr. 33, 772.
Interrelations b e t w e e n V i t a m i n s A and E 5. GF, E E N
Walton Oaks ExTerimental ,Station, Vitamins Ltd., Tadworth Su~'zey, England Thel~ arc important interrelations between vitamins A and E which manifest t h e m s e l v e s i n v a r i o u s w a y s . I n t h e die~, t h e p r v s c n c c o f ~:itamin ]~ is of mrrjor i m p o r t a n t e i a p r o t ~ c t l n g small q u a n t i t i e s o f v i t a m i n A f r o m o x i d a t i o n . U n d e r p h y s i o l o g i c a l c o n d i t i o n s , v i t a m i n I~ h a s a s p a r i n g e f f e c t o n vitatLain A a n d o n t h e earot, e n o i d s . i n e x p e r i m e n t a l arfimals t h a t iLt~ve b e e n gig-on d i e t s o n l y m a r g i n a l l y s u f f i c i e n t in v i t a m i n s A a n d ]E, c a r e m u s t be t a k e n t h a t d e f i c i e n c y s y m p L o m s d u e t o t h e abse.nee o f o n e v i t a m i n ~re n o t c o m p l i c a t e d b y s y m t . o m s p r o d u c e d b y a d e f i c i e n c y o f t h e o t h e r .
V i t a m i n s A a n d E are both m e m b e r s of the fat-soluble group of v i t a m i n s a n d , f r o m t h e n u t r i t i o n i s t ' s p o i n t of view, t h e r e are several in tcrcstlng a n d often i m p o r t a n t interrelations b e t w e e n them. These m a y be considered as arising, a t least p a r t l y , from differences in the c h e m i s t r y of the two substances. V i t a m i n A is a h i g h l y u n s a t u r a t e d alcohol and, as such, is sensitive to d e s t r u c t i o n by o x i d a t i o n , espccially in the presence of pcroxidizing lipids, as m a y occur u n d e r physiological conditions. S u b s t a n t i a l a m o u n t s of v i t a m i ~ A, therefore, t h a t m a y be originally preserlt in foodstuffs can be lost a t various stages in the process by which the v i t a m i n is t r a n s f e r r e d from the diet of an a n i m a l to its final d e s t i n a t i o n , t h a t is, the u l t i m a t e site of a c t i o n in t h e tissues of the a n i m a l itself. I n a s m u c h as a large p r o p o r t i o n of the loss of v i t a m i n A during this process is b y o x i d a t i o n , it c a n be p r e v e n t e d or delaycd by the presence of f a t a n t i oxidants. Of these, t h e m o s t i m p o r t a n t n a t u r a l l y - o c c u r r i n g s u b s t a n c e is v i t a m i n E. V i t a m i n E, in fact, is n o t a single s u b s t a n c e b u t a complex of several c o m p o u n d s , t h e tocopherols, which are s t r u c t u r a l l y closely related. These substances are w i d e l y dist r i b u t e d in h a t , ire in v a r y i n g a m o u n t s a n d h a v e s o m e w h a t differing a n t i o x i d a n t properties. However, one of ~hese, ~-tocopherot, is the m o s t widespread, a n d is u s u a l l y the one w i t h which we are concerned in a n i m a l n u t r i t i o n , Since it is the one m o s t r e a d i l y absorbed b y animals. a-Tocopherol is f o u n d in t h e ]ipids of all green pIants, from the unicellular organisms such as E~glena gracilis t o t h e higher flowering species. Chemically, it is a h i g h l y s u b s t i t u t e d phenol, r e n d e r e d far-soluble b y v i r t u e of its i s o p r e n o i d - t y p e side chain. I n c o n t r a s t to vitazrdn A, i t is a powerful reducing s u b s t a n c e a n d loses its p h e n o l i c h y d r o g e n a t o m , by a one-electron o x i d a t i o n reaction, to give a t o e o p h e r y l radical, which is stable enough to a c t as c h a i n - b r e a k e r in radical chain-reactions. The processes b y which u n s a t u r a t e d lipids become oxidized arc o u t l i n e d below: [ . . . . * R " + O2 [ lZH + RO0"
-->ROO" --> ~ O 0 ~ . t
+ R" I
I n t h e presence of small a m o u n t s o£ a n a n t i o x i d a n t such as t o c o p h e r o l (TH), i n h i b i t i o n of ttle process t a k e s place, for t h e radicaI 1-¢00" a b s t r a c t s a h y d r o g e n a t o m m o r e readily f r o m a t o c o p h e r o l molecule t h a n fi'om a n o t h e r molecule of R H . RO0" + TH-* l%00H + T'. 388
IN'.i'I,:I~IC,],ILATIO:NS :BI~TWEEbI
VITAMI:NS
A AND
]~
389
F r o m the Iipids ol plants, ~-tocopherol finds its w a y into the tissues of animals, a n d s u b s t a n t i a l a m o u n t s occur in fish liver oils, which supply us a n d m a n y of our domestic animals with m u c h of our vit~min A. These oils are, therefore, ~lready stabilized to some degree against~ t h e processes of oxidation. Moore (1939) was t h e first worker to report a connection between the v i t a m i n A. reserves of an a n i m a l a n d the v i t a m i n l~ content of its diet a n d showed t~hat r a t s given v i t a m i n E in their food stored up to 10 times a s much v i t a m i n A in their livers as those w i t h o u t v i t a m i n E. D a v i e s a n d Moore (1941) later d e m o n s t r a t e d t h a t a t least p a r t of the effect was due to proteetipn of v i t a m i n A t h a t h a d a l r e a d y been stored before the e x p e r i m e n t wag. Started. S t a r t i n g with rat~ containing a b o u t 20,000 I U of vitamin A in their livers, t h e y found t h a t 7 weeks of diet deficient in b o t h vitamins was sut Iieient to reduce t h e r e s e r v e s a l m o s t t o zero, whereas r a t s ~ v c n v i t a m i n E still contained a b o u t 5,000 I U of v i t a m i n A a f t e r this time. H i c k m a n , K a l e y and H a r r i s (1944) carried out. several refined e x p e r i m e n t s in order to disclose more a b o u t the precise n a t u r e of t h e protective effect. T h e y showed t h a t if v i t a m i n A, in a m o u n t s j u s t sufficient to l~eep t h e m alive a n d growing, was given to rata, smalI amotlnts of tocopherol, a d m i n i s t e r e d a t t h e same time, significantly increased their growth r a t e s a n d survival times. T h e y concluded t h a t t h e role of vit,umln E was mainly due to it,s action in p r e v e n t i n g v i t a m i n A becoming oxidized in the gut, although, t h e y did n o t r~lle o u t the possibility t h a t protection would tal~e place a t s e c o n d a r y sites in the tissue.s. I t was suggested b y Clausen, McCoord, Golf and L a v e n d e r (1946) t h a t the effect of v i t a m i n E was g r e a t e r in m a l e t h a n in female rats. L a t e r studies, however, b y several groups of workers, has t e n d e d to suggest t h a t t h e v i t a m i n A - v i t a m i n E relationship m a y be m o r e c o m p l e x t h a n at first a p p e a r e d . Thus, if v i t a m i n A p a l m : t a t s and ~-tocophcroI are adminisLered, in Tween-based a q u e o u s ~iispersions, to weanling, v i t a m i n A-deficient ruts, no s p a r i n g effect on v i t a m i n A could be observed, a l t h o u g h this was clearly observed w h e n b o t h v i t a m i n s were given in oily solutions (Esh a n d B h a t t a c h a r y ~ , 1953). P o p p e r a n d Volk (:[944) m a d e a carei'ul exambaation of r a t L~testine b y fluorescent microscopy a n d r e p o r t e d t h a t t h e y could, in fact, find no izmrease in the a b s o r p t i o n of v i t a m i n A a f t e r tocophcrol a d m i n i s t r a t i o n . Lemley, :Brown, :Birch. and E m m e t t (1947) found t h a t soltibilJzed toeopherol given pa.renterally to ratz h a d the same effect in s u p p l e m e n t i n g v i t a m i n A a c t i v i t y as when g:-ven orally. This suggested to t h e m t h a t t h e action of tocophcrol could n o t be limited to the gut. H o w c v c r , these result'~ m u s t be viewed in t h e light of r e c e n t evidence t h a t ~-tocopherol is itself excreted a n d absorbed ~da the intestine in e eylical mam~er (Sternberg a n d P a s c o z - D a w s o n , 1959). h[ore reccr~ly, Dicks et el. (1959) h a v e described some e x p e r i m e n t s in d o u b l y deficient male calves; t h e y found a s ~ e r g i s t i c relationship between vitamins A a n d E only when either v e r y low or v e r y high levels of v i t a m i n A were given, i n t e r m e d i a t e levels showing no effect: F u r t h e r , ][)range, S S n d e r g a a r d a n d D a m (1952) have s h o w n tha~ a third, p a r a m e t e r in the relationship is the n a t u r e of the f~t in t h e to~al diet. T h u s , i n rats, the sparing .effec~ of v i t a m i n E eouid only be observed if there was ConsiderabIe u n s a t u r a t e d fat present: w h e n a s a t u r a t e d f a t was used, no s y n e r g i s m was o b s e r v e d . I t seems, therefore, t h a t m a n y factors can influence the v i t a m i n A - v i t a m i n :E relationship a n d these h a v e n o t so f a r been precisely defm.ed: The o b s e r v a t i o n of Cox, Deuel e n d :Ershoff (1957) is a case in point. T h e y f e d r a t s a diet containing 3 × 10 ~ I U v i t a m i n A / k g t6 p r o d u c e hypervit~minosis A a n d w e r e a b l e to de2~ress t h e s y m p t o m s b y giving s u p p l e m e n t a r y tocopherol. Several reports ha.re a p p e a r e d t h a t pose t h e questio n as t o : w h e t h e r there is in f a c t ~. synergism in t h e action of the two v i t a m i n s a t a more f u n d a m e n t a l biochemical
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level. It, is k n o w n , for example, t h a t v i t a m i n E deficiency a n d v i t a m i n A deficiency m a y induce c r e a t i n u r i a in ra~s. B u t t u r i n i (1943) claimed t h a t although 1-10 m g of a-tocopherol m u s t be given e v e r y other d a y for 2 wee]~s t o n o r m a l r a t s to reduce the excretion of creathae to zero, as little as 0.1 m g is effective if 12,000 I U of v i t a m i n A is given a t the s a m e time. Similar results were f o u n d with v i t a m i n A-deficient r a t s , a l t h o u g h t h e c r e a t i n u r i a of v i t a m i n E deficiency was m o r e difficult to p r e v e n t . A n o t h e r possibility of s y n e r g i s m a t t h e cellular level spr!ngs f r o m the f a c t t h a t both v i t a m i n A a n d a-tocopherot h a v e high surface tensior~ a c t i v i t y a n d c a n f o r m m o n o l a y c r s on hydrophilic surfaces in vitro. B o t h v i t a m i n s can p e n e t r a t e and stabilize m e m b r a n e s of various kinds, especially the e r y t h r o c y t e m e m b r a n e , Ttnm, b o t h vitamins can, u n d e r differing conditions, protect r a b b i t e r y t h r o c y t e s f r o m haemolysis, a n d a synergistic action between t h e two s u b s t a n c e s has been d e m o n s t r a t e d in this s y s t e m (Dingle, ]964). Effects a t o t h e r m e m b r a n e s , especially vasctflar ones, m a y occur; Weitzel, Sch0n a n d Gey (1955), for example, h a v e shown t h a t a combination of v i t a m i n A a n d v i t a m i n E w a s exceptionally active in reduc~ug plaque f o r m a t i o n a n d t h e f a t a n d cholesterol c o n t e n t s of the a o r t a of hens on a n atherosclerotogenic diet. As m i g h t be expected, a f t e r the n u t r i t i o n a l relationship between v i t a m i n A a n d E had been d e m o n s t r a t e d , it was soon shown t h a t a similar connection could be obesrved b e t w e e n vif~amin ]~J a n d carotene utilization, I-Iowever, certain differences, still of a r a t h e r puzzling kind, h a v e emerged. Z'Ioore (1940) first r e p o r t e d t h a t v i t a m i n E h a d less effect on t h e storage of v i t a m i n A derived f r o m carotene ~han on the s t o r a g e of t h e p r e f o r m e d v i t a m i n . Harris, K a l e y a n d t ~ c k m a n (1944) confirmed t h a t small daily doses of 0"5 m g of mLxed tocopherols increased t h e g-rowth response of r a t s t o m a r g i n a l doses of fl-carotene, b u t larger doses of tocopherols reversed t h e eifec~, which could also be ir~fluenced b y a complex i n t e r p l a y of o t h e r d i e t a r y factors. I t seems, therefore, that,, in the case of carotenc, t h e a b s o r p t i o n process itself m a y be affected b y t h e presence of tocopherol in t h e gut. This seern~ to be s u p p o r t e d b y the work of Bieri (1955), who foo_nii t h a t the utilization of single, small (13/zg) doses of fl-carotene b y d o u b l y deficient r a t s was unaffected b y tocopherol, if t h e f o r m e r was given in solubilized form. I-Iowevcr, o t h e r workers h a v e s u g g e s t e d t h a t tocopheroI m i g h t affect a step in the actual conversion of fl-carotene to vitamin A. Thus, Johnson and B a u m a n n (1948) f o u n d t h a t if 40 t~g of carotene a d a y w a s given to v i t a m i n A-deficient rats, increased a m o u n t s of tocopherol could lead to m a r k e d l y diminished storage of v i t a m i n A itself in t h e liver a n d the effect persisted, a l t h o u g h it was less if the tocopherol was g i v e n i n ~ a p e r i t o n e a l l y . I t seems almost c e r t a i n t h a t there are in f a c t two opposing effects of v i t a m i n E on c a r o t e n e metabolism. F r o m t h e p r a c t i c a l point of view, therefore, it is possible to s u m m a r i z e as foUows. VChile the full natltre of the t o e o p h c r o l - - v i t a m i n A - - c a r o t e n e interreh-.ti~nships is p r o b a b l y n o t y e t understood, it is clear t h a t e x p e r i m e n t s w i t h v i t a m i n A, especially those involving m a r g i n a l deficiency o r sufficiency, c a u be m a r k e d l y influenced b y t h e v i t a m i n E s t a t u s of t h e animals concerned. I t is even possible t h a t t h e signs of one deficiency m a y be confused w i t h t h e signs of t h e o t h e r if botda v i t a m i n supplies are marginal. M a n y reports in the l i t e r a t u r e indicate t h a t v i t a m i n A deficiency signs, p a r t i c u l a r l y x e r o p h t h a l m i a , are m a r k e d l y accelerated in animals low in v i t a m i n ~E reserves, especially on low protein diets (McLaren, 1959). Conversely, several descriptions of ocular lesions ascribed to ~dtamin E deficiency h a v e been reported. Th.u s, Corrado (1953) f o u n d interstitial keratitis of t h e u r e a a n d alterations in t h e lens in x~itamin E-deficient r a b b i t s , a n d effects of v i t a m i n E deficiency on t h e e m b r y o n i c d e v e l o p m e n t of t h e t u r k e y eye h a v e been described (I~erguson, Y a u g h t , Cross,/-tander
[ N T E R R E L A T I O N S BETWI~EN VITAMINS A AND ]:]
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and 0ouch, 1955; Ferguson, Rigdo a and Couch, 1956a, b). It is necessary for the vitamin A stratus of the ahimals to be ]~nown before certain of these effects can be positively ascribed to the vitamin1 E-deficient state. Knowledge about the cellular levels of both vitamins is often, lmfortunatcly, not to be obtained from the literature. Practical experience has shown that urdess care is taken with the composition and storage o f laboratory diets, or un]ess positive steps are taken to stabilize vitamin A or to administer this vitamin separately from the diet, a deficiency of vitamin E may be induced in the ration and this in turn o,~n precipitate vitamin A deficiency symptoms. F, E F E R E N C E S ]3ierl, J. G. (1955). Prec, Soc. exl±. Biol., N.Y. 88, 482, .Bufturini, H. (1943). G. Cli~,. reed. 24, (16), 30. Ctausen, S. W., MeCoord, A. B., Goff, B. L. and Lavender. C. F. (1946). Abstr. Amer. Chem. Soc. 110th Meeting. 2913. Chicago, September 194G. Corrrtdo, M. (1953). Ann. Ottal. 79, 211. Cox, R. P., Dcuel, A. J. and Ershoff, B. A. (1957). Exp. reed. Surg. 35, 328. Davies, A. W. and Moore, T. (1941). Nature, Lend. 147, 794. Dicks, ]~1.W., l~0ussoau, J. E., Eaton, H. D., TMchamn, R., Grlfo, A. P. ~nd Kommerer, I-I, A. (1959). J. Da~rySci. 42, 501. Dingle, J. T. (1964). Biochem. J. 90, 36. Esh, G. C. and Bhattacharya, S. (1953). Indian J. Pt~ysiol. 7, 153. Ferguson, T. M., Rigdon, R. El. and Couch, J. P~. (1956a). Arch.. Ophthal., Chicago, 55, 346. Fcrguson, T. M., Rigdon, R. H. and Couch, J. R. (1956b)..Fed. Prec. 15, 550. Ferguson, T. M., Vaught, I-I..P., Cross, M. S., Hander, O. if. and Couch, J. R. (1955). Poultriji~ci 34, 1193. Harris, P. L., KMev, bi,-W, and Hickm~n, K. C. D. (1944). J. bloI Chem. 152, 313. Hickman, K: C. D., Kaley, M. W. and Harris, P. L. (1944). J. biol. GheVi.:~S2, 303. Johnson, ~. M. and ]3aumann. C. A. (1948). J. blol. Chem, 175, 881. Lemley, J. M., Brow~, R. A., Bird, O. D. and Emmctt, A. D. (1947). J. NuSr. 34, 205. McLarcn, D. 8. ([959). Brit. J. Ophthal. 43, 234. Moore, T, (1939). Chem. & Ind. (Roy.) 58, 65. ~[ooro, T. (19~t0). Biochem. J. 34, 1321. Popper, H. and Volk, B. W. (1944), Arch. Path., Chicago. 38, 71. Pran ge, t., SSndergaard, E. and ]')am, H. (1952). zI bstr. Communs. 9rid I~¢. Coheir. 13iochem.(Paris) 1952, 330. 8ternberg, J. and Pascoe-Dawson, E. (1959). Canad. meg. Ass. J. 80, 266. Weitzel, G., Sch6n, R. and Gcy, ~.~.(1955). Kl¢n. Wschr. 33, 772.