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Vitamin E status of infant formulas. O'Leary, Lillian Patricia 1971

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VITAMIN E STATUS OF INFANT FORMULAS by LILLIAN PATRICIA O'LEARY B.A., University of Western Ontario, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE. OF MASTER OF SCIENCE i n the Div i s i o n of HUMAN NUTRITION SCHOOL OF HOME ECONOMICS We accept this thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLOMBIA A p r i l , 1971 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permission f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Depa rtment The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada ABSTRACT I n e a r l y Infancy, l i m i t e d i n g e s t i o n of v i t a m i n E, e s p e c i a l l y the a _ t o c o p h e r o l form, and/or a high, i n t a k e of polyunsaturated f a t t y a c i d s are r e f l e c t e d as a low serum tocopherol l e v e l . This v i t a m i n E d e f i c i e n c y s t a t e i s probably r e s p o n s i b l e f o r the development of an anemia c h a r a c t e r i z e d by abnormal e r y t h r o c y t e hemolysis. Edema and s k i n changes may a l s o be present. In order to a s c e r t a i n the tocopherol s t a t u s of i n f a n t formulas a v a i l a b l e i n Canada, t h i r t y - s i x r e p r e s e n t a t i v e formulas were ch e m i c a l l y analyzed f o r v i t a m i n E and polyunsaturated f a t t y a c i d contents. For the tocopherol a n a l y s i s , a combination of the Emmerie-Engel procedure and two-dimensional t h i n - l a y e r chromatography was used. An a l k a l i n e isom-e r i z a t i o n technique was chosen f o r the polyunsaturated f a t t y a c i d a n a l y s i s . The v i t a m i n E s t a t u s of the v a r i o u s formulas was assessed i n r e l a t i o n to three c r i t e r i a of t ocopherol adequacy - the c t - t o c o p h e r o l - t o -PUFA r a t i o , the a-tocopherol content, and the l e v e l of I n t e r n a t i o n a l U n i t s of v i t a m i n E. With the exception of one formula which was supplemented w i t h v i t a m i n E, the twenty-one formulas based on f r e s h or m o d i f i e d cow's m i l k o f t e n contained i n s u f f i c i e n t t o c o pherol to meet the v a r i o u s c r i t e r i a of adequacy. A l a r g e per cent of the v i t a m i n E content i n these formulas was present as a-tocopherol. The polyunsaturated f a t t y a c i d content was r e l a t i v e l y low. In g e n e r a l , the f i f t e e n p r o p r i e t a r y formulas had much higher l e v e l s of v i t a m i n E and polyunsaturated f a t t y a c i d s than the formulas based on f r e s h or m o d i f i e d cow's m i l k . For many of the p r o p r i e t a r y products, a-tocopherol c o n s t i t u t e d only a s m a l l p a r t of the v i t a m i n E content. Four i of the p r o p r i e t a r y formulas ( i n c l u d i n g three which had been f o r t i f i e d w i t h v i t a m i n E) contained s u f f i c i e n t amounts of v i t a m i n E to s a t i s f y the three standards of adequacy. On the b a s i s of the chemical a n a l y s i s r e s u l t s , i t i s recommended tha t a l l i n f a n t formulas be supplemented w i t h s u f f i c i e n t amounts of v i t a m i n E. to meet the v a r i o u s c r i t e r i a of adequacy. In t h i s way, the r i s k of v i t a m i n E d e f i c i e n c y i n e a r l y childhood should be minimal. The present study i s a l s o concerned w i t h p a s t , p r e s e n t , and f u t u r e trends i n i n f a n t feeding p r a c t i c e s . In the 1960's, the ready-to-feed h o s p i t a l feeding systems, which are u s u a l l y based on a p r o p r i e t a r y preparation,were i n t r o d u c e d i n Canada. A survey conducted among Canadian h o s p i t a l s i n d i c a t e d t h a t the use of such systems has become widespread. At the same time, the p o p u l a r i t y of the h o s p i t a l - p r e p a r e d formula system has d e c l i n e d . Consequently, the use of m o d i f i e d cow's m i l k formulas has d e c l i n e d i n Canadian h o s p i t a l s . However, such formulas are f r e q u e n t l y used f o r home-feeding of i n f a n t s . No d e f i n i t e trends were noted i n the frequency o f - b r e a s t - f e e d i n g . . i i ACKNOWLEDGEMENT I s i n c e r e l y thank Dr. I.D. Desai whose knowledge has been i n v a l u a b l e i n the completion of the f o l l o w i n g work. Thanks are extended to a l l members of the D i v i s i o n of Human N u t r i t i o n , e s p e c i a l l y Dr. C. Raychaudri. I a l s o would l i k e to thank the Vancouver General H o s p i t a l and the Ch i l d r e n ' s H o s p i t a l f o r k i n d l y s u p p lying some of the formula samples. F i n a l l y I would l i k e to thank David Agnew f o r h i s a s s i s t a n c e w i t h the drawings and h i s encouragement. i i i TABLE OF CONTENTS Page ABSTRACT i ACKNOWLEDGEMENT ' i i i LIST OF TABLES v LIST OF FIGURES v i I. INTRODUCTION 1 I I . REVIEW OF LITERATURE 1. History of the Tocopherols 3 2. B i o l o g i c a l A c t i v i t i e s of the Tocopherols 5 3. Vitamin E Deficiency - Occurrence 6 4. Hemolytic Anemia, Vitamin E Deficiency and Infant Feeding Practices 9 5. Tocopherols and the Prevention of Erythrocyte Hemolysis... 15 I I I . -MATERIALS AND METHODS 1. Formulas Used for Analysis 17 2. Fat Extraction and Saponification of the Extracted Fat.... 17 3. Total Tocopherol Determination 21 4. Individual Tocopherol Determination 23 5. Polyunsaturated Fatty Acid Determination 24 IV. RESULTS AND DISCUSSION 1. Fresh Cow's Milk Used for Infant Formulas 27 2. Cow's Milk Formulas 31 3. Proprietary Formulas 37 4. Hospital Survey - Infant Feeding Practices i n Canada 44 V. SUMMARY AND RECOMMENDATIONS 51 LITERATURE CITED 54 i v LIST OF TABLES Table No. Page 1 Structure and Nomenclature of the Tocopherols 4 2 Formulas Used for Analysis 18 - 3 Vitamin E and Polyunsaturated Fatty Acid Contents of Fresh Cow's Milk 28 4 Vitamin E Content i n Amounts of Fresh Cow's Milk Normally Ingested by Infants at One, Three and Six Months of Age 30 5 Vitamin E and Polyunsaturated Fatty Acid Content of Cow's Milk Formulas 32 6 Vitamin E Content i n Amounts of Cow's Milk Formulas Normally Ingested by Infants at One, Three and Six Months of Age 35 7 Vitamin E and Polyunsaturated Fatty Acid Contents of Proprietary Formulas 38 8 Vitamin E Content i n Amounts of Proprietary Formulas Normally Ingested At One, Three and Six Months of Age 41 9 Vitamin E Status of Infant Formulas 45 10 Hos p i t a l Response to Survey Questionnaire 46 11 Formulas Currently Used i n Canadian Hospitals 48 12 Frequency of Breast-Feeding 49 v LIST OF FIGURES Figure No. P a 8 e l a Standard Curve f o r d l a-Tocopherol (520 my).,. 22a lb Standard Curve f o r 8-Carotene (450 my) 22b l c Standard Curve f o r 6-Carotene (520 my) 22c 2 Two-Dimensional Thin Layer Chromatogram of Tocopherol.. 25 v i CHAPTER I INTRODUCTION The importance of vitamin E in infant nutrition has become evident only in recent years. Vitamin E deficiency may be defined as serum tocopherol levels of less than 0.5 mg per 100 ml. On the basis of this c r i t e r i a , new-born infants exhibit vitamin E deficiency (1-3). It now seems very probable that, in neonates, a syndrome characterized by hemolytic anemia, edema and skin changes (4,5), is related to this vitamin E-deficient state. Premature and low birth weight infants are most l i k e l y to manifest vitamin E deficiency. The tocopherol level, as well as the ratio of vitamin E to poly-unsaturated fatty acids (PUFA) of human milk are adequate to maintain normal levels of serum tocopherol in neonates. In consequence of this,.the vitamin E deficiency syndrome is not l i k e l y to occur i n infants who are nourished on human milk (6,7). However, breast-feeding i s not always preferred. A wide variety of simulated products, fresh cow's milk formulas or modified cow's milk formulas may serve as substitutes. These formulas do not always possess adequate vitamin E and/or an a-tocopherol-to-PUFA ratio sufficient to eliminate the risk of vitamin E deficiency in early infancy. This has been shown by a number of investigators (4,5,7,8). To date, the tocopherol and PUFA contents of infant formulas available in Canada have not been reported. Consequently, chemical analysis of commonly used formulas was undertaken to determine these parameters. On the basis of these results, the vitamin E status of the formulas, and any need for tocopherol supplementation, could be determined. Furthermore, a survey was conducted among Canadian hospitals in order to ascertain current trends in infant feeding practices. Information obtained from t h i s survey along w i t h the chemical analyses of the formul was used to estimate the tocopherol adequacy of Canadian i n f a n t s . 3 CHAPTER I I REVIEW OF LITERATURE 1. H i s t o r y of Tocopherols In the 1920's v i t a m i n E was discovered as a f a t - s o l u b l e f a c t o r necessary f o r normal r e p r o d u c t i o n i n the r a t (9-13). I t was subsequently revealed t h a t v i t a m i n E, a term synonymous w i t h t o c o p h e r o l , was concentrated i n the n o n - s a p o n i f i a b l e f r a c t i o n of vegetable o i l s (14-16). In the l a t e 1930's, i s o l a t i o n of pure tocopherol (17), e l u c i d a t i o n of i t s chemical s t r u c t u r e (18) , and i n i t i a l s y n t h e s i s were achieved numerous i n v e s t i g a t o r s (20-27). To date, e i g h t s t r u c t u r a l l y s i m i l a r forms, d e r i v e d from the parent compound t o c o l or 2-methyl-2-(4,8,12-trimethyldecyl)-6 chromanol, as i l l u s t r a t e d below, have been dis c o v e r e d . (19). Since t h a t time, s t u d i e s of v i t a m i n E have been conducted by OH N A A NA According to W h i t t l e and Pennock (28), these compounds may be d i v i d e d i n t o two groups designated as the tocopherols and the t o c o t r i e n o l s . T h e i r b a s i c s t r u c t u r e and new suggested nomenclature (29) are shown i n Table 1. TABLE 1 Structure and Nomenclature of the Tocopherols TOCOPHEROL TOCOTRIENOL 5,7,8-trimethyl (a-tocopherol) 5,8-dimethyl (^-tocopherol) . 7,8-dimethyl (y-tocopherol) 8-methyl (6-tocopherol) 5,7,8-trimethyl (a-tocotrienol) 5,8-dimethyl (6-tocotrienol) 7,8-dimethyl (y-tocotrienol) 8-methyl (6-tocotrienol) 2. B i o l o g i c a l A c t i v i t i e s of The Tocopherols The d i f f e r e n t forms of tocopherol are known to possess v a r i a b l e b i o l o g i c a l a c t i v i t y . This a c t i v i t y i s dependent on the species of t e s t animal used and the method of bioassay. Bunyan et a l . (30) used i n v i t r o t e s t s to study the a c t i o n of the tocopherols i n preventing d i a l u r i c acid-induced e r y t h r o c y t e hemolysis. They found t h a t , when d1-a-tocopherol was assigned an a c t i v i t y of 100, n a t u r a l d-a-tocopherol had a potency of 133. The a c t i v i t i e s of s y n t h e t i c 3-, Y-» and 6.-tocopherols were 54, 67 and 26 r e s p e c t i v e l y . Rose and Gyorgy (31) conducted s i m i l a r t e s t s and concluded that 3-, Y-, and 6-tocopherols possessed 40, 30 and 20 per cent of the a c t i v i t y of d-a-tocopherol. For i n v i v o b i o a s s a y s , f a c t o r s such as a b s o r p t i o n and t r a n s p o r t become important (30,32). Bunyan et a l . (30) used such t e s t s and assigned d l - a - t o c o p h e r o l acetate a potency, of 100. S y n t h e t i c specimens of 3-, Y-and 6-tocopherols were shown to have b i o l o g i c a l a c t i v i t i e s of 27, 20, and 0.3 r e s p e c t i v e l y . Rose and Gyorgy (31) determined t h a t , r e s p e c t i v e l y , the i n v i v o potencies of 3-, Y- and 5-tocopherol were 20, 4 and 3 percent of t h a t f o r d-a-tocopherol. Friedman et a l . (33) reported t h a t Y-tocopherol possessed 21.7 per cent of the a c t i v i t y of a-tocopherol. This value i s i n agreement w i t h that of Bunyan et a l . (30). The 1-tocopherols are much l e s s b i o l o g i c a l l y a c t i v e than the d-epimers (32). Various authors have demonstrated that d-a-tocopherol possessed an a c t i v i t y at l e a s t 1.3 times g r e a t e r than s y n t h e t i c forms (31, 33, 34). In these s t u d i e s , the p r e v e n t i o n of d i a l u r i c acid-induced e r y t h r o c y t e hemolysis was used to judge tocopherol a c t i v i t y . Other bioassay methods, such as g e s t a t i o n - r e s o r p t i o n , weight gain during pregnancy, u t e r i n e pigmentation, t e s t i c u l a r degeneration, and muscular dystrophy p r e v e n t i o n have a l s o been used to evaluate the b i o l o g i c a l a c t i v i t i e s of the d i f f e r e n t tocopherols (20, 35-44). Such t e s t s confirmed that d-a-tocopherol was the most potent. The dimethyl compounds had i n t e r -mediate a c t i v i t i e s . Monomethyl d e r i v a t i v e s e x h i b i t e d the lowest p o t e n c i e s . 3. V i t a m i n E D e f i c i e n c y - Occurrence  Animals According to H e r t i n g (47) v i t a m i n E d e f i c i e n c y i n animals i s accompanied by a wide v a r i e t y of d i s o r d e r s . Most systems of the body are adversely a f f e c t e d i n some way. The m a n i f e s t a t i o n s of v i t a m i n E d e f i c i e n c y i n animals may i n c l u d e f o e t a l r e s o r p t i o n (9,12), t e s t i c u l a r degeneration (48,49), d e f e c t i v e embryonic development (50,51), s k e l e t a l muscular dystrophy (44,52,53), c a r d i v a s c u l a r d i s o r d e r s (54,55), c e r o i d pigment d e p o s i t i o n i n muscle (56,57), e r y t h r o c y t e hemolysis, decreased e r y t h r o c y t e s u r v i v a l time, and anemia (3, 58-66), exudative d i a t h e s i s (67,68) and encephalomalacia (69,70). D e t a i l s of the development of v i t a m i n E d e f i c i e n c y i n animals are a v a i l a b l e i n s e v e r a l e x c e l l e n t reviews (71-74). Human A d u l t s To date, no v i s i b l e s i g n s of v i t a m i n E d e f i c i e n c y i n human a d u l t s have been recognized. On the b a s i s of e r y t h r o c y t e hemolysis t e s t s , i t has. been suggested that serum tocopherol l e v e l s below 0.5 mg per 100 ml could be c l a s s i f i e d as d e f i c i e n t (6,75). Various authors (31,75-78) have reported t h a t , i n North America, v i t a m i n E d e f i c i e n c y , as d e f i n e d by these c r i t e r i a , i s not common. Yet, Desai (76) cautioned t h a t d e f i c i e n t s t a t e s may be found i n c e r t a i n segments of the p o p u l a t i o n depending upon t h e i r d i e t a r y p e c u l i a r i t i e s . V itamin E d e f i c i e n c y has a l s o been reported i n other r e g i o n s , such as P a k i s t a n (79). Horwitt and a s s o c i a t e s (80-83), by means of d i e t a r y r e s t r i c t i o n , induced v i t a m i n E d e f i c i e n c y i n a d u l t men. E r y t h r o c y t e a b n o r m a l i t i e s , such as decreased s u r v i v a l time and i n c r e a s e d s u s c e p t i b i l i t y to hemolysis were noted. Fat malabsorption syndromes appear to promote tocopherol d e p l e t i o n i n a d u l t s . Thus, i t has been shown that the d i s o r d e r s which accompany s t e a t o r r h e a (such as c h r o n i c p a n c r e a t i t i s , xanthamous b i l i a r y c i r r h o s i s n o n - t r o p i c a l sprue, and b i l i a r y a t r e s i a ) were f r e q u e n t l y c h a r a c t e r i z e d by c l a s s i c signs of v i t a m i n E d e f i c i e n c y (84,96). C h i l d r e n and I n f a n t s S u f f e r i n g from Steatorrhea or M a l n u t r i t i o n Prolonged f a t malabsorption i n c h i l d r e n may be accompanied by tocopherol d e f i c i e n c y (97-105). In recent y e a r s , emphasis has been d i r e c t e d towards a c o r r e l a t i o n between the accepted signs of tocopherol d e f i c i e n c y and the c h a r a c t e r i s t i c s of s t e a t o r r h e a (103). V i t a m i n E d e f i c i e n c y and/or anemia which i s c h a r a c t e r i z e d by low hemoglobin values and abnormal e r y t h r o c y t e hemolysis have been reported i n cases of kwashiorkor (106-115). In a number of these s t u d i e s (107-111) v i t a m i n E proved to be the s o l e t h e r a p e u t i c agent capable of i n d u c i n g a p o s i t i v e hematopoietic response. However, Majaj (110) cautioned that many f a c t o r s are i n v o l v e d i n the anemia of kwashiorkor, and that v i t a m i n E therapy may not always be e f f e c t i v e . Such a s i t u a t i o n was encountered by Asfour and F i r z l i (116) and Baker et a l . (117). Furthermore, serum tocopherol l e v e l s are not n e c e s s a r i l y low i n a l l cases of kwashiorkor (118). E a r l y Infancy Because of l i m i t e d p l a c e n t a l t r a n s f e r of t o c o p h e r o l s , newborns, e s p e c i a l l y prematures, have d e f i c i e n t l e v e l s of serum tocopherol (1-3,75,100, 120-123). In a d d i t i o n , the e r y t h r o c y t e s of newborns are p a r t i c u l a r l y s u s c e p t i b l e to l y s i s i n the presence of hemolyzing agents (2-5,7,75,119). Oski and Barness (4) were among the f i r s t to recognize t h a t t h i s tendency . towards abnormal e r y t h r o c y t e hemolysis i n neonates was d i r e c t l y r e l a t e d to the v i t a m i n E - d e f i c i e n t s t a t e . The hemolytic anemia bas f u r t h e r been c h a r a c t e r i z e d by a b n o r m a l i t i e s such as marked r e t i c u l o c y t o s i s , p y k n o c y t o s i s , and decreased e r y t h r o c y t e s u r v i v a l time (4,5). A decreased hemoglobin l e v e l (122), edema, s k i n l e s i o n s , e l e v a t e d p l a t e l e t count, and morphological changes i n the e r y t h r o c y t e have a l s o been reporte d (5,7,119). The v i t a m i n E d e f i c i e n c y syndrome may be c l a s s i f i e d as t r a n s i e n t . I t reaches i t s peak at s i x to ten weeks a f t e r b i r t h and u s u a l l y disappears w i t h i n s e v e r a l months (4). The s e v e r i t y and d u r a t i o n of the hemolytic anemia of v i t a m i n E d e f i c i e n c y are very dependent on the mode of i n f a n t f e e d i n g . This w i l l be d i scussed l a t e r . -Goldbloom (123) was not able to demonstrate a decreased e r y t h r o c y t e s u r v i v a l time i n i n f a n t s w i t h low serum tocopherol l e v e l s . F i l e r (124) r e p o r t e d that the i n c r e a s e d s u s c e p t i b i l i t y to e r y t h r o c y t e hemolysis r e f l e c t e d b i o c h e m i c a l immaturity at an enzyme l e v e l , and not tocopherol d e f i c i e n c y . Panos et a l . (125) have a l s o s t a t e d that the hemolytic anemia found i n neonates was not r e l a t e d to tocopherol d e f i c i e n c y . 9 4. Hemolytic Anemia, Vitamin E Deficiency and Infant Feeding P r a c t i c e s  Dietary Factors It has been suggested that c e r t a i n dietary f a c t o r s may be r e l a t e d to tocopherol metabolism and thereby a l t e r the amount needed to prevent d e f i c i e n c y . These d i e t a r y factors may include increased l e v e l s of i r o n (5,61,100,126-128) and ascorbic acid (4) or suboptimal intakes of selenium and/or methionine (129,130), f o l i c a c i d , p r o t e i n , vitamin (131) and vitamin B, (132). D However, an elevated l e v e l of d i e t a r y polyunsaturated f a t t y acids (PUFA) w i l l increase tocopherol requirements more than any known f a c t o r . Studies conducted i n man (81,133,134), monkeys (135), rats (136-138), and chickens (139), have demonstrated that, as the d i e t a r y PUFA was increased vitamin E d e f i c i e n c y was more probable. The syndrome was prevented by tocopherol supplementation. Harris and Embree (133) have compiled data from various experiments and from f a t consumption figures f or North America. They concluded that 0.6 mg of a-tocopherol per g of PUFA ( r a t i o 0.6) was necessary to eliminate the r i s k of vitamin E d e f i c i e n c y . Human Milk i n Infant N u t r i t i o n For many years, i t has been common p r a c t i c e to breast-feed young i n f a n t s . Various i n v e s t i g a t o r s have analyzed human milk f o r tocopherols, PUFA, and vitamin-E-to-PUFA r a t i o s . In an e a r l y study, Quaife (140) found values ranging from 76 to 1800 ug t o t a l tocopherols per g of human milk l i p i d (0.4-3.6 mg per 100 ml). These samples were examined within the f i r s t week of p a r t u r i t i o n . In 10 samples analyzed one to e i g h t months a f t e r p a r t u r i t i o n , the t o t a l tocopherol content v a r i e d from 37 to 58 )xg per g of l i p i d . H a r r i s et a l . (141) determined t o t a l tocopherol l e v e l s that ranged from 0.10 to 0.45 mg per 100 ml of human m i l k (average of 8 0 ^ per g of l i p i d ) . Composite samples of p a s t e u r i z e d human m i l k had t o t a l tocopherols values ranging from 0.14 to 0.46 mg per 100 ml. The average value was 49 ug per g of m i l k l i p i d . Tarjan and a s s o c i a t e s (142) conducted s i m i l a r s t u d i e s and found an average v i t a m i n E l e v e l of 0.28 mg (range 0.11 to 0.86) per 100 ml. In a d d i t i o n , these authors reported l i t e r a t u r e values f o r v i t a m i n E l e v e l s i n human m i l k that ranged from 0.21 to 3.60 mg per 100 ml. Hashim and Asfour (3), Woodruff and a s s o c i a t e s (143), and H e r t i n g and Drury (144) reported l e v e l s o f 324, 50 and 40 Ag a-tocopherol per g of m i l k l i p i d . The PUFA content of human m i l k has a l s o been i n v e s t i g a t e d . D i e t a r y f a t has been shown to i n f l u e n c e the PUFA l e v e l (145-147). However, values u s u a l l y range from 5 to 11 g per 100 g of human m i l k l i p i d (145-148). H e r t i n g and Drury (144), assuming l e v e l s of 8 per cent PUFA and 40 .Mg a-tocopherol per g of l i p i d , c a l c u l a t e d the a-tocopherol-to-PUFA r a t i o to be 0.5. Woodruff et a l . (143), D i c k s - B u s h n e l l and Davis (149), and R i t c h i e et a l . (5) c i t e d a-tocopherol-to-PUFA r a t i o s of 0.49, 0.4 or g r e a t e r and approximately 1 r e s p e c t i v e l y . In f a c t , D i c k s - B u s h n e l l and Davis (149) proposed t h a t , f o r purposes of i n f a n t n u t r i t i o n , the c r i t i c a l r a t i o should be 0.4, and not 0.6 (133). The above data suggest that b r e a s t - f e d i n f a n t s should not manifest v i t a m i n E d e f i c i e n c y and the a s s o c i a t e d hemolytic anemia. Nitowsky et a l . (6), Gordon et a l . (100), Wright et a l . (150), Hashim and Asfour (3), Woodruff et a l . (143), and Sugahara (151) have a l l shown that tocopherol l e v e l s of • 11 b r e a s t - f e d i n f a n t s (though d e f i c i e n t at b i r t h ) soon exceeded the c r i t i c a l l e v e l of 0.5 mg per 100 ml serum. E r y t h r o c y t e hemolysis was minimal. In another study, Gerloczy (152) found that the s e v e r i t y of sceleredema neonatorum, a type of edema common i n the premature i n f a n t , could be g r e a t l y a l l e v i a t e d by tocopherol therapy. Cow's M i l k i n I n f a n t N u t r i t i o n Although human m i l k has a very s a t i s f a c t o r y tocopherol content and an a-tocopherol-to-PUFA r a t i o adequate to prevent hemolytic anemia, i t i s not always the p r e f e r r e d mode of fe e d i n g . Formulas based on f r e s h cow's m i l k , or evaporated whole, p a r t l y skimmed, or w h o l l y skimmed m i l k o f t e n serve as s u b s t i t u t e s . In e v a l u a t i n g the tocopherol s t a t u s of these formulas, two f a c t s are of prime concern. F i r s t l y , the v i t a m i n E content of cow's m i l k c o n s i s t s l a r g e l y of the b i o l o g i c a l l y important a-tocopherol. Secondly, summer and autumn samples of cow's m i l k tend to be r i c h e r i n tocopherols than w i n t e r and s p r i n g m i l k s . This i s - a r e f l e c t i o n of seasonal v a r i a t i o n i n the tocopherol content of forage crops (153,154). H a r r i s e t a l . (155) reported l e v e l s of 42 and 23 yg t o t a l . t o c o p h e r o l s per g of l i p i d f o r summer and w i n t e r m i l k s . H e r t i n g and Drury (144) noted values of 30 and 4 j*g a-tocopherol per g of l i p i d i n autumn and s p r i n g m i l k s . Macy et a l . (156) determined the average a-tocopherol value to be 0.06 mg per .100 ml of sample, w h i l e Bunnell et a l . (157) found an average t o t a l t ocopherol content of 0.093 mg per 100 ml m i l k . Of t h i s , only 0.036 mg appeared as a-tocopherol. In an e a r l i e r study, H a r r i s and a s s o c i a t e s (141) c i t e d l i t e r a t u r e value f o r tocopherols (not s p e c i f i e d as a-tocopherol) i n cow's m i l k that 12 ranged from 0.02 to 0.17 mg per 100 ml sample (5-45 ug per g of l i p i d ) . Such v a r i a t i o n s suggest that analyses of the m i l k samples were conducted at d i f f e r e n t times of the year. In t h e i r own s t u d i e s , H a r r i s et a l . (141) examined samples of evaporated m i l k and whole powdered m i l k . They found that the tocopherol content (presumably a-tocopheral) v a r i e d from 23 to 40 ug per g of l i p i d . H a r r i s et a l . (154), i n studying evaporated m i l k , found an average tocopherol l e v e l of 40 ug per g of l i p i d . Woodruff et a l (143) showed th a t 100 ml of u n d i l u t e d evaporated m i l k contained 0.28 mg t o t a l t o copherols. This value i s equal to 35 ug t o t a l tocopherols per g of m i l k l i p i d (144). H e r t i n g and Drury (144) a s c e r t a i n e d t h a t depending on the time of canning, evaporated m i l k samples contained from 10 to 25 ug a-tocopherol per g of l i p i d . Macy et a l . (156) and others (144) have shown that the PUFA content of b u t t e r f a t averaged about 4 per cent of the t o t a l l i p i d s . H e r t i n g and Drury (144) reported an a-tocopherol-to-PUFA r a t i o of 0.21 f o r f r e s h cow's m i l k obtained i n the s p r i n g . Evaporated cow's m i l k canned i n e a r l y s p r i n g and l a t e f a l l had a-tocopherol-to-PUFA r a t i o s of 0.51 and 0.67 r e s p e c t i v e l y . D i c k s - B u s h n e l l and Davis (149) c i t e d an a-tocopherol-to-PUFA r a t i o of 0.20 f o r evaporated m i l k . Non-fat dry cow's m i l k has a l s o been analyzed. An a-tocopherol val u e of 29 ug. per g of l i p i d and an a-tocopherol-to-PUFA r a t i o of 0.60 were reported (144). These values seem very adequate f o r n u t r i t i o n of the young i n f a n t . However, the absolute q u a n t i t y of a v a i l a b l e tocopherol (20 ug per quart) i s so low that v i t a m i n E d e f i c i e n c y i s l i k e l y to develop. E r i c k s o n et a l . (158) have demonstrated that the tocopherol c o n c e n t r a t i o n i n c r e a s e s i n the l i p i d f r a c t i o n remaining i n skimmed m i l k products. Thus, i n g e n e r a l , the a-tocopherol-to-PUFA r a t i o i n formulas based 13 on cow's m i l k i s more v a r i a b l e and o f t e n l e s s s a t i s f a c t o r y than t h a t of human m i l k . The tocopherol content i s a l s o more l i m i t e d . H a r r i s et a l . (141) and W i l l i a m s (159) concluded that on the average, human m i l k contained two to four and up to ten times more tocopherol than cow's m i l k . H a r r i s ( 8 ) , and Gordon et a l . (100) reported that neonates fed f r e s h cow's m i l k o f t e n maintained d e f i c i e n t l e v e l s of serum tocopherol and manifested hemolytic anemia. Wright et a l . (150) noted b o r d e r l i n e v i t a m i n E d e f i c i e n c y i n i n f a n t s who were nourished w i t h evaporated m i l k during the f i r s t months of l i f e . I n v a r i o u s s t u d i e s (6,75,122,150,160) i t was shown that young i n f a n t s , e s p e c i a l l y prematures, who were fed p a r t l y skimmed m i l k formulas had d e f i c i e n t l e v e l s of serum t o c o p h e r o l . A m u l t i p l i c i t y of v i t a m i n E d e f i c i e n c y symptoms were noted. Thus, i t i s evident that i n f a n t s who are maintained on cow's m i l k formulas, e s p e c i a l l y the skimmed v a r i e t y , may manifest v i t a m i n E d e f i c i e n c y much more r e a d i l y than the b r e a s t - f e d baby. Simulated Formulas i n Infant N u t r i t i o n An i n c r e a s i n g l y popular s u b s t i t u t e f o r human m i l k i s simulated m i l k . In these p r e p a r a t i o n s the source of l i p i d i s a vegetable o i l . Such formulas have been found to be b e t t e r t o l e r a t e d (161) and more e a s i l y absorbed than formulas based on cow's m i l k (162,163). Furthermore, they are i n d i s -pensable i n the feeding of i n f a n t s a l l e r g i c to animal m i l k (163). H e r t i n g and Drury (164) noted that r e f i n e d , unhydrogenated cotton-seed, c o r n , s a f f l o w e r , and soybean o i l s contained, on an average, 0.31, 0.19, 0.31, and 0.16 ug. a-tocopherol per g of l i p i d . These values o f t e n represented only a f r a c t i o n of the t o t a l t o c o pherol content. The authors observed that only cottonseed o i l had an a-tocopherol-to-PUFA r a t i o greater than the c r i t i c a l 0.6. Herting and Drury (144) analyzed seventeen simulated milks and found a-tocopherol levels ranging from 85 to 224 ug per g of l i p i d . Thirteen of these formulas had a-tocopherol-to-PUFA r a t i o s less than 0.6 while the rat i o s of eight were less than 0.4, Herting and Drury (144) noted that the a-tocopherol f r a c t i o n was often a small part of the t o t a l tocopherol content. They also observed that twelve of these formulas were f o r t i f i e d with vitamin E. In an e a r l i e r study by the same authors, only eight formulas had been tocopherol-supplemented (165). However, i n spite of tocopherol supplementation, various formulas s t i l l had a-tocopherol-to-PUFA r a t i o s less than 0.6 (144). Dicks-Bushnell and Davis (149) studied f i v e undiluted milks and found t o t a l tocopherols ranging from 2.36 to 3.86 mg per 100 ml sample. Thirteen to 45 per cent of the vitamin E content was present as a-tocopherol. The a-tocopherol-to-PUFA r a t i o s for two of the formulas were given to be 0.23 and 0.29. In an e a r l i e r study, Harris et a l . (154) reported a range of 29 to 171 ug a-tocopherol per g of l i p i d for samples of three simulated milks. Oski and Barness (4) cited a-tocopherol levels of 0.73 to 5.2 mg per quart of reconstituted formula. Owen (166) stated that the tocopherol content of simulated milks may range from a trace to ten International Units. Hassan et a l . (7) nourished premature infants with a formula containing 50 per cent PUFA and 0.32 mg a-tocopherol per 100 ml sample. These infants showed various signs of vitamin E deficiency as serum toco-pherol levels became very depleted. When the diet contained 27 per cent PUFA and 0.35 mg a-tocopherol per 100 ml, vitamin E deficiency s t i l l prevailed. Ri t c h i e et a l . (5), Oski and Barness (4), Wright et a l . (150), Hanna et a l . (167), and Hashim and Asfour (3) a l l noted that infants fed various simulated formulas had c h a r a c t e r i s t i c vitamin E deficiency during the f i r s t months of l i f e . Thus, j u s t as with cow's milk formulas, simulated formulas may not contain adequate tocopherol and/or a vitamin E-to-PUFA r a t i o s u f f i c i e n t to avoid the r i s k of the vitamin E deficiency syndrome. 5. Tocopherols and the Prevention of Erythrocyte Hemolysis It has been w e l l established that vitamin E deficiency i s charac-terized by abnormal erythrocyte hemolysis. To understand the roles of vitamin E and PUFA i n erythrocyte hemolysis, two facts are important. F i r s t l y , the erythrocyte contains PUFA. An increase i n dietary PUFA i s manifested as an increased l e v e l of PUFA i n the erythrocyte (168-170). Secondly, a-tocopherol probably serves as a major, i f not main, b i o l o g i c a l antioxidant (171-173). In view of these f a c t s , i t i s reasonable to assume that oxidative changes w i l l occur i n the erythrocyte i f vitamin E levels become depleted. In severe deficiency, abnormal hemolysis, i n the presence of a weak hemolyzing agent, w i l l occur. This was f i r s t shown by Rose and Gyorgy (31,174,175) and was l a t e r confirmed by other investigators (58-61,64,176). In vitamin E deficiency, the oxidative changes that characterize the erythrocyte have been shown to render i t less stable. Consequently, there i s greater s u s c e p t i b i l i t y to hemolysis. These changes have included increased levels of l i p i d peroxides (83,177-179), Heinz Body and methemoglobin formation (180-183), reduced levels of detoxifying agents such as glutathione peroxidase (180), and increased levels of reduced glutathione (122,180,184). The erythrocyte has been shown to be less stable i n the presence of oxygen (178,185,186). 16 In many s t u d i e s , tocopherol i n h i b i t e d the formation of o x i d a t i o n products, e s p e c i a l l y l i p i d peroxides i n the er y t h r o c y t e s (31,58-61,174-176, 178,187). Consequently, the e r y t h r o c y t e remained s t a b l e i n the presence of hemoli z i n g agents. Thus, i n the feeding of neonates w i t h formulas high i n PUFA and/or low i n a-tocopherol content, the s t a b i l i t y of the e r y t h r o c y t e may be lessened. In such a s i t u a t i o n , the c h a r a c t e r i s t i c hemolytic anemia of v i t a m i n E d e f i c i e n c y i s manifested. CHAPTER I I I MATERIALS AND METHODS 1. Formulas Used for Analysis The formulas to be analyzed were purchased l o c a l l y or supplied by the Vancouver General Hospital and Children's Hospital i n Vancouver. The formulas were obtained i n vacuum-pack containers and were analyzed i n duplicate immediately after opening. Further storage of the formulas, i f necessary, was carried out at -20°C. A description of the formulas i s presented i n Table 2. Except where otherwise stated, a l l chemicals necessary for analysis of the formulas were purchased from Fisher S c i e n t i f i c Company Ltd. Most of the chemicals used were a n a l y t i c a l grade reagents. . A l l analyses were carried out as quickly as possible. Samples were excluded from l i g h t and where possible, the different procedures were conducted under an atmosphere of nitrogen. These precautions helped to l i m i t tocopherol destruction. 2. Fat Extraction and Saponification of the Extracted Fat The method of Folch et a l . (188) was used for extraction of the l i p i d from the infant formulas. Powdered Samples A suitable sample (2-3 g)' was accurately weighed and transferred to a. 250 ml dark brown erylenmyer flask. For each g of sample, 20 ml of chloroform-methanol solution (2:1 v/v) were added c a r e f u l l y with gentle s w i r l i n g . Mixing at low speed on a magnetic s t i r r e r apparatus was continued 18 TABLE 2 Formulas Used f or Analysis Formula Fat Source Manufacturer or Supplier D e s c r i p t i o n Dairyland Butterfat Dairyland, Vancouver Fresh whole cow's milk Lucerne Butterfat Safeway, Vancouver Fresh whole cow's milk Carnation Butterfat Carnation Company Evaporated whole cow's milk P a c i f i c B u tterfat P a c i f i c Milk Co. Evaporated whole cow's milk Farmer's Wife #1 Butterfat Cow & Gate Ltd. Evaporated whole cow's milk Farmer's Wife #1, Added CHO, v i t a -mins Butterfat Cow & Gate Evaporated whole cow's milk, added CHO and vitamins Dairyland Butterfat Dairyland, Vancouver Fresh p a r t l y skimmed cow's milk Lucerne Butterfat Safeway, Vancouver Fresh p a r t l y skimmed cow's milk Carnation Morning' But t e r f a t Carnation Company i Evaporated p a r t l y skimmed cow's milk Delta P a c i f i c Butterfat P a c i f i c Milk Co. Evaporated p a r t l y skimmed cow's milk Farmer's -Wife #2 Butterfat Cow & Gate Ltd. Evaporated p a r t l y skimmed cow's milk Farmer's Wife #2, Added CHO v i t a -mins Butterfat Cow & Gate Ltd. Evaporated p a r t l y skimmed cow's milk, added CHO and vitamins Dairyland Butterfat Dairyland, Vancouver Fresh skimmed cow's milk Lucerne Butterfat Safeway, Vancouver Fresh skimmed cow's milk Farmer's Wife #3 Butterfat Cow & Gate Ltd. Evaporated skimmed cow's milk Klim Butterfat Borden Company Powdered whole cow' milk Lactogen Butterfat Nestle Company Powdered skimmed cow's milk Probana Butterfat Mead Johnson Co. Powdered low f a t , high p r o t e i n Pelargon Butterfat Nestle Company Powdered low f a t Dryco Butterfat Borden Company Powdered low f a t (Table 2 continued ) 19 (Table 2 Continued) Formula Fat Source Manufacturer or Supplier Description Lonalac Coconut O i l Mead Johnson Co. Powdered low sodium Olac • Corn O i l Mead Johnson Co. Powdered hypoallergenic formula Nutramigen Corn O i l Mead Johnson Co. Powdered hypoallergenic formula Prosobee Corn O i l Mead Johnson Co. Liquid hypoallergenic formula Lofenalac Corn O i l Mead Johnson Co. Powdered PKU formula Soyalac Soybean O i l Loma Linda Ltd. Powdered hypoallergenic formula Neo-Mull-Soy Soybean O i l Borden Company Liquid hypoallergenic formula CHO-free Soybean O i l Borden Company Liquid CHO-free formula Similac Corn, Coco-nut, o l i v e o i l s Ross Lab Inc. Liquid standard formula Similac + Iron Corn, Coco-nut, o l i v e o i l s Ross Lab Inc. Liquid standard formula, added iron Isomil Corn, Coco-nut , o l i v e o i l s Ross Lab Inc. Liquid with added elec-tr o l y t e s Portagen Coconut, saf-flower o i l s Mead Johnson Company Powdered with added v i t a -mins and minerals Formula 4 Corn, coco-nut o i l s Cow & Gate Ltd. Liquid standard formula SMA Oleo, coco-nut, corn, soybean o i l s J. Wyeth & Co. Powdered standard formula for several minutes. The remainder of the method was carried out according to Folch's directions ( 1 8 8 ) . Liquid Samples Twenty ml of chloroform-methanol solution per ml of diluted milk sample were transferred to a 2 5 0 ml dark broxm erlenmyer flask. With constant, gentle swirling the milk sample ( 2 - 3 ml) was added drop by drop from a pipette. The remainder of the procedure was identical to that for powdered samples. Recovery of Total Fat The f i n a l weight of the flask minus the i n i t i a l weight equalled the amount of fat recovered. To test for per cent recovery, fat was extracted from two samples of the same milk. The f i r s t sample (A) was unaltered. To the second sample ( B ) 1 . 0 g of fat was added. Upon analysis, the f i r s t sample yielded . 5 2 9 6 g of fat. A total of 1 . 4 9 9 3 g of fat was recovered from the second sample. Using these data, per cent recovery of fat from the infant formulas could be calculated as follows: „ wt. of l i p i d from B - wt. of l i p i d from A % recovery = c x 1 0 0 1 . 0 g The per cent recovery of l i p i d was 9 6 . 9 . Saponification of the Extracted Fat The saponification procedure of The Committee on Vitamin E Determination ( 1 8 9 ) was used in this study. The extracted fat was saponified in the presence of pyrogallol and potassium hydroxide. The non-saponifiable fraction was then extracted with diethyl ether according to the Committee's specifications. 21 3. T o t a l Tocopherol Determination For t o t a l tocopherols d e t e r m i n a t i o n , the Emmerie-Engel procedure as o u t l i n e d by B i e r i et a l . (78) was employed. The u n s a p o n i f i a b l e f r a c t i o n was d i s s o l v e d i n petroleum ether and q u a n t i t a t i v e l y t r a n s f e r r e d to a glass-stoppered c e n t r i f u g e tube covered w i t h f o i l . The petroleum ether was evaporated using a stream of n i t r o g e n and a 50°C water bath. The n o n - s a p o n i f i a b l e e x t r a c t was then t r e a t e d according to B i e r i ' s d i r e c t i o n s (78) . Carotenoids and t o t a l tocopherols were determined at 450 and 520 my r e s p e c t i v e l y on a Beckman spectrophotometer. C a l c u l a t i o n s Standard curves f o r mg tocopherol per 100 ml were e s t a b l i s h e d u s i n g d l - a - t o c o p h e r o l ( D i s t i l l a t i o n Products I n d u s t r y , Rochester, New York) The average slope of the curve at 520 my was found to be 10 .29 . An i l l u s t r a t i o n of t h i s standard curve i s shown i n Figure l . \ S i m i l a r i l y , standard curves f o r g-carotene (Sigma Chemical Co. Ltd.) were determined at 450 and 520 mp ( i l l u s t r a t e d i n Figures 2b and 2 c ) . Color due to g-carotene was c a l c u l a t e d as f o l l o w s : mg % g-carotene = A O . D . ^ ^ Q X slope of curve = A0.D. / c_ x 2.52 450 At 450 my 1.0 mg % g-carotene has A0.D. = 0 . 3 8 4 At 520 my 1.0 mg % g-carotene has A0.D. = 0.084 Per cent absorbancy at 520 my due to g-carotene i n a tocopherol g-carotene mixture i s equal to ^ ' ^ ^ x 100 = 21 117 0.384 l i - . i i k . A O.D.xlO A 24.0--Ffg. 1a STANDARD CURVE FOR dl a-TOCOPHEROL f 520 mp ) 16.0 8.04 0 0.5 -4-1.0 mg dl cr-tocopherol per 100 ml 2.0 ethanol A 0. D. x W 75.(4 70.0+ Fig. 1c STANDARD CURVE FOR J3- CAROTENE (520 mjj) 5.0 f 0 —f-0.5 -4-1.0 mg B-Carotene per 100 ml N .2.0 Pet. Ether fi> 23 Total tocopherols were then calculated as follows: Total tocopherols (mg per 100 ml of reconstituted formula) = [AO.D.520 - (AO.D.450 x .2177) x slope of tocopherol curve at 520 mp = [AO.D.520 - (AO.D.450 x .2177) x 10.29 In the preliminary laboratory work, known amounts of dl-a-tocopherol were added to stripped lard (tocopherol-free). The tocopherol was re-extracted and the per cent recovery calculated. I t was found that a 93 per cent recovery could be obtained. 4. Individual Tocopherol Determination The dif f e r e n t tocopherols were determined by the thin-layer chromatography method of Whittle and Pennock (28). The unsaponifiable l i p i d f r a c t i o n was dissolved i n cyclohexane. I t was then applied to a 20 x 20 cm thin layer plate covered with activated s i l i c a gel G. Two dimensional development of the chromatogram i n solutions of chloroform and of diisopropyl ether followed. This procedure and detection of the i n d i v i d u a l tocopherols with ethanol flourescein solution was carried out as described by the authors (28). Having ascertained the tocopherol pattern, chromatograms were s i m i l a r i l y developed for quantitative tocopherol analysis. Areas of s i l i c a gel, corresponding to each tocopherol, were transferred by vacuum suction to the upper section of a graduated cylinder covered with f o i l . The tocopherols were eluted from the glass wool on which they were suspended with p u r i f i e d ethanol. A t o t a l of 4 ml of eluate was collected. Tocopherol-free areas of s i l i c a gel were treated s i m i l a r i l y . These were used for blank determinations. The remainder of the procedure was conducted 24 according to the authors' s p e c i f i c a t i o n s (28). An average tocopherol loss of 10 per cent was encountered. A s i m i l a r s i t u a t i o n had been reported by the authors (28). Consequently, i n the c a l c u l a t i o n s , a 10 per cent c o r r e c t i o n f a c t o r to compensate for t h i s loss was employed. ' The d i s t r i b u t i o n pattern of the tocopherols a f t e r two-dimensional t h i n layer chromatography i s presented i n Figure 2. Because of l i m i t e d space, only that portion of the t h i n layer chromatogram containing the tocopherols i s shown. In general, the solvent fronts t r a v e l l e d 16 cm i n both d i r e c t i o n s . For the f i r s t dimension, Rf values for a-, 3-> Y~ a n <3 ^-tocopherol were .70, .50, .50, and .30 r e s p e c t i v e l y . Second dimension Rf values for a-, g-, Y~> a n d 6-tocopherol were ca l c u l a t e d to be .44, .40, .32, and .24 r e s p e c t i v e l y . 5. Polyunsaturated Fatty Acid Analysis The method of Holman and Hayes (190) was used for t h i s determination. The l i p i d was dissolved i n petroleum ether. Background absorption was determined upon t h i s s o l u t i o n i n the U.V. range at wavelengths of 375, 346, 315, 268 and 233 mu. A Beckman spectrophotometer was used. The sample was then isomerized with an ethylene glycol-potassium hydroxide s o l u t i o n . Absorbance of t h i s s o l u t i o n at the same wavelengths was then determined. E x t i n c t i o n c o e f f i c i e n t s were calculated for the samples before and a f t e r isomerization at a l l f i v e wavelengths. The d i f f e r e n c e s were subs t i t u t e d i n t o the s e r i e s of equations outlined by Holman and Hayes (190) f o r c a l c u l a t i o n of polyenoic f a t t y a c i d contents. These were expressed as per cent of t o t a l l i p i d . 25 TWO-DIMENSIONAL THIN LAYER CHROMATOGRAM OF TOCOPHEROLS Fig. 2 o1 30 02 Run 7 t Run 2 1. a - tocopherol 2. B - tocopherol 3. J - tocopherol 4. 6 - tocopherol 5. Source 26 In p r e l i m i n a r y a n a l y s i s , known amounts of polyunsaturated f a t t y a c i d s were used to t e s t f o r accuracy of the method. Recoveries of p o l y -unsaturated f a t t y a c i d s were 5 to 13 per cent low. Holman and Hayes (190) reported l o s s e s of 2 to 10 per cent. Despite i t s shortcomings, t h i s method was s t i l l used f o r c e r t a i n formulas. 27 CHAPTER IV RESULTS AND DISCUSSION R e s u l t s of the chemical analyses of i n f a n t formulas are presented i n Tables 3 to 9. Values are arranged i n descending order of mg t o t a l tocopherols per l i t r e of r e c o n s t i t u t e d formula. For f r e s h cow's m i l k and cow's m i l k products, and two m i l k s u b s t i t u t e s , d i f f i c u l t y was encountered w i t h the PUFA determination. I t i s probable that the i s o m e r i z a t i o n process was not s e n s i t i v e enough to detect the very low contents of PUFA i n the formulas. A n a l y s i s of the PUFA l e v e l of these formulas by gas l i q u i d chromatography was a n t i c i p a t e d . U n f o r t u n a t e l y , the necessary chromatography columns were not r e a d i l y a v a i l a b l e . Consequently, t h e i r PUFA contents were estimated according to Fomon's f i g u r e s (191) or from i n f o r m a t i o n s u p p l i e d by the manufacturer. 1. Fresh Cow's M i l k Used as I n f a n t Formulas As can be seen i n Table 3, some 78.3 to 90.8 per cent (average 84.2) of the v i t a m i n E present i n f r e s h cow's m i l k samples c o n s i s t e d of the b i o l o g i c a l l y important form - a - t o c o p h e r o l . The average t o t a l t ocopherol and a - t o c o p h e r o l contents per l i t r e of formula were 0.79 mg (range 0.46 to 1.18) and 0.63 mg (range 0.36 to 0.89) r e s p e c t i v e l y . R e l a t i v e l y h i g h l e v e l s of a-tocopherol i n f r e s h cow's m i l k have been p r e v i o u s l y reported (75,119). Gamma-tocopherol was the only other tocopherol found. I t represented 9.2 to 21.4 per cent (average 15.73) of the t o t a l t ocopherol content. These m i l k samples were obtained and analyzed i n the l a t e autumn. T h e i r r e l a t i v e l y h i g h tocopherol contents are suggestive of the TABLE 3 Vitamin E and Polyunsaturated F a t t y A c i d Contents of Fresh Cow's M i l k Formula % Fat TOCOPHEROLS I.U. 3 V i t a m i n E / l i t r e PUFA 4 (g/g Fat) T o t a l Tocopherol /PUFA R a t i o (mg/g) a-Tocopherol /PUFA R a t i o (mg/g) m g / l i t r e jjg/g Fat . % Of T o t a l 2 a 2 6 2 2 Y o2 T o t a l 1 2 a a2 2 Y <? T o t a l1 a 6 Y 6 D a i r y l a n d 3.2 0.89 0.0 0.24 0.0 1.18 25.34 0.0 6.93 0.0 33.60 78.3 0.0 21.4 0.0 1.37 0.04 0.84 0.63 ' Whole Lucerne 3.9 0.81 0.0 0.10 0.0 0.93 20.85 0.0 2.43 0.0 24.09 90.8 0.0 9.2 0.0 1.23 0.04 0.60 0.52 Whole D a i r y l a n d 2.0 0.44 0.0 0.14 0.0 0.60 22.17 0.0 6.86 0.0 30.09 81.4 0. 18.6 0.0 ; 0.69 0.04 0.75 0.55 P t . Sk. Lucerne 2.0 0.36 0.0 0.07 0.0 0.46 17.95 0.0 3.69 0.0 23.23 86.3 0.0 13.7 0.0 0.55 0.04 0.58 0.45 P t . Sk. D a i r y l a n d Sk. Undetectable Lucerne Sk. Average 2.9 0.63 0.0 0.14 0.0 0.79 21.58 0.0 4.98 0.0 27.75 84.2 0.0 15.7 0.0 0.96 0.04 0.69 0.54 Values (exclud i n g l a s t two formulas) 1. F i g u r e s are based on t o t a l t o c o pherol a n a l y s i s r e s u l t s . 2. F i g u r e s are based on T.L.C. r e s u l t s . 3. I.U. v i t a m i n E = mg a-tocopherol x l . 4 9 p l u s mg y-tocopherol * 0.20. 4. PUFA f i g u r e s are estimated according to Fomon (191). 29 seasonal v a r i a t i o n i n tocopherol content of cow's milk. (156,157). The average t o t a l tocopherol and a-tocopherol contents.were 27.75 and 21.58 jig per g of milk l i p i d . These values are i n accord with previously published results when seasonal v a r i a t i o n i s considered (144,155). Fomon (191) reported that the average infant consumed 750 and 1000 ml of formula at ages one and s i x months respectively. He further stated that d a i l y a-tocopherol intakes of 0.34 and 0.45 mg would be necessary at these ages. Vitamin E deficiency i s most l i k e l y to occur during the f i r s t three months of l i f e . Therefore vitamin E intake at t h i s age must be considered. Extrapolation of Fomon's figures indicates that the three month-old infant consumes 875 ml of formula d a i l y and w i l l require 0.40 mg of a-tocopherol. The 1968 edit i o n of the Recommended Daily Allowances of the National Academy of Sciences (U.S.) suggested an intake of 5 I.U. of vitamin E for infants up to one year of age (192). No d a i l y allowance for vitamin E has ever been cit e d i n the Canadian Dietary Standards (193). Comparison of the calculated r a t i o s of a-tocopherol-to-PUFA with the c r i t i c a l r a t i o of 0.4 (149) may also be used as a means for assessing the tocopherol status of infant formulas. On the basis of the above c r i t e r i a and the values shown i n Table 3 and 4, the vitamin E status of fresh cow's milk samples may be evaluated. A l l of the samples displayed adequate a-tocopherol-to-PUFA r a t i o s . At the three levels of formula intake, only whole cow's milk formulas s a t i s f i e d Fomon's standards of vitamin E adequacy (191). None of the milks contained s u f f i c i e n t vitamin E to meet the requirements of the National Academy of Sciences (U.S.) (192). In f a c t , even at the 1000 ml l e v e l of intake, a l l formulas s t i l l contained less than 30 per cent of the recommended intake. TABLE 4 Vi t a m i n E Content In Amounts of Fresh Cow's M i l k Normally Ingested by I n f a n t s a t One, Three and S i x Months of Age FORMULA V I T A M I N E C O N T E N T In 750 ml 1 In 875 ml In 1000 ml mg Tocopherols I.U. % of mg Tocopherols I.U. % of mg Tocopherols I.U. % of a T o t a l V i t a m i n E R.D.A. a T o t a l V i t a m i n E R.D.A. a T o t a l V i t a m i n E R.D.A. D a i r y l a n d Whole 0.67 0.89 1.03 20.6 0.78 1.03 1.20 24.0 0.89 1.18 1.37 27.4 Lucerne Whole 0.61 0.71 0.93 18.6 0.71 0.82 1.08 21.6 0.81 0.93 1.23 24.8 D a i r y l a n d P t . Sk. 0.33 0.45 0.51 10.2 0.39 0.53 0.60 12.0 0.44 0.60 0.69 13.8 Lucerne P t . Sk. 0.27 0.35 0.41 8.2 0.31 0.41 0.48 9.6 0.36 0.46 0.55 ' 11.0 Average Values 0.47 0.60 0.72 14.4 0.55 0.70 0.84 16.8 0.63 0.79 0.96 19.3 1. % of R.D.A. based on the Recommended D a i l y Allowance of 5 I.U. of v i t a m i n E as set by the N a t i o n a l Academy of Sciences (U.S.) (192). O 31 No f a t could be obtained from the f u l l y skimmed samples. Because of t h i s , and since vitamin E i s f a t - s o l u b l e , i t seems very probable that these samples would contain only very l i m i t e d l e v e l s of tocopherol. The s i g n i f i c a n c e of the vitamin E contents of the fresh milk samples i n r e l a t i o n to hemolytic anemia of ea r l y infancy w i l l be discussed l a t e r . 2. Cow's Milk Formulas F i f t e e n formulas based on cow's milk were chemically analyzed for tocopherols. Results of the analyses are presented i n Table 5. The t o t a l tocopherol content of the formulas ranged from 0.03 to 7.25 mg per l i t r e . The average t o t a l tocopherol content was 0.82 mg per l i t r e . Probana i s f o r t i f i e d with ten I.U. of vitamin E and consequently contained a very high l e v e l of a-tocopherol. No other formula appeared to be supple-mented with tocopherol. When Probana was excluded, the range and average t o t a l tocopherol content were 0.03 to 0.77 and 0.36 mg per l i t r e r e s p e c t i v e l y . For the f i f t e e n formulas, a range of 9.15 to 363.0 jag t o t a l tocopherols per g of milk l i p i d was found. The average t o t a l tocopherol content was 38.84 ug per g of milk l i p i d . Exclusion of Probana indi c a t e d a range and average t o t a l tocopherol content of 9.15 to 23.23 and 15.69 iig per g of milk l i p i d . The presence of v a r i a b l e tocopherol contents (with the exception of Probana) suggests that the products were canned at d i f f e r e n t times of the year or that some of the tocopherol was destroyed during processing or storage. Tocopherol destruction under these conditions has been previously reported (149,192). The a-tocopherol content ranged from 0.02 to 7.22 mg and 0.02 to 0.70 mg per l i t r e with and without the i n c l u s i o n of Probana r e s p e c t i v e l y . The average a-tocopherol contents i n these two cases were 0.77 and 0.31 mg N3 TABLE 5 Vitamin E and Polyunsaturated Fatty Acid Contents of Cow's Milk Formulas Formula^ TOCOPHEROLS I.U.4 PUFA5 T o t a l a-ToocoDherol ^ Fat mg/litre Pg/g Fat % Of T o t a l3 Vitamin E / l i t r e (g/g Fat) Tocopherols /PUFA /PUFA. Ratio 2 a 8 2 2 Y 6 2 T o t a l 3 2 a 6 2 2 Y 62 Total 1 a B Y 6. Ratio' (mg/g) . (mg/g) Probana 2.0 7.22 0.0 0.0 0.0 7.25 361.0 0.0' 0.0 0.0 363.0 100.0 0.0 0.0 0.0 10; 76 0.04 9.07 9.02 P a c i f i c WEM 3.9 0.70 0.0 0.05 0.0 0.77 17.87 0.0 1.35 0.0 19.76 93.6 0.0 6.4 0.0 1.05 0.04 0.49 0.45 F. Wife WEM + CHO, v i t a -mins 3.2 0.52 0.0 0.06 0.0 0.64 16.16 0.0 1.98 0.0 20.13 89.1 0.0 10.9 0.0 0.78 0.04 0.50 0.40 F. Wife WEM 4.0 0.42 0.0 0.05 0.0 0.49 10.56 .0. 1.32 0.0 12.14 88.9 0.0 11.1 0.0 0.64 0.04 0.30 0.26 P a c i f i c PSEM 2.0 0.44 0.0 0.0 0.0 0.46 22.18 0.0 0.0 0.0 23.23 100.0 0.0 0.0 0.0 0.67 0.04 0.58 0.55 Carnation WEM 3.9 0.29 0.0 0.07 0,0 0.38 7.31 0.0 1.90 0,0 9.75 83.7 0.0 16.3 0.0 0.44 0.04 0.24 0.18 F. Wife PSEM 2.0 0.34 0.0 0.02 0.0 0.38 16.89 0.0 1.05 0.0 19.01 94.2 0.0 5.9 0.0 0.51 0.04 0.48 0.42 F. Wife PSEM + CHO, v i t a -mins 2.0 0.26 0.0 0.10 0.0 0.37 13.19 0.0 4.75 0.0 18.48 73.5 0.0 26.5 0.0 0.41 \' 0.04 0.46 0.33 Klim 3.3 0.33 0.0 0.0 0.0 0.35 9.92 0.0 0.0 0.0 10.56 100.0 0.0 0.0 0.0 0.49 0.04 0.26 • 0.25 (Table 5 continued ) (Table 5 continued) (' Formula "/ TOCOPHEROLS I.U.4 PUFA5 T o t a l a-Tocopherol Fat m g / l i t r e ug/g Fat % Of T o t a l 3 Vitamin E / l i t r e (g/g Fat) Tocopherols /PUFA /PUFA Ratio a 2 3 2 Y 2 62 Total 1 a 2 8 2 Y 2 o 2 Total 1 a Y <5 Ratio (mg/g) (mg/g) Lactogen 3.2 0.32 0.0 0.0 0.0 0.33 9.90 0.0 0.0 0.0 10.23 100.0 0.0 0.0 0.0 0.48 0.04 0.25 0.25 Pelargon 2.3 0.24 0.0 0.0 0.0 0.26 10.56 0.0 0.0 0.0 11.48 100.0 0.0 0.0 0.0 0.36 0.04 0.29 0.26 Carnation PSEM 2.0 0.20 0.0 0.04 0.0 0.26 10.03 0.0 2.11 0.0 13.12 82.6 0.0 17.4 0.0 0.31 0.04 0.33 0.25 F. Wife Cone. 1.0 0.18 0.0 0.0 0.0 0.20 17.95 0.0 0.0 0.0 20.06 100.0 0.0 0.0 0.0 0.26 0.04 0.48 0.43 SM Dryco 1.5 0.11 0.0 0.0 0.0 0.14 7.04 0.0 0.0 0.0 9.15 100.0 0.0 0.0 0.0 0.16 0.04 0.23 0.18 P a c i f i c NFMS 0.14 0.02 0.0 0.0 0.0 0.03 15.09 0.0 0.0 0.0 22.57 100.0 0.0 0.0 0.0 0.03 0.04 0.56 0.38 Average 0.77 0.0 0.03 0.0 0.82 36.38 0.0 0.96 0.0 38.84 93.7 0.0 6.3 0.0 1.16 0.04 0.97 0.91 Values Average Values (Exclud- 0.31 0.0 0.03 0.0 0.36 13.19 0.0 1.03 0.0 15.69 9326 o.o 6.75 0.0 0.47 0.04 0.39 0.33 ing Pro-bana) 1. WEM, PSEM and NFMS represent whole evaporated milk, p a r t l y skimmed evaporated milk and non-fat milk s o l i d s r e s p e c t i v e l y . 2. Based on t o t a l tocopherol a n a l y s i s . 3. Based on T.L.C. a n a l y s i s . 4. I.U. vitamin E = mg a-tocopherol x 1.49 plus mg y t o c o p h e r o l x 0.20. 5. PUFA content based on Fomon's figures (191). 6. F o r t i f i e d with 10 I.U. vitamin E per quart. 34 per l i t r e . When expressed as jag per g of milk l i p i d , the average a-tocopherol content for a l l f i f t e e n formulas was 36.38 (range 7.04 to 361.0). Without Probana, the average a-tocopherol decreased to 13.19 jag per g of l i p i d . The range was 7.04 to 22.18 jag of a-tocopherol per g of l i p i d . The amount of vitamin E present as a-tocopherol averaged 93.7 per cent. Gamma-tocopherol averaged 6.3 per cent of the t o t a l tocopherols. Its range was 0.0 to 26.5 per cent. No other forms of tocopherol were found. Tables 5 and 6 serve to i l l u s t r a t e the tocopherol status of the f i f t e e n cow's milk formulas. With the exception of Probana, a l l of the formulas contained less than 22 per cent of the Recommended Daily Allowance (U.S.) for vitamin E (192). Comparison with Fomon's standards (191) indicated that only Probana, P a c i f i c WEM and F. Wife WEM with added CHO and vitamins contained adequate vitamin E at the 750, 875, and 1000 ml levels of formula consumption. In fact, some formulas were quite inadequate i n vitamin E when judged by Fomon's standards (191). The a-tocopherol-to-PUFA ratios for the cow's milk formulas approximated the t o t a l tocopherol-to-PUFA r a t i o s . Of the f i f t e e n formulas, s i x had a-tocopherol-to-PUFA ratios greater than the c r i t i c a l r a t i o of 0.4 (149). The average a-tocopherol-to-PUFA r a t i o for a l l formulas was 0.91. However, when Probana was excluded, the r a t i o decreased to 0.33. The chemical analyses of fresh cow's milk and cow's milk products indicated a considerable v a r i a t i o n i n tocopherol content. In many cases, the tocopherol l e v e l s , as w e l l as the a-tocopherol-to-PUFA r a t i o s were less than optimum. Similar observations have been made by other authors (141,144). Such facts would explain the variable occurrence of hemolytic anemia i n infants maintained on fresh cow's milk or cow's milk products. TABLE 6 V i t a m i n E Content i n Amounts of Cow's M i l k Formulas Normally Ingested by Infants at One, Three and S i x Months of Age FORMULA V I T A M I N E C O N T E N T In 750 m l 1 In 875 ml In 1000 ml mg Tocopherols I.U. % of mg Tocopherols I.U. % of mg Tocopherols I.U. % of a T o t a l V i t a m i n E R.D.A. a T o t a l Vitamin E R.D.A. a T o t a l V itamin E R.D.A. Probana 5.42 5.44 8.07 161.4 6.32 6.35 9.42 188.4 7.22 • 7.25 10.76 215.2 ' P a c i f i c WEM 0.52 0.58 0.78 15.6 0.61 0.67 0.91 18.2 0.70 0.77 1.05 21.0 F. Wife WEM + 0.39 0.48 0.59 11.8 0.45 0.56 0.68 13.6 0.52 0.64 0.78 15.6 CHO, v i t -amins F. Wife WEM 0.32 0.36 0.48 9.6 0.37 0.43 , 0.56 11.2 0.42 0.49 0.64 12.8 P a c i f i c PSEM 0.33 0.35 0.50 10.0 0.39 0.41 0.58 11.6 0.44 0.46 0.67 13.4 F. Wife PSEM 0.25 0.29 0.38 7.6 0.30 0.33 0.44 8.8 0.34 0.38 0.51 10.2 Carnation WEM 0.21 0.29 0.33 6.6 0.25 0.33 0.39 7.8 0.29 0.38 0.44 8.8 F. Wife PSEM + CHO, v i t - 0.20 0.28 0.31 '6.2 0.23 0.32- • 0.36- 7.2 • '0.26 0.37 0.41 8.2 amins (Table 6 continued ) (Table 6 continued) FORMULA V T T A M I N . . E C O N T E N T In 750 m l 1 In 875 ml In 1000 ml mg Toco pherols I.U. % of mg Tocopherols I.U. % of mg Tocopherols I.U. % of a T o t a l Vitamin E R.D.A. a . T o t a l Vitamin E R.D.A. a T o t a l Vitamin E R.D.A. Klim 0.25 0.26 0.36 7.2 0.29 0.30 0.43 8.6 0.33 0.35 • 0.49 9.8 Lactogen 0.24 0.25 0.36 7.2 0.28 0.29 0.42 8.4 0.32 0,33 0.48 9.6 Pelargon 0.18 0.20 0.27 5.4 0.21 0.23 0.31 6.2 0.24 0.26 0.36 7.2 Carnation PSEM 0.15 0.20 0.23 4.6 0.18 0.23 0.27 5.4 0.20 0.26 0.31 6.2 F. Wife Cone. 0.13 0.15 0.20 4.0 0.16 0.18 0.23 4.6 0.18 0.26 0.26 5.2 SM Dryco 0.08 0.10 0.12 2.4 0.16 0.12 0.14 2.8 0.11 0.14 0.16 3.2 P a c i f i c NFMS 0.02 0.02 0.02 0.4 0.02 0.03 0.03 0.6 0.02 0.03 0.03 0.6 Average Value 0.58 0.62 0.87 17.33 0.68 0.72 1.01 20.22 0.77 0.82 1.16 23.13 Average Value 0.23 0.27 0.35 6.53 0.28 0.32 0.41 8.21 0.31 0.36 0.47 9.41 (Excluding Probana) 1. % of R.D.A. based on the Recommended Dai l y Allowance of 5 I.U. of vitamin E as set by the National Academy of Sciences (U.S.) (192). CO Thus, vitamin E d e f i c i e n c y may or may not be present i n infants maintained on these types of formulas (6,75,100,194). Premature i n f a n t s , who are born with the most l i m i t e d stores of tocopherol (195) are perhaps most susceptible to the vitamin E d e f i c i e n c y . Both Fomon (191) and the Food and N u t r i t i o n Board of the National Academy of Sciences (U.S.) (192) have reported that formulas based on cow's milk may not contain s u f f i c e i n t vitamin E for neonates. Thus, ample evidence i s a v a i l a b l e to warrant tocopherol supplement-t a t i o n of formulas based on cow's milk. This supplementation i s e s p e c i a l l y important for low-fat formulas. R e a l i z a t i o n of t h i s recommendation would probably minimize the r i s k of vitamin E de f i c i e n c y i n inf a n t s maintained on such formulas. 3. Proprietary Formulas Data from the chemical analyses of f i f t e e n synthetic formulas are presented i n Table 7. Three formulas, Portagen, Neo-Mull Soy and CHO-Free Formula have each been supplemented with 10 I.U. of vitamin E. A notable v a r i a t i o n i n tocopherol content among the various simulated preparations was seen. In comparison with fresh cow's milk and cow's milk products, the synthetic products contained considerably more vitamin E and PUFA. Va r i a b l e amounts of a-, y~» a n c * 6-tocopherol were found i n these proprietary formulas. In general, a-tocopherol accounted for much le s s of the t o t a l tocopherol content (average 50.43 per cent) than i n the fresh cow's milk or cow's milk formulas. No g-tocopherol was detected. The amount of t o t a l tocopherols per l i t r e of the simulated pre-parations ranged from 0.42 to 19.97 mg and averaged 9.81 mg. When the three f o r t i f i e d formulas were excluded, the t o t a l tocopherol content TABLE 7 Vi t a m i n E and Po l y u n s a t u r a t e d F a t t y A c i d contents of P r o p r i e t a r y Formulas Formula % TOCOPHEROLS "3 T o t a l Toc- a-To-r e t c m g / l i t r e ug/g Fat < i Of T o t a l 2 LU J Vila PUFA-(g /g) Fat) -oph e r o l /PUFA R a t i o (mg/g) -coph - e r o l /PUFA R a t i o (mg/g) B2 Y 2 62 T o t a l 1 2 a B2 2 Y 62 T o t a l 1 a B Y 6 -min /E litre Soyalac 2.9 4.41 0.0 12.35 2.89 19.75 152.10 0.0 426.0 99.62 573.0 22.4 0.0 62.8 14.7 9.12 0.5C 1.15 0.30 Olac 2.7 2.87 0.0 11.94 0.0 15.13 106.62 0.0 440.73 0.0 553.0 19.4 0.0 80.5 0.0 6.66 0.46 1.23 0.23 Lof e n a l a c 2.7 2.59 0.0 7.69 3.46 14.57 96.13 0.0 285.20 127.74 540.21 18.8 0.0 56.0 25.1 5.49 0.44 1.23 0.29 CHO-Free Formula 4 3.5 10.82 0.0 1.28 0.0 12.37 309.42 0.0 36.90 0.0 353.31 89.4 0.0 10.5 0.0 16.38 0.5C 0.71 0.62 SMA 3.6 4.67 0.0 6.71 0.63 12.25 126.63 0.0 186.51 0.0 342.10 38.3 0.0 56.4 0.0 8.53 0.23 1.47 0.55 Nutramigen 2.6 2.18 0.0 7.47 0.40 10.71 83.80 0.0 281.62 15.0 405.33 22.0 0.0 74.0 3.9 4.73 0.51 0.79 0.16 Prosobee 3.4 4.04 0.0 3.89 1.95 9.92 118.92 0.0 116.47 57.66 405.34 40.5 0.0 39.7 19.6 6.85 0.43 0.94 0.28 En f a l a c 3.7 3.57 0.0 5.03 0.0 9.27 96.37 0.0 135.84 0.0 241.36 41.4 0.0 58.5 0.0 6.33 0.26 0.93 0.37 4,5 Portagen 3.2 8.32 0.0 0.0 0.0 8.70 260.0 0.0 0.0 0.0 271.84 100.0 0.0 0.0 0.0 12.40 0.03 8.76 8.38 Neo-Mull Soy 4 3.5 7.89 0.0 0.33 0.0 8.30 225.36 0.0 0.0 9.35 237.51 96.0 0.0 4.0 0.0 1235 0.48 0.50 0.48 S i m i l a c + Iron 3.4 3.47 0.0 3.48 0.0 7.19 102.36 0.0 101.47 0.0 212.34 50.2 0.0 49.7 0.0 5.87 0.22 0.98 0.47 S i m i l a c 3.4 3.25 0.0 3.53 0.0 7.04 95.51 0.0 103.82 0.0 207.66 47.9 0.0 .52.1 0.0 5.55 0.31 0.66 0.31 Formula 4 3.5 1.38 0.0 5.32 0.0 6.90 39.46 0.0 152.0 0.0 197.71 20.5 0.0 79.4 0.0 3.12 0.20 0.96 0.19 I s o m i l 3.5 4.00 0.0 0.45 0.0 4.55 114.41 0.0 14.43 0.0 130.36 88.8 0.0 11.1 0.0 6.04 0.29 0.43 0.38 Lonalac^ 3.4 0.24 0.0 0.16 0.0 0.42 7.14 0.0 4.65 0.0 12.42 60.8 0.0 39.1 0.0 0.39 0.01 1.24 0.70 Average Value 3.3 4.25 0.0 4.64 0.62 9.81 129.90 0.0 152.34 20.62 312.23 50.43 0.0 44.92 4.22 7.31 0.32 1.47 0.91 Average Value CExcludins 3.2 3.06 0.0 5.67 0.78 9.83 94.95 0.0 187.40 23.75 318.40 39.25 0.0 51.69 5.28 5.71 0.32 1.00 0.35 y Neo -Mul 1. Soy CH0-F ree Fr jrmula and Poi ctagi (Table 7 continued) ' 1. Based on t o t a l tocopherols a n a l y s i s . 2. Based on T.L.C. r e s u l t s . 3. I.U. v i t a m i n E = mg a-tocopherol x 1.49 plu s mg y-tocopherol x 0.20 plu s mg 6-tocopherol x 0.03. 4. F o r t i f i e d w i t h 10 I.U. of v i t a m i n E. 5. PUFA content obtained from Mead Johnson Co. Ltd.. 6. PUFA content estimated according to Fomon's f i g u r e s (191). w v£> AO averaged s l i g h t l y higher (9.38 mg per l i t r e of formula). The range of t o t a l tocopherol content did not change. Further analyses r e s u l t s , as shown i n Table 7, indicate an average and t o t a l tocopherols range of 312.32 and 12.42 to 573.0 ug per g of l i p i d respectively. Exclusion of the three vitamin E-supplemented products gave an i d e n t i c a l range of t o t a l tocopherol content i n a s l i g h t l y higer average value. The a-tocopherol content of the f i f t e e n synthetic formulas varied from 0.24 to 10.82 mg per l i t r e (7.14 to 309.42 yg per g of l i p i d ) . An average a-tocopherol content of 4.25 mg per l i t r e of formula (128.90 yg per g of l i p i d ) was found. When the three vitamin E - f o r t i f i e d synthetic formulas were not included, the a-tocopherol content ranged from 0.42 to 4.67 mg per l i t r e of formula or expressed otherwise 7.14 to 152.10 yg per g of l i p i d . Average a-tocopherol contents i n mg per l i t r e and yg per g of l i p i d decreased to 3.06 and 94.95 respectively. Herting and Drury (144) and others (141,149) have reported s i m i l a r trends i n the vitamin E content of simulated formulas. In considering the vitamin E requirement for infants maintained on proprietary formulas, Fomon (191) suggested d a i l y a-tocopherol intakes of 1.3 and 1,8 mg at ages one and s i x months respectively. Extrapolation of t h i s data would s i g n i f y a d a i l y requirement of 1.5 mg a-tocopherol for the three month-old infant. As shown i n Table 8, only Lonalac and Formula 4 f a i l e d to meet this standard at the various levels of formula consumption. Comparison with the Recommended Daily Allowance of 5 I.U. for vitamin E (192) indicated that at the 750 ml l e v e l of formula intake eight preparations (Lofenalac, Nutramigen, Enfalac, Similac, Similac with Iron, Formula 4, Isomil and Lonalac) contained i n s u f f i c i e n t vitamin E. On the TABLE 8 Vi t a m i n E Content i n Amounts of P r o p r i e t a r y Formulas Normally Ingested at One, Three and S i x Months of Age FORMULA V I T A M I N E C 0 N T E N T In 750 m l 1 In 875 ml In 1000 ml mg Tocopherol I.U. % of mg Tocopherol I.U. % of mg Tocopherol I.U. % of a T o t a l V i t a m i n E R.D.A. a T o t a l V i t a m i n E R.D.A. a T o t a l V i t a m i n . E . .R.D.A. Soyalac 3.31 14.97 6.85 137.0 3.86 17.46 7.99 159.8 4.41 19.96 9.13 182.6 Olac 2.15 11.35 5.00 100.0 2.51 13.24 5.83 116.6 2.87 15.13 6.66 133.2 L o f e n a l a c 1.94 10.93 4.12 8.24 2.26 12.75 4.80 96.0 2.59 14.57 5.49 109.8 CHO-Free Formula 8.12 9.27 12.28 245.6 9.47 10,82 14.33 286.6 10.82 12.37 16.38 327.6 SMA 3.50 9.19 6.26 125.2 4.08 10.71 7.31 146.2 4.67 12.25 8.35 167.0 Nutramiger 1.63 8.03 3.55 71.0 1.90 9.73 4.14 82.8 2.18 10.71 4.73 94.6 Prosobee 3.03 7.44 5.14 102.8 3.54 8.68 6.00 120.0 4.04 9.92 6.85 137.0 E n f a l a c 2.68 6.95 4.74 94.8 3.12 8.11 5.53 . 110,6 3,57 9.27 6.33 126.6 Portagen 6,24 6.53 9.30 186.0 7.28 7.61 10.85 217.0 8.32 8.70 12.40 248.0 Neo-Mull Soy 5.92 6.22 9.32 186.4 6.90 7.27 10.88 217.6 7.89 8.30 12.43 248.6 S i m i l a c + I r o n 2.61 5.39 4.40 88.0 3.04 6.29 5.14 102.8 3.47 7.19 5.87 117.4 (Table 8 continued ) (Table 8 continued) FORMULA V I T / t M I N E C 0 N T E N T In 750 m l 1 In 875 ml In 1000 ml mg Tocopherol I.U. % of mg Tocopherol I.U. % of mg Tocopherol I.U. % of a T o t a l V itamin E R.D.A. a T o t a l V i t a m i n E R.D.A. a T o t a l V i t a m i n E R.D.A. S i m i l a c 2.44 5.28 4.12 82.4 2.85 6.16 4.86 97.2 3.25 7.04 5.55 111.0 Formula 4 1.03 5.17 2.34 46.8 1.05 6.03 2.73 54.6 1.38 6.90 3.12 62.4 I s o m i l 3.00 3.41 4.53 90.6 3.50 3.98 5.29 105.8 4.00 4.55 6.04 120.8 Lonalac 0.18 0.32 0.29 5.8 0.21 0.37 0.34 6.8 0.24 0.42 0.39 7.8 Average Value 3.19 7.36 5.48 109.7 3.70 8.59 6.40 128.03 4.25 9.81 7.31 146.3 Average Value (Exclud-i n g Neo-M u l l Soy, CHO-Free Formula and Por-tegen) 2.29 7.37 4.28 85.57 2.66 8.60 5.00 99.93 ) \ \ 3.06 9.83 5.71 114.18 1. % of R.D.A. i s based on the Recommended D a i l y Allowance of 5 I.U. of v i t a m i n E as set by the N a t i o n a l Academy of Sciences (U.S.) (192). v average, the f i f t e e n formulas contained 109.7 per cent of the Recommended Allowance for vitamin E. When th.e three f o r t i f i e d formulas were excluded, the average value decreased to 85.57 per cent. Lofenalac had inadequate vitamin E at the 875 ml l e v e l of formula consumption. With and without inclu s i o n of the supplemented formulas, 128.03 and 99.93 per cent respective of the Recommended Allowance for vitamin E was contained. Only two formulas Formula 4 and Lonalac - f a i l e d to meet th i s c r i t e r i o n of adequacy when the 1000 ml l e v e l of intake was considered. More than 100 per cent of the Recommended Allowance was contained i n the simulated formulas whether or not the f o r t i f i e d preparations were included i n the analysis. The-a-tocopherol-to-PUFA r a t i o s of nine formulas (Table 7) f a i l e d to exceed the c r i t i c a l r a t i o s of 0.4. A notable discrepancy between the total-tocopherol-to-PUFA and a-tocopherol-to-PUFA r a t i o s were apparent for the synthetic formulas. This was not seen i n the fresh cow's milk and cow's milk formulas. However, si m i l a r decreases i n the average t o t a l tocopherols-to-PUFA and a-tocopherol-to-PUFA ratios were noted when the supplemented synthetic .formulas were excluded. Several workers (4,7,125,160) have shown that simulated milks containing 2.8, 3.3, 4.4 or even 11.5 mg a-tocopherol per quart did not prevent vitamin E deficiency i n young infants. In view of these pertinent findings, and the frequent occurrence of low tocopherol levels i n some of the proprietary formulas, i t i s recom-mended that the necessary vitamin E supplementation of simulated infant formulas be undertaken. The presence of iron i n certain formulas may enhance oxidative destruction of tocopherol (5-7). Therefore, vitamin E f o r t i f i c a t i o n of such formulas i s especially important. 44 A summary of the vitamin E status of a l l the formulas i s presented i n Table 9. In addition to four formulas which were supplemented with 10 I.U. of vitamin E only one unsupplemented commercial proprietary formula (SMA) contained an adequate l e v e l of vitamin E. A variety of nineteen formulas, including fresh cow's milk, cow's milk products, and simulated formulas contained some, but not enough tocopherol to be c l a s s i f i e d as adequate. Ten formulas, of which only one was a proprietary preparation, did not contain s u f f i c i e n t vitamin E to s a t i s f y any c r i t e r i a of tocopherol adequacy. On the basis of these f a c t s , i t seems probable that low fat formulas based on cow's milk would be most l i k e l y to promote vitamin E deficiency i n neonates. 4. Hospital Survey - Infant Feeding Practices i n Canada In August 1970, a l e t t e r was circulated to some 850 Canadian hospitals. Information regarding past, present and anticipated trends i n infant feeding practices was requested. The o v e r - a l l response to the survey questionnaire was very good. As seen i n Table 10, 55.4 per cent of the hospitals (range 40-63 per cent for the i n d i v i d u a l provinces) supplied pertinent data. In Canada, two hospital-feeding systems (Table 11) predominate. In the formula room system, a l l of the formulas prescribed by the doctors are prepared within the h o s p i t a l . In the ready-to-feed systems, the formulas are prepared commercially and then delivered to the h o s p i t a l . A single system ( i . e . Mead Johnson Beniflex, Ross Laboratories or John Wyeth system) i s usually employed i n any given h o s p i t a l . Some hospitals i n Ontario used the National Baby Formula Service. This enterprise can usually supply hospitals with a l l commonly used brands of formulas. 45 TABLE 9 Vitamin E Status of Infant Formulas Vitamin E Status Formulas Adequate 1 (5 formulas) 4 4 4 Probana , CHO-Free Formula , Meo-Mull Soy, SMA, 4 Portagen 2 Partly Adequate (19 formulas) Dairyland whole fresh cow's milk, Lucerne whole fresh cow's milk, P a c i f i c WEM, F. Wife WEM + CHO and vitamins, Dairyland PSEM, Lucerne PSEM, F. Wife PSEM, P a c i f i c PSEM, F. Wife Cone. Sk. milk, Soyalac, Olac, Prosobee, Enfalac, Similac, Similac with i r o n , Isomil, Lonalac, Nutramigen, Lofenalac. 3 Inadequate (10 formulas) Carnation WEM, F. Wife WEM, F. Wife PSEM + CHO and vitamins, Klim, Lactogen, Pelargon, Carnation PSEM, Dryco, P a c i f i c NFSM, Formula 4. 1. A l l of the c r i t e r i a for vitamin E adequacy were s a t i s f i e d for ages 1, 3, and 6 months. 2. Some of the c r i t e r i a for vitamin E adequacy were s a t i s f i e d for ages 1, 3, and 6 months. 3. None of the c r i t e r i a for vitamin E adequacy were s a t i s f i e d for ages 1, 3, and 6 months. 4. Supplemented with 10 I.U. of vitamin E. 46 TABLE 10 Hospital Response to Survey Questionnaire Province or Territory H o s p i t a l s 1 Total Response Small Medium Large /Province No. % 2 No. % 2 No. % 2 No. % 2 B r i t i s h Columbia 30 60 16 70 9 64 55 63 Alberta 50 62 6 66 5 63 61 62 Manitoba 34 62 5 50 2 50 41 59 Saskatchewan 57 50 7 78 4 67 68 52 Ontario 49 61 30 53 34 61 113 57 Quebec 17 52 20 44 24 ' 56 61 50 New Brunswick 12 60 7 54 2 50 21 57 Nova Scotia 16 73 9 50 1 50 26 62 Newfoundland 4 33 7 47 1 33 12 40 Prince Edward Island 3 60 1 33 - - 4 50 Yukon; N.W.T. 3 60 1 50 - - 4 57 Total According to Size 275 57.5 109 53.5 82 54.9 Total 466 55.4 1. Size based on the number of l i v e b i r t h s i n 1969 according to the Canadian Hospital. Directory for that year: Small = 0 - 200 b i r t h s ; medium = 200 - 700 b i r t h s ; large = > 700 b i r t h s . 2. % response = no. of re p l i e s received/no. of questionnaires mailed. 47 The ready-to-feed systems were introduced i n Canada i n the 1960's. Since that time, they have become very popular (Table 11). This trend has also been noted i n the United States (196). Advantages of such systems include convenience, improved sanitation, less labour costs, and better use of ho s p i t a l space. Re l a t i v e l y few of the medium or large hospitals currently employ the formula room system. In f a c t , of those that do, many expressed a desire to switch to the ready-to-feed systems i n the near future. For many of the small h o s p i t a l s , such a switch would not be economically fea s i b l e . I t seems reasonable to anticipate that, with the exception of very small hospitals, the ready-to-feed systems w i l l be used by the majority of- Canadian hospitals i n the future. A t o t a l of 192 hospitals commented on the practice of breast-feeding. The range i n the frequency of breast feeding i n Canadian hospitals i s shown i n Table 12. As can be seen, the incidence of breast-feeding varied greatly. Sixteen hospitals reported that the frequency of breast-feeding had increased i n recent years. Eleven hospitals reported a decline. Previous studies (6-7) have indicated that the breast-fed infant would not be vitamin E-deficient. By combining the hospital survey data with the chemical analysis data, i t i s possible to estimate the tocopherol status of hospital-fed infants. For t h i s purpose only the c r i t e r i a for vitamin E adequacy at one month of age w i l l be used. SMA served as the standard formula i n approximately one-third of those hospitals that responded to the questionnaire. When compared to the three c r i t e r i a of tocopherol adequacy (149,191,192), t h i s formula appears to be excellent (Tables 7 and 8). I t i s not l i k e l y that infants maintained oo si-TABLE 11 Formulas Currently Used i n Canadian Hospitals Formulas 1 Hospitals Employing Formula Room System Hospitals Employing Ready-to-Feed Systems Total Small No Medium Large No Small Medium Large Small Medium Large Total No No No No No % No " % No ' % No % SMA mainly 18 4 • 1 53 30 25 71 26 34 31 26 32 131 28 Enfalac mainly 16 3 ~ 1 19 22 15 35 13 25 23 16 20 76 16' Similac mainly 19 6 2 29 22 13 48 17 28 26 15 18 91 20 W.E.M. mainly 15 2 - - - - 15 5 2 2 - - 17 4 SMA + Enfalac 11 1 3 6 4 - 17 6 5 5 3 4 25 5 SMA + Similac 9 - 2 5 3 - 14 5 3 3 2 2 19 4 SMA + W.E.M. 8 1 1 - - - 8 3 1 1 1 1 10 2 Enfalac + Similac 5 - 1 5 - - 10 4 - - 1 1 11 2 Enfalac + W.E.M. 8 - 1 — - - 8 3 - - 1 1 9 2 Similac + W.E.M. .. 9 - 2 - • - 9 3 - - 2 2 11 2 Any combination of the above and/or other formulas 31 10 9 9 1 62 40 15 11 9 , 15 , 18 , 66 , 15 1. Most hospitals reported that special-purpose formulas such as described i n Table 2 were kept 2. Hospitals employing the National Baby Formula Service. TABLE 12 Frequency of Breast-Feeding HOSPITALS F R E Q U E N C Y No. % Small 104 11--75 . Medium 61 16--53 Large 27 5--49 Total 192 10--59 50 on SMA would manifest vitamin E d e f i c i e n c y . Similac was used as the primary formula i n some 20 per cent of the h o s p i t a l s . Another 20 per cent of the h o s p i t a l s employed Enfalac as t h e i r main formula. These preparations contained s u f f i c i e n t vitamin E only when judged by Fomon's c r i t e r i a (191), Similac with i r o n i s commonly fed to premature i n f a n t s . This preparation contained adequate vitamin to s a t i s f y two of the three c r i t e r i a of tocopherol adequacy. Variable vitamin E contents i n the Similac products have been reported elsewhere (5). On the basis of such f a c t s , i t i s very d i f f i c u l t to predict whether or not infants nourished with Similac or Enfalac would be vitamin E - d e f i c i e n t i n ea r l y l i f e . The r i s k would probably be greater than for in f a n t s fed SMA. Evaporated cow's milk constituted the main formula i n approximately 5 per cent of the h o s p i t a l s . As already noted, v a r i a b l e tocopherol contents, which often tend to be low, were c h a r a c t e r i s t i c of cow's milk formulas. On such grounds, i t i s very p o s s i b l e that neonates maintained on cow's milk formulas would be vitamin E - d e f i c i e n t . The special-purpose formulas (Table 2) depending on f o r t i f i c a t i o n p r a c t i c e s , may or may not promote vitamin E def i c i e n c y i n young babies. The h o s p i t a l stay of a normal i n f a n t i s usually l e s s than two weeks. Therefore, home-feeding p r a c t i c e s during e a r l y infancy are important i n assessing the p o s s i b i l i t y of tocopherol d e f i c i e n c y . The h o s p i t a l survey data ind i c a t e d that although many mothers maintained t h e i r i n f a n t s on the proprietary formula used i n the h o s p i t a l (SMA, Enfalac of S i m i l a c ) , a large number switched to some form of evaporated cow's milk. Depending on the f a t content and time of canning, i t i s possible that vitamin E def i c i e n c y could p r e v a i l i n in f a n t s nourished on these products. 51 CHAPTER V SUMMARY AND RECOMMENDATIONS Summary 1. Six fresh cow's milk formulas of variable l i p i d content had t o t a l tocopherol contents ranging from 0.46 to 1.18 mg per l i t r e (average 0.79). Alpha and y-tocopherol averaged 84.2 and 15.7 per cent of the t o t a l tocopherols. No other form of vitamin E was detectable. The a-tocopherol-to-PUFA r a t i o , a-tocopherol content, and I.U. of vitamin E l e v e l of the formulas served as three basic c r i t e r i a for judging the tocopherol status of the formulas. These parameters were compared with the set standards. No cow's milk formula s a t i s f i e d a l l three c r i t e r i a of tocopherol adequacy. 2. Fifteen cow's milk products of varying l i p i d content had an average t o t a l tocopherol content of 0.82 mg per l i t r e (range 0.03 to 7.25). One of the formulas was supplemented with vitamin E. No form of vitamin E other than a- and y-tocopherol were detectable. Alpha tocopherol averaged 93.7 of the t o t a l tocopherols. Only the supplemented formula contained s u f f i c i e n t tocopherol to s a t i s f y a l l three c r i t e r i a of vitamin E adequacy. It i s very probable that infants maintained on u n f o r t i f i e d cow's milk or cow's milk products would be susceptible to vitamin E deficiency i n early l i f e . 3. Analyses of f i f t e e n proprietary preparations showed higher levels of t o t a l tocopherols (average 9.81 mg per l i t r e ) than i n fresh cow's milk or cow's milk products. The t o t a l tocopherol content varied from 0.42 to 19.97 mg per l i t r e of formula. Alpha-tocopherol averaged only 50.43 per cent of the t o t a l tocopherols. Lesser amounts of y- and 6-tocopherol 52 Were found. The PUFA l e v e l was also considerably greater i n the simulated formulas. Five of the f i f t e e n synthetic formulas (including three which were tocopherol-supplemented) contained s u f f i c i e n t vitamin E to s a t i s f y a l l three c r i t e r i a of adequacy. Infants fed n o n - f o r t i f i e d synthetic formulas would probably r i s k vitamin E deficiency. 4. Information obtained from 466 Canadian hospitals indicated that the ready-to-feed formula systems are replacing the formula-room method. Approximately 30 per cent of the hospitals employed SMA as the i r standard preparation. This formula contains adequate vitamin E to eliminate the r i s k of a deficiency. Similac, Enfalac, and evaporated cow's milk served as standard formulas i n approximately 20, 20 and 5 per cent of the hospitals respectively. These formulas contain less adequate amounts of tocopherol than SMA and t h e i r use could possibly promote vitamin E deficiency i n young infants. No d e f i n i t e trends i n the frequency of breast-feeding were noted. For home feeding of infants, some form of evaporated cow's milk was often preferred. Recommendations 1. Where necessary, infant formulas should be s u f f i c i e n t l y f o r t i f i e d with vitamin E to eliminate the r i s k of vitamin E deficiency i n young infants. This supplementation would probably vary from 5 to 10 I.U. of vitamin E per l i t r e of reconstituted formula. 2. Infants and children suffering from prolonged fat malabsorption are known to manifest vitamin E deficiency. In such cases, water-soluble vitamin E preparations should be added to any special formulas being used. 3. Infants and children suffering from kwashiorkor often manifest a p a r t i c u l a r megaloblastic anemia. Vitamin E has frequently been shown to 53 be an e f f e c t i v e therapeutic agent. In view of t h i s , i t i s recommended that a l l skimmed cow's milk products used l o c a l l y i n Canada and sent to developing countries should be supplemented with adequate amounts of tocopherol. A. F i n a l l y , i t i s recommended that a d a i l y tocopherol allowance of 5 I.U. for young in f a n t s be included i n the Canadian Dietary Standards. 54 LITERATURE CITED 1. Moyer, W.T. 1950 Vitamin E levels i n term and premature Infants. Pediatrics 6:893. 2. Nitowsky, H.M., M. Cornblath and H.H. Gordon 1956 Studies of tocopherol deficiency in infants and children. II. Plasma tocopherol and erythrocyte hemolysis i n hydrogen peroxide. Amer. J. Dis. Child. 92:164. 3. Hashim, S.A., and R.H. Asfour 1968 Tocopherol in infants fed diets rich in polyunsaturated fatty acids. Amer. J. Clin. Nutr. 21:7. 4. Oski, F.A. and A.L. Barness 1967 Vitamin E deficiency. A previously unrecognized cause of hemolytic anemia in the premature infant. J . Pediat. 70:211. 5. . Ritchie, J.H., B.F. Mathews, V. McMasters, and M. Crossman 1968 Edema and hemolytic anemia in premature infants. New Eng. J. Med. 279:1185. 6. Nitowsky, H.M., M. Cornblath, and H.H. Gordon 1956 Studies of tocopherol deficiency in infants and children. II. Plasma tocopherol and erythrocyte hemolysis i n hydrogen peroxide. Amer. J. Dis. Child. 92:164. 7. Hassan, H., L.A. Hashim, T.B. Van I t a l l i e , and W.H. Sebrell 1966 Syndrome in premature infants associated with low plasma vitamin E levels and high polyunsaturated diet. Amer. J. Clin. Nutr. 19:147. 8. 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