Vitamin a Requirements of Animal Species

Vitamin A Requirements of Animal Species SAUL H. RUBIN

AND

ELMER DE RITTER

Nutrition Laboratories, HoQinann-La Roche Inc., NutEey, New Jerseg Page

1. Rat . . . . . . . a. Requirement of Vitamin A in Relation to: Growth. ............................ Blood Levels, Liver Storage, and Retina . . . . . . . . . . . . . 103 Longevity ............. Tooth Development. . . . . . . . . b. Summary of Vitamin A Studies.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

a. Chicken and Turkey.. . . . . . . . . . . . . . . . . . . . . . . . .

6. Cattle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . 114

10. Human Being.. . . . . . . . . a. Infant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................................................

118

. . . . . . . . . . 120

. . . . . . . . . . 122 4. Source of Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

a. Mineral Oil. . . .

...................................

125

.......................................... 126 . . . . . . . . . . . . . . . 127 8. Stress Factors..

101

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SAUL H.,, RUBIN AND ELMER DE RITTER

Paye

a. Cold., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . b. Thyroxine.. . . . . . . . . . . ........................ V. Utilization of Other Forms of 1. Vitamin A z . . . ........... 2. Neovitamin A . . .................................. 3. Other Isomers and Role in Vision.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary ......................... ............. ............ References.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

127 127

123 130 131

I. INTRODUCTION In considering the requirement of an animal for a vitamin, a prime question concerns the criteria t o be used. I n general, the minimum requirement of a species for vitamin A has been considered to be that level which gives satisfactory growth and prevents lesions or dysfunctions due to vitamin A deficiency. This is, perhaps, a somewhat arbitrary standard which does not consider other important factors influenced by vitamin A intake: tooth development, longevity, and maintenance of adequate liver stores and blood levels of vitamin A, particularly during pregnancy and lactation. The amount of vitamin A required to provide optimal conditions with regard to these latter considerations may be widely different from the minimum requirement established on the usual basis noted above. I n other words, the daily intake needed to correct or prevent a frank clinical deficiency may be quite different from the amount necessary to correct or prevent a deficiency the existence of which can be elicited only by examination with special means or by special analyses. Vitamin A requirement is also dependent on various factors affecting utilization, such as the medium or diluent in which the vitamin is given, other constituents of the diet, or disease conditions in which absorption is impaired. The efficiency of a species in converting provitamins such as p-carotene into vitamin A also influences the dietary requirement. Since the precise intracellular and perhaps enzymic function of vitamin A is not known, as in the case of some of the other vitamins, it would seem desirable to use the most sensitive criteria of vitamin A deficiency as guides in judging the daily requirement. Many of the studies of vitamin A requirement have been concerned with the problem of animal growth. The experimental approach to this problem of determining growth requirement is inherently difficult. General principles and procedures for evaluating biological assays with laboratory animals have been summarized by Coward (1938) and more recently by Guerrant (1951) and Bliss and Gyorgy (1951). In contrast to the bio-assay, where growth is measured in the sensitive and linear portion of the log dose-response curve, the determination of growth require-

VITAMIN A REQUIREMENTS O F ANIMAL SPECIES

103

ment necessitates study of the extreme portions of the curve, where the likelihood of error is considerably greater. Hegsted (1948) has pointed out that the prevailing type of error in such experiments leads to a low estimate of requirement, which is considered a more serious error than the less likely overestimation, since requirements should be set high enough t o cover all normal individual animals adequately. In the following paragraphs brief descriptions of vitamin A deficiency symptoms and data on the daily requirement of each individual species for vitamin A are presented. For the larger animals and some of the small animals only a limited number of studies have been made. Studies of rats and poultry are discussed in greater detail, since extensive data are available which provide much basic knowledge regarding relative requirements for various physiological functions and the factors influencing vitamin A or carotene utilization. 11. DAILYREQUIREMENTS OF INDIVIDUAL SPECIES 1. Rat

Since the rat has been used most extensively for nutrition studies, the subject of requirement for vitamin A and the factors related thereto are best illustrated by a discussion of rat experiments. The criteria on which requirement studies are based include many of the symptoms of vitamin A deficiency. A high degree of similarity exists in the symptoms shown by various animal species. In the rat, deprivation of vitamin A results in reduction and cessation of growth and ultimately in early death. Cornification of epithelial cells, xerophthalmia, reduction of erythrocyte count, hemoglobin and hematocrit, severe anemia, infertility and fetal resorption have all been attributed to vitamin A deficiency in the rat. Young rats born from vitamin A-deficient mothers show gross dental and eye abnormalities (Warkarny, 1945). a. Requirement of Vitamin A in Relation to Growth. Lewis et al. (1942) fed various levels of U.S.P. cod liver oil and of a vitamin A concentrate to rats receiving a purified ration low in vitamin A. The growth rates of these rats are shown in Fig. 1. At a level of 2 I.U. per day (20 I.U.per kilogram of body weight), good growth was obtained, and no gross or histological signs of vitamin A deficiency were observed. Thus ordinarily a value of 2 I.U. of vitamin A per day would be assigned as the minimum requirement of the rat. However, it is obvious that if optimal growth were considered the criterion, a level of 25 I.U. per rat per day would be necessary. Blood Levels, Liver Storage, and Retinal Concentration. These authors also determined blood levels, liver storage, and retinal concentrations of

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SAUL H. RUBIN AND E L M E R D E R I T T E R

vitamin A as shown in Table I. Maximal blood levels of about 100 I.U. per milliter of plasma were found in rats receiving 50 I.U. of vitamin A per day. As regards liver storage, the authors obtained what they considered adequate reserves in rats fed 100 I.U. of vitamin A per day. Retinal levels, on the other hand, are maintained well, even a t the low levels of vitamin A intake. It is apparent that the level of 2 I.U. of vitamin A per day, which satisfies the usual definition for minimum requirements, is not the level which provides optimal conditions for the rat.

I

I

WMkS 1

I

2

I

3

I

4

I

5

I

6

1

FIG.1. The relation of daily vitamin A intake to growth of rats (Lewis et al., 1942).

Longevity. The relation between vitamin A intake and longevity in rats has been investigated by Sherman et aL. (1945; 1949a; 1949b). Doubling of the already adequate level of 3 I.U. per gram of food resulted in longer life for both sexes (5% for males and 10% for females). At the 12 I.U. level the gain over the 3 I.U. level was 10% for males and 12 % for females. At both the 6 and 12 I.U. levels there was an even longer proportionate prolongation of the reproductive period in the females. Some longevity responses were less favorable a t the 24 I.U. level. Liver storage was slight at 3 I.U. (Caldwell et al., 1945; Sherman et al., 194913) but increased progressively as the intake was raised to 6, 12, and 24 I.U. per gram of diet. The level of 3 I.U. per gram of diet appeared to be near the minimum limit of adequacy.

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105

Paul and Paul (1946) determined the amount of vitamin A required for maximum life span, normal growth, tooth structure, and eyes. Fourweek-old rats were fed the U.S.P. XI1 vitamin A-free diet until weight loss or eye symptoms were observed. Vitamin A was then fed by syringe at four levels based on body weight. The longevity data are presented in Table 11, from which it is apparent that the highest level fed, 20 I.U. per 100 g. of body weight, provided the longest life span for the rats. TABLE I RELATIONOF VITAMINA INTAKE I N THE RAT TO BLOOD LEVEL,LIVER STORAGE AND CONCENTRATION I N THE RETINA* Vitamin A given daily for 6 weeks,

Vitamin A Animals in group

I.U.

0

13 11 31 31 16 15 21 26

1

2 10 25 50 103 1000

In blood plasma, I.U./100 ml. 0

7

14 35 69 100 112 110

In liver I.U./g.

In retina I.U./g.

0 0 0 0 3 34 113 1270

1-1 20 25 20 26 25

*Lewis et al. (19423.

-4VERAGE AGE

AT

TABLE I1 DEATHO F RATS FEDVARIOUS LEVELSO F VITAMIN A*

Group

Vitamin A per 100 g . body weight daily, U.S.P. units

No. of animals

Average age at death all animals, days

A B C D

1 2 4 20

23 19 20 20

80 2 234 18 521 k 28 649 I 30

* Paul and Paul (1946).

The growth data for these experiments are plotted in Fig. 2. The level of 1 I.U. per 100 g. of body weight was insufficient for prolonged maintenance of life or growth; moderate growth was obtained a t 2 I.U. and progressively increased growth, a t the 4 and 20 I.U. levels.

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SAUL H. RUBIN AND ELMER DE RITTER

Tooth Development. The tooth conditions of the rats after 26 weeks at the above levels of vitamin A are illustrated in Fig. 3. Paul and Paul concluded that the minimum requirement for optimal growth and longevity of the rat is about 10 I.U. per 100 g. of body weight and that the requirement for normal teeth and eyes lies in the same general range. I n the weight range studied (80 to 360 g.), this would correspond to about 8 to 36 I.U. per day. Such levels of intake would be somewhat below the 3 I.U. per gram of food, which Sherman et al. (1945) considered close to the minimum adequate level, but in the range of the

FIG.2. Body weight curves of rats receiving different amounts of vitamin A per 100 g. of body weight. The columns at the top show the number alive in each group at intervals of 100 days. A : 1 I.U.; B : 2 I.U.; C: 4 I.U.; and D:20 I.U. (Paul and Paul, 1946).

value of 25 I.U. per day a t which Lewis et al. (1942) obtained maximal growth. b. Summarg of Vitamin A Studies. A summary of the results of various workers who have reported rat requirements for vitamin A or carotene is presented in Table 111. It is obvious that even for such a well-studied animal as the rat, a wide range of vitamin A requirement is encountered, depending upon the criterion used. In general, if good growth and prevention of deficiency signs are the criteria, a range of 20 to 100 I.U. per kilogram of body weight is estimated. However, as far as blood levels, liver storage, and longevity are concerned, much higher levels are required.

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Utilization of 0-Carotene. It is interesting to note that the rat is one of the most efficient utilizers of carotene as a source of vitamin A. By definition 1 I.U. is equivalent to 0.6 microgram of @-carotene. Pure vitamin A alcohol has a theoretical equivalence of 0.3 microgram per I.U.; hence twice as much 0-carotene is required to meet the vitamin A requirement of the rat. Koehri (1948) has shown, however, th a t in the presence of adequate a-tocopherol the rat can convert 0-carotene quantitatively into c.

FIG.3. Teeth of rats receiving various levels of vitamin A for 26 weeks. Normal, translucent, orange-pigmented teeth were seen only in t h a t group receiving 20 I.U. per day per 100 g. of body weight (rat 0). Rats 1 and 2, receiving 4 I.U., had opaque, light yellow teeth, with slight black mottling in rat 2. Rats 3 and 4, receiving 2 I.U., had chalky-white teeth, with black mottling in rat 4 (Paul and Paul, 1946).

two molecules of vitamin A, so that on a weight basis the activity of 0-carotene becomes equal to that of vitamin A. Hove (1952) pointed out that Koehn had omitted yeast from the diet and found 0-carotene to have 70% t o 90% of the activity of vitamin A on a weight basis in the absence of yeast, but only 40% to 50% of the activity of vitamin A on the same basis when 8% dried yeast was incorporated into the rat diet. Yeast allowed greater response to vitamin A but diminished the growth response to 0-carotene. The effect of tocopherol as well as the influence of other factors on vitamin A requirements is discussed in a subsequent section.

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TABLE 111 VITAMIN A REQUIREMENTS OF RATSPER KILOGRAMOF BODYWEIGHT Vitamin A, Carotene, I.U. micrograms micrograms

10 30 18-22 200

3 9 3.8-4.6 12 60

25 20 250 500

7.7 6 76 152

40

1000 302 Greater than 20 120 100

38 30

40

12

15-20

40

Criterion

Reference

Cure xerophthalniis Teeth color Vaginal smears Cure xerophthalmia Maintain normal blood level Growth Prevent deficiency signs Optimum growth Maintain normal blood level Adequate liver storage

Baumann et al. (1934a, b) Irving & Richards (1939) Gnilbert et al. (1940) Horton et al. (1941) Horton et al. (1941)

Growth Longevity Longevity, growth, and teeth color Growth

Braude et al. (1941) Lewis et al. (1942) Lewis el al. (1942) Lewis et al. (1942) Lewis et al. (1942) Callieon & Knowles (1945) Sherman et al. (1945) Paul & Paul (1946) Brown & Stnrtevant (1949)

2. Mouse, Guinea Pig, Rabbit, Hamster, and Cotton Rat

I n a comprehensive study of the vitamin A requirement of the mouse, McCarthy and Cerecedo (1952, 1953) determined the effect of vitamin A intake upon growth, longevity, blood constituents, liver storage, and deficiency signs. It was noted by these authors th a t more stringent conditions were required t o produce vitamin A deficiency symptoms in the mouse than in the rat. I n young mice from mothers on a deficient diet, death occurred in some cases before extensive changes in the epithelium or structural changes in the eye. A severe eye condition was observed, characterized by a thick, clear, colorless exudate. Diarrhea, tremors, and unkempt fur were also noted and detailed histopathological findings reported. I n the growth experiments, albino mice were placed a t weaning on a casein-sucrose vitamin A-free diet without yeast until 36 days of age, after which the diet was supplemented with 1, 2, or 3 I.U. of vitamin A per day (U.S.P. Reference Standard). An additional group received vitamin A in the diet at a high level which provided an intake of 210 to 300 I.U. per day. The growth of the mice at the low levels is shown in Fig. 4. Maximum growth was obtained a t 1 I.U. per day, which was also ade-

VITAMIN A REQUIREMENTS OF ANIMAL SPECIES

109

quate for maintenance of life over long periods of time and for prevention of vitamin A deficiency symptoms. It was noted that the reproductive function in the male mouse is particularly sensitive to a deficiency of vitamin A; 1 I.U. daily allows for a completely normal exercise of this function and also for significant storage in the adult mouse. Hence, it appears that the requirement is somewhat less than 1 I.U. per day.

WEEKS OF SUPPLEMENTATION

FIG.4. Average growth rates of male and female mice on the various levels of vitamin A supplementation (McCarthy and Cerecedo, 1953).

The guinea pig has been shown by numerous investigators to require vitamin A, as evidenced by typical deficiency symptoms similar to those described for other species (Mannering, 1949). No quantitative studies of vitamin A requirements for growth have been reported, but Bentley and Morgan (1945) found that at least 2 mg. of carotene per kilogram of body weight were required to give significant liver storage of vitamin A. Preformed vitamin A appeared to be more than six times as effective in promoting storage as carotene. No information is available on the requirements of the cotton rat or hamster for vitamin A. The rabbit has been shown to require vitamin A (Pirie and Wood, 1946), but no quantitative estimate of the requirement has been made. Deficiency symptoms noted by these authors include impairment of growth, corneal lesions, and lowered ascorbic acid content of the aqueous humor. 9. Dog and Monkey In a study of puppies on a vitamin A-deficient diet, Frohring (1935) observed loss of appetite to be one of the first signs of deficiency. Other

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symptoms which developed included eye infections, a peculiar, divergent strabismus, ataxia, nervous running around the cage in circles, skin lesions, and corneal opacities. The limited data available on the quantitative requirements of the dog for vitamin A are summarized in Table IV. TABLE IV OF T H E DOG A N D MONKEY VITAMIN A REQUIREMENTS (Per kilogram of body weight) Carotene, Vitamin A, I.U. micrograms micrograms Dog

Monkey

100

30

22-47

7-14

Greater than 800 66

Greater than 240 20

Not determined

Criterion

Reference

Depletion of liver stores

Frohring (1937) Crimm & Short (1937)

40-60

Optimum health Estimate

Morgan (1940) Michaud & Elvehjem (1944) Truscott & Van Wagenen (1952)

Frohring (1937) estimated the daily requirement as 100 I.U. per kilogram by determining the rate of decrease of vitamin A stores in the liver of young pups. Crimm and Short (1937), using a similar technique with adult dogs, found the rate of utilization to be 22 to 47 I.U. of vitamin A per kilogram per day. Morgan (1940) places the optimum requirement of the dog for vitamin A above 800 I.U. per kilogram per day. I n a review of the nutritional requirements of the dog, Michaud and Elvehjem (1944) calculated that 66 I.U. of vitamin A or 40 to 60 micrograms of carotene per kilogram per day is a generous iQtake, even for the growing dog. These calculations were based on the reported requirements of dogs and other species and on the conclusion of Guilbert et al. (1937) that animals differing widely in body weight show almost identical requirements of vitamin A or carotene per kilogram of body weight. A description of the results of vitamin A deficiency in the monkey has been given by Day (1944). Diarrhea is the most consistent deficiency sign, but other common symptoms have been reported, including xerophthalmia, night blindness, edema, cessation of menstruation, mild gingivitis, diminished secretion of bile, and slight increase in white blood cells. Although the monkey is known to require vitamin A (Truscott and Van Wagenen, 1952), no quantitative estimate is available.

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111

4. Poultry a. Chicken and Turkey. Hogan (1950) has written an excellent critical review of the vitamin requirements of poultry, including a description of vitamin A deficiency symptoms. For the chicken, these include growth failure, ataxia, and muscle incoordination. Soreness develops around the eyes, and typical ophthalmia may occur after a longer depletion period. Other signs include accumulation of urates in the renal tubules, degeneration of the kidneys, and a high uric acid content in the blood. Rotation or retraction of the head and degeneration of the nictitating membrane were reported by Almquist and Mecchi (1939). A characteristic change in the older chicks was sinusitis and lesions resembling pustules in the mouth and esophagus. According to Jungherr (1943), “histopathologic examination of the nasal passages constitutes a delicate method for the recognition of subtotal vitamin A deficiency.” Most of the symptoms for turkey poults are the same as for chicks; Scott (1937) reported additional symptoms occurring as spasms and hemorrhagic enteritis in the advanced stage of vitamin A deficiency. The table on vitamin A requirements taken from Hogan’s paper is reproduced in Table V. Most of the values are reported on the basis of 100 g. of feed, which is the usual method of expressing poultry requirements. Comparison with other species on a body weight basis is made in a subsequent section. It is evident that there is considerable variation among the various authors’ estimates; this may be due to differences in the criteria used as well as to differences in the breeds, diets, and test conditions. I n general the requirements of turkeys expressed as units of vitamin A per 100 g. of diet are slightly higher than for chickens. The recommended allowances of the National Research Council are one-third more than the minimum required for normal growth without liver storage. Some recent estimates of poultry requirements are given in Table VI. Castano et al. (1951) found the minimum requirement for growth of chicks to be in the range of 110 to 220 I.U. per 100 g. of diet. Wharton et aE. (1949) fed a natural ration low in vitamin A to Broad-Breasted Bronze turkeys, a heavy breed, and supplemented the diet with various levels of vitamin A using cod liver oil. Optimum growth and significant liver storage were obtained at 1700 I.U. per 100 g. of feed. Van Reen et al. (1951) fed Jersey Buff turkeys, a light breed, a purified ration plus various levels of purified natural vitamin A ester. They estimated the requirement for growth at 450 I.U. per 100 g. of ration. Good growth was given by 300 I.U., but the higher level was necessary to clear all deficiency signs such as unnatural gait. Gurcay et al. (1950) found a similar level (440 I.U. per

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SAUL H . RUBIN AND ELMER DE RITTER

100 g. diet) as the minimum requirement for growth of white Holland turkey poults, a medium size breed. TABLE V SELECTEDESTIMATESOF VITAMINA REQUIREMENTS OF POULTRY AND NATIONAL RESEARCHCOUNCIL RECOMMENDED ALLOWANCES*

Species

Vitamin A, I.U. per 100 g.

Chicken Baby Chick

1,aying Hen

diet

300 833 265 355 440 t 105 3 210 $ 450 1000 917 440

Turkey Poult

730 t 1733 500 1000 600 500

Laying Hen

880 t

* From Hogan (1950).

Criterion of adequacy Growth Liver storage Growth Growth, liver storage

Gray and Robinson (1941) Bolin et al. (1943) Taylor and Russell (1947) Johnson el al. (1948) Cravens et al. (1946) With & Wanscher (1943) With & Wanscher (1943) Almquist & Mecchi (1939)

Growth Liver storage Hatchability, chick survival Egg production, fertil- Sherwood & Fraps (1940) ity and hatchability of eggs Liver storage, egg pro- Rubin & Bird (1942) duction, hatchability, chick survival Egg production, hatch- Taylor et al. (1947) ability, and mortality Cravens et al. (1946)

Liver storage, signs of deficiency, mortality Growth, mortality Not stated Growth, mortality Growth, signs of deficiency

t National Research Council Recommended Allowances-these $ I.U.per kilogram live weight.

Authority

Hinshaw & Lloyd (1934) Scott (1937) Asmundson & Jukes (1939) Wilgus (1940) Russell et al. (1949) Cravens et al. (1946) estimates preferred by Hogan.

b. Bobwhite Quail and Duck. Nestler et al. (1948) studied the vitamin A requirements of Bobwhite Quail and found 660 I.U.per 100 g. of feed necessary for maximum growth. The duck is known to require vitamin A (Wolbach and Hegsted, 1952), but the exact requirement has not been determined.

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113

TABLE VI SOME RECENTESTIMATES O F VITAMIN A REQUIREMENT0 Vitamin A,

Species Chicks Turkeys Broad-Breasted Bronze Jersey Buff White Holland Bobwhite quail Duck

I.U.per 100 g. diet

110-220 1700

450 440 660

Criterion of adequacy

OF

POULTRY

Reference

Growth

Castano et al. (1951)

Optimum growth, liver storage Growth, signs of deficiency Growth Maximum growth Not determined

Wharton et a2. (1949) Van Reen et al. (1951) Gurcay et al. (1950) Nestler et al. (1948) Wolbach & Hegsted (1952)

c. Utilization of @-Carotene.The chick is an efficient utilizer of p-carotene. Expressed as International Units, carotene is as active as vitamin A when the criterion is growth (Bethke et al., 1939; Reynolds et al., 1948; Castano et al., 1951). For liver storage of vitamin A, conflicting claims have been made. Johnson et al. (1948) and Rubin and Bird (1943) reported the storage of proportionately more vitamin A in the liver when the diet contained carotene, than when it contained a comparable amount of vitamin A. On the other hand, Castano et al. (1951) found vitamin A acetate more efficient than 0-carotene in promoting liver storage as well as higher blood levels of vitamin A. Russell et aE. (1942) reported that the absorption of carotene is depressed when the diet is deficient in fat, in contrast to vitamin A, which is absorbed as well on a low-fat as on a normal diet. In turkeys, Gurcay et al. (1950) compared vitamin A acetate, blackcod liver oil, and 0-carotene in growth and liver storage studies with white Holland poults. Vitamin A acetate was four times as effective as P-carotene and twice as effective as black-cod liver oil in supporting growth. For liver storage, vitamin A acetate was 20 to 30 times as effective as &carotene and about twice as active as black-cod liver oil. Some of the factors affecting the utilization of vitamin A and carotene are discussed in a subsequent section. 6 . Swine

Studies of farm animals by Guilbert et al. (1937, 1940) revealed that night blindness is the first detectable symptom of vitamin A deficiency. Swine occasionally develop partial posterior paralysis before defective vision can be demonstrated in semidarkness. Continued depletion of

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vitamin A causes diarrhea, infertility of sows, muscular incoordination, and an unthrifty appearance. The estrus cycle becomes irregular, and abortionpl, the birth of dead pigs, and congenital malformations occur. The requirements of swine for vitamin A or carotene are summarized in Table VII. These data show a progressively increasing requirement to TABLE VII OF SWINEPER KILOGRAM OF BODYWEIGHT VITAMINA REQUIREMENTS Vitamin A, Carotene, I.U. micrograms micrograms 18-24 54-72 25 57

4-6 13-17 8 17

25-39 125-195 40 88

Criterion

Reference

Night blindness Reproduction and lactation Growth Growth, reproduction plus safety factor

Guilbert et al. (1940) Guilbert et a2. (1940) Braude et al. (1941) Guilbert & Loosli (1951)

meet the needs for (1) prevention of night blindness, (2) growth, and (3) reproduction and lactation. Guilbert et at. (1940) and Braude et al. (1941) found no liver storage at the minimum levels required to support adequate growth or prevent night blindness during growth. I n both of these studies it was found that swine utilized vitamin A more efficiently than carotene. The last values in the table represent the National Research Council’s Recommended Allowances, which include safety margins for reproduction and loss in storage of feeds. On a weight basis, the carotene requirement is about five times that of vitamin A. 6. Cattle

The results of vitamin A deficiency in cattle have been described by Guilbert et al. (1937) and by Lewis and Wilson (1945). In addition to night blindness, keratinization of the epithelium is found as well as severe xerophthalmia. A low-grade type of pneumonia may ensue. The effects of prenatal deficiency have been reviewed by Warkarny (1945) ;these include birth of dead calves or weak calves with or without eye lesions and suffering from diarrhea. Table VIII presents data on cattle requirements for vitamin A and carotene. Minimum daily needs for growth and prevention of deficiency signs have been estimated in the range of 21 to 32 I.U. of vitamin A per kilogram of body weight. For maximum growth, however, Lewis and Wilson (1945) found 64 units necessary, and for high blood levels, 512 units. They concluded that 250 units per kilogram of body weight per day would provide for optimal growth and sufficient liver storage to allow

VITAMIN A REQUIREMENTS OF ANIMAL SPECIES

115

TABLE VIII VITAMINA REQUIREMENTS OF CATTLEPER KILOQRAM OF BODYWEIGAT Vitamin A,

Carotene,

I.U. micrograms micrograms 21-27

5-6

73-182

22-55

32

10

64 512

19 156

250

76

66

20

Criterion

Reference

26-33

Night blindness Guilbert et al. (1940) Calves: Birth to 6 Months Growth, signs of deficiency Converse & Meigs (1939) Calves Growth and deficiency Lewis & Wilson (1945) signs Optimum growth Lewis & Wilson (1945) Maximum blood concen- Lewis & Wilson (1945) tration of vitamin A Growth and liver storage Lewis & Wilson (1945) Dairy and Beef Cattle: Birth to Maturity 132 Growth, reproduction Guilbert & Loosli (1951) plus safety factor

for reproduction and milk production. The last set of values in Table VIII represent the National Research Council’s Recommended Allowances for vitamin A and carotene which permit only a small margin of safety for reproduction. The value for vitamin A corresponds to the level that Lewis and Wilson (1945) claim to be necessary for optimum growth only and approximates the lower level of the estimate of Converse and Meigs (1939). It is evident from the data given that cattle utilize carotene less efficiently than vitamin A. On a weight basis, the National Research Council’s Recommended Allowance for carotene is 6.6 times that for vitamin A. In a recent study of beef cattle by Baker et al. (1953), the N.R.C. recommended level of 132 micrograms of carotene per kilogram was insufficient to maintain liver stores or plasma levels of vitamin A. A t an intake during lactation of 730 micrograms per kilogram of body weight, liver stores and plasma levels of vitamin A were increased.

7’. Sheep The symptoms of vitamin A deficiency in sheep are very similar to those described for cattle or swine, but sheep develop partial optical impairment of a permanent nature in a shorter time than cattle after development of night blindness (Guilbert et al., 1937). Sheep requirements for vitamin A or carotene are given in Table IX. The minimum levels necessary to prevent night blindness are not adequate to provide for storage, reproduction, or other special demands by the body. The National Re-

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SAUL H. RUBIN AND ELMER DE RITTER

search Council recommendation (Pearson et al., 1945; Guilbert and Loosli, 1951) calls for three to five times this amount to provide for moderate storage and the demands for reproduction and lactation. These allowances are the same as for cattle. The relative activity of carotene also appears to be about one-sixth that of vitamin A on a weight basis. TABLE IX VITAMINA REQUIREMENTS OF SHEEPPER KILOGRAM OF BODYWEIGHT Vitamin A, Carotene, I.U. micrograms micrograms 17-26 66-92

4.3-6.3 20-28

25-35 100-140

66

20

132

Reference

Criterion

Night blindness Guilbert et al. (1940) Growth, reproduction, and Pearson et al. (1945) lac tation Growth, reproduction, and Guilbert & Loosli (1951) lactation

8. Horse

Guilbert et al. (1940) determined the vitamin A requirement of horses, using night blindness as the criterion, and obtained the results shown in Table X. These values are in line with those given for sheep and cattle, TABLE X VITAMIN A REQIJIREMENTS OF HORSES PER KILOGRAM OF BODYWEIGHT ~

Vitamin A, Carotene, I.U. micrograms micrograms 17-22 66

4.2-5.3 20

20-30 132

Criterion

Reference

Night blindness Growth, reproduction, and lactation

Guilbert et al. (1940) Guilbert & Loosli (1951)

including the fivefold higher requirement for carotene. The National Research Council’s Recommended Allowances (Guilbert and Loosli, 1951) are also the same as for sheep and cattle. Although night vision is particularly good in horses, night blindness is the first detectable symptom of vitamin A deficiency. On continued depletion, the horse’s hair becomes rough, corneas become clouded, and daylight vision is impaired. Excessive lachrymation was found by Guilbert et al. (1940) rather than xerophthalmia. Diarrhea and loss of weight were also observed. 9. Mink and Fox

‘

Estimates of the quantitative requirement of foxes for vitamin A are given in Table XI. The value of 15 to 25 I.U. per kilogram of body weight

VITAMIN A REQUIREMENTS O F ANIMAL SPECIES

117

daily, which Smith (1942) found necessary to prevent deficiency signs, is in the same range as reported for other animal species. The requirement for satisfactory growth is a t the top of this range, but 50 I.U. per kilogram per day are necessary for good blood levels and some liver storage (Bassett et al., 1948). These authors also reported mink to require vitamin A, but no quantitative estimate of the requirement is available. TABLE XI VITAMIN A REQUIREMENTS OF FOX PER KILOGRAM O F BODYWEIGHT Vitamin A, I.U. micrograms 1 5-25 25 50

4.2-7.8 6.2 12.4

Criterion Prevent deficiency Growth and deficiency signs Growth, blood level, and liver storage

Reference Smith (1942) Bassett et al. (1948) Bassett el al. (1948)

The first sign of vitamin A deficiency in the fox found by Smith (1942) was an irreversible nervous derangement, accompanied by loss of balance. Later symptoms included coma, xerophthalmia, abortion, stratification and keratinization of the epithelium, and vaginal cornification. 10. Human Being

An excellent review of the effects of vitamin A deficiency in the human being has been given by Clements (1946). The earliest clinical sign of established vitamin A deficiency, namely cornified epithelial cells, does not appear until bodily stores of vitamin A are completely exhausted and vitamin A has disappeared from the blood. Epithelial changes are widespread throughout the body. The classical signs of xerophthalmia and keratomalacia are comparatively late manifestations. Night blindness precedes the objective eye symptoms. Bitot’s spots are found when xerosis conjunctiviae is far advanced. Diarrhea and bronchopneumonia are the principal causes of death in infants with vitamin A deficiency. a. Infant. Some of the studies of vitamin A requirements of human beings are illustrated in Table X I I . Lewis and Haig (1939), using dark adaptation as a criterion, found that a t a daily intake of 18 to 20 I.U. of vitamin A per kilogram the infants appeared normal and grew well but had low blood levels of vitamin A. The authors considered this level as the minimum requirement. I n later experiments Lewis and Bodansky (1943) studied blood levels of vitamin A in infants under seven months and reported the normal range as 40 t o 114 I.U. per 100 ml. of blood. It was found that 100 t o 200 I.U. per kilogram of body weight were required t o maintain normal blood levels. This is five to ten times the amount re-

118

S A U L H. RUBIN AND E L M E R DE R I T T E R

quired when dark adaptation is used as the criterion. Infants placed on a diet devoid of vitamin A exhibit definitely lowered plasma concentrations of vitamin A before any disturbance in dark adaptation is manifest. TABLE XI1 VITAMIN A REQUIREMENTS OF HUMAN BEINGS Vitamin A, Carotene, I.U. micrograms micrograms Infants Adults

Per Kilogram Body Weight 20 6 100-200 30-61 25-55

7-16.5

26-62

18

5.5

43

81

24.5

2500

Per Adult per Day 760 4500

5000 Vitamin A Activity* *Average 1000 I.U. of vitamin A

Criterion Dark adaptation Blood levels Growth, dark adaptation Growth, dark adaptation Dark adaptation Dark adaptation, plasma levels, liver storage Recommended Allowance

Reference Lewis & Haig (1939) Lewis & Bodansky (1943) Booher & Callison (1939) Wagner (1940) Batchelder & Ebbs (1944) Hume & ICrebs (1949)

N.R.C. (1953)

+ 4000 I.U. of carotene.

b. Adult. Booher et al. (1939) showed that normal dark adaptation in adults required 25 to 55 I.U. of vitamin A or 26 to 62 micrograms of 0-carotene per kilogram of body weight. Batchelder and Ebbs (1944), using a different instrument for determining dark adaptation, found a higher requirement of 81 I.U. of vitamin A per ki1ogra.m of body weight necessary to maintain a normal visual threshold. Several studies have been reported in which human subjects were fed vitamin A-deficient diets for prolonged periods. The development of deficiency signs in such cases is dependent on existing liver stores of vitamin A, which are subject t o change with changing dietary habits and intake of vitamin supplements. Wagner (1940) maintained normal adult males on a vitamin A low diet for 188 days; all showed loss of weight, visual dysadaptation, and change in blood cells. Half were given p-carotene in oil and half vitamin A concentrate. Twenty-five hundred International Units of vitamin A or 5000 I.U. of &carotene per day were reported sufficient to meet normal requirements. However, since a ‘‘ Vogan” vitamin A concentrate was used, which was overvalued in potency by a

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VITAMIN A REQUIREMENTS OF ANIMAL SPECIES

factor of 2 (Moll and Reid, 1939) the vitamin A value should be 1250 I.U. These values are given in Table XI1 on the basis of body weight. TABLE XI11 MINIMUMDAILYREQUIREMENTS OF VITAMIN A SET FORTHBY U.S. FOODAND DRUG ADMINISTRATION * U.S.P. units 1500 Infants Children, 1-11 years 3000 Children, 12 years and over 4000 Adults 4000 RECOMMENDED DAILYALLOWANCES OF VITAMIN A, FOODAND NUTRITION BOARD, NATIONAL RESEARCH COUNCILt A ~Daily ~vitamin ~ A allowance ~ ~ Age, weight, years kg. I.U. I.U./kg. (approx.) Infants under 1 av. 7 . 5 1500 200 Children 1-3 12 2000 167

Girls BOY8 Women Pregnancy, latter half Lactation Men

4-6 7-9 10-12 13-15 16-20 16-20

* Federal Register, section 403 (j), Nov. 22, 1941.

18 27 35 49 54 63 55 65 55 65

2500 3500 4500 5000 5000 6000 5000 6000 8000 5000

140 130 130 100 95 95 90 90 145

75

t Recommended Dietary Allowances, Revised 1953, Publication 302. National Academy of Sciences -National Research Council, Washington, D.C.

In England a large-scale experiment was undertaken to determine the vitamin A or carotene requirements of young adults (Hume and Krebs, 1949). In a group of 16, no unequivocal signs of depletion appeared within the first year these subjects were given the depletion diet, but plasma levels of vitamin A decreased. Few subjects were suitable for test dosing. A second group of subjects were given a diet supplying 2500 units of vitamin A daily; none of these developed any evidence of vitamin A deficiency, as measured by dark dysadaptation or lowered concentration of plasma vitamin A. From these results, from prophylactic studies, and from analyses of livers of persons killed in accidents, a figure of 1300 I.U. of vitamin A was put forth as the minimum daily protective dose for a healthy adult. This figure was doubled to create a safety margin and a figure of 2500 I.U. of vitamin A proposed as the minimum daily requirement for healthy adults.

.

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SAUL H. RUBIN AND ELMER DE RITTER

The requirement for p-carotene was more difficult to assess, since factors such as the carrier vehicle and extent of absorption entered the picture. A figure of 1500 I.U. of p-carotene was used with the assumption of complete absorption. This was doubled to 3000 I.U. t o allow for a safety margin. ’However, experiments with human beings on the absorption of carotene from various sources showed that an intake of 4000 to 12,000 I.U. was necessary to get an absorption of 3000 I.U. of &carotene. A compromise figure of 7500 I.U. of p-carotene was chosen as the minimum daily requirement. There are in the United States at the present time two sets of dietary allowances, those of the Food and Drug Administration and those of the Food and Nutrition Board, National Research Council. Both sets of values are given in Table X I I I ; the former are deemed sufficient to prevent deficiency signs and the latter are pointed a t maintenance of good nutritional status. The National Research Council allowances are “based on the premise that two thirds of the vitamin A value of the average diet in this country is contributed by carotene and that carotene has half or less than half the value of vitamin A” on a unitage basis.

111. COMPARISON OF THE REQUIREMENTS OF VARIOUS SPECIES Guilbert and Hart (1935) reported that the vitamin A requirement of animals is dependent upon body weight rather than food consumption. On this basis the data reported above for the various species are compared in Fig. 5. The daily requirements for vitamin A are divided into two categories, namely (1) the minimum amount required to give good growth and prevent outward signs of deficiency, and (2) the higher amount needed to maintain good nutritional status, to allow for storage in the body, and to provide for increased needs during reproduction and lactation. In both cases the requirements of all mammalian species per kilogram of body weight fall in the same general range. It is interesting to note, however, that both minimum and recommended allowances for poultry on the same body weight basis are about five times greater than for mammals. The poultry requirements, which are usually expressed per weight of feed, have been converted to a body weight basis with the use of tables of average weight and food consumption given by Ewing (1943). IV. FACTORS AFFECTINQ VITAMIN A REQUIREMENTS 1. Alpha-Tocopherol

It has been shown by Moore (19.20) that the absorption of vitamin A and storage in the liver is dependent upon the vitamin E intake. Figure 6 gives recent data by Moore and Sharman (1951) which demonstrate that

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121

as the a-tocopherol intake increases up to an optimum level, the liver stores of vitamin A increase in rats receiving a constant amount of vitamin A. The sparing action of tocopherols on vitamin A has been shown by Hickman et al. (1944a) in rate growth and survival studies. These authors (Harris et al., 1944) conducted similar studies with carotene and found small amounts of tocopherols to be synergistic with small and moderate intakes of carotene. Larger quantities of tocopherols are neutral or antagonistic toward small intakes of carotene but enhance the utilization of large

0 OPTIMUM

0 MINIMUM

k:

FIQ. 5. Comparison of minimum and recommended vitamin A requirements of various species.

quantities of carotene. The three tocopherols, a, 0,and y, were reported by Hickman et al. (1944b) to be equally effective in sparing vitamin A. As previously noted, Koehn (1948) found that in the presence of adequate a-tocopherol the rat can convert @-carotenequantitatively into two molecules of vitamin A. Hebert and Morgan (1953) studied the effect of a-tocopherol on liver storage of vitamin A at higher levels of intake of vitamin A or carotene. They found that the addition of 0.5 mg. of a-tocopherol daily to the diet of partially vitamin A-depleted rats receiving 35 to 129 micrograms of vitamin A produced no significant change in liver stores of vitamin A. The same amount of tocopherol given with 87 to 174

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SAUL H. RUBIN AND ELMER DE RITTER

micrograms of carotene in oil produced a significant increase in liver stores of vitamin A. Above and below these levels of carotene intake, tocopherols had no effect on vitamin A storage. Carotene in aqueous solutions made with Tween 40 produced larger amounts of liver vitamin A than carotene given in oil. Tocopherols did not increase vitamin A storage when given with the aqueous solution of carotene but actually appeared to have a depressant effect. Increased growth and storage of vitamin A in the livers of chicks has been reported by Dam et al. (1952) when synthetic tocopherol was added to a diet containing 10% cod liver oil. Free tocopherol was more effective than tocopherol acetate. Methylene blue, thiodiphenylamine, and

-

DL%-tocopherol

Adequate

3mg.

Img.

Inadequate I

03mg.

,

A

0.1 mg.

*

Omg.

1

C

f

FIG.6. Storage of vitamin A in the liver by female rats given 1000 I.U. vitamin A weekly for about 6 months, in conjunction with doses ranging from 0 t o 3 mg. dl-atocopherol weekly (Moore and Sharman, 1951 .)

Antabuse had similar activity. When cod liver oil was replaced by lard or when fat was omitted from the diet, d,l-a-tocopherol acetate or methylene blue had no important effect on vitamin A storage. 6. Aqueous Dispersion Vitamin A has been found in animals and man to be absorbed and utilized much more rapidly when dispersed in aqueous solutions than from an oily medium. Lewis et al. (1947) reported higher blood levels and storage of vitamin A in rats, guinea pigs, and children following ingestion of aqueous preparations than after oil solutions of vitamin A. Sobel and his associates (Kramer et al., 1947; Sobel et al., 1948; Sobel et al., 1949) reported similar findings in rats, children, and adults-a review of which has been given by Sobel (1952). Typical data from these authors comparing blood levels of vitamin A in a normal child after ingestion of aqueous dispersions and fish liver oil are illustrated in Fig. 7. Considerably higher blood levels are found after the aqueous dispersions than after

VITAMIN A REQUIREMENTS OF ANIMAL SPECIES

123

the oil. This rapid absorption of vitamin A from aqueous dispersions is particularly helpful in such disease states as celiac disease, pancreatic fibrosis, and bile duct obstruction, where absorption is impaired.

FIG.7 . A comparison of the vitamin A tolerances in a normal child, using fish liver oil, aqueous dispersion I, and aqueous dispersion I1 (Krarner et al., 1947).

Halpern and Biely (1948) have reported a similar finding in chicks. When fed orally by pipet, vitamin A oils, whether fresh or oxidized, had higher growth activity in water emulsion than in vegetable oil. 3 . Form of Vitamin A : Alcohol or Esters

Week and Sevigne (1949) compared the utilization of vitamin A alcohol, vitamin A acetate, and vitamin A natural esters by the chick. Dosages of 30,000 I.U. were fed in various oils in a three-day period. In corn oil and castor oil and a low level of jojoba seed oil the alcohol and acetate gave equivalent storage of vitamin A in the chick liver, but the natural esters gave significantly lower storage. High levels of jojoba seed oil or ethyl laurate (12 ml. fed over the three-day dosing period) led to lower storage in the case of acetate as compared to vitamin A alcohol. This effect was attributed to interference of the carrier with ester hydrolysis. Kagan et al. (1950) gave equal single doses of either vitamin A alcohol or palmitate in aqueous dispersion to normal children and found no difference in serum vitamin A concentration. Week and Sevigne (1950) fed men and women 134,000 micrograms of vitamin A alcohol, acetate, or natural esters in 50 g. of margarine and measured blood serum levels of vitamin A in the following 24 hours to determine relative utilization. For the male subjects, vitamin A alcohol showed greater efficacy than the acetate, which, in turn, was superior to the natural esters. For the female

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SAUL H. RUBIN AND ELMER DE RITTER

group, the alcohol was superior to the acetate and to one natural ester preparation but not to a second. It is possible th a t these relations are influenced by the margarine used as a carrier, since Deuel et al. (1951) have shown by rat growth tests that margarine increases the physiological availability of carotene relative to vitamin A derived from high-potency fish oils or distilled natural esters. I n contrast t o the findings of Week and Sevigne (1950) of superior absorption of vitamin A alcohol, Popper et al. (1950) reported higher plasma leveIs of vitamin A in hospital patients after the intake of vitamin A esters than after equal doses of vitamin A alcohol. This difference was found when the esters and alcohol were given in oily as well as in aqueous medium.

4. Source of Carotene The source of carotene in the diet is an important factor as regards its utilization by the animal and is consequently also an important consideration in determining dietary requirements. The relative efficiency of utilization of carotenoids in various foods varies considerably. T o cite an example, Booher and Callison (1939) determined the amount of carotene required daily by human beings, using night blindness as criterion, to be 47 and 57 I.U. when two subjects were given cooked green peas and 77,87, and 100 I.U. when three subjects were fed cooked spinach. I n studies of chick liver storage, Frey and Wilgus (1949) found the carotene in fresh alfalfa t o be utilized more efficiently than that in dehydrated alfalfa meal. One factor responsible for such differences in utilization is the different nature of the carotenoids in different foods and feeds. For example, in rat liver and kidney storage tests, Johnson and Baumann (1947a) reported the relative efficiencies of all-trans-P-carotene, neo-0-carotene B, neo-0carotene U, and all-trans-a-carotene to be 100 :48 :33 :25. The first three are in excellent agreement with the results obtained by Deuel et al. (1945) in rat growth studies, but a-carotene is about half as active as 0-carotene in promoting growth. Cryptoxanthine is also half as active as @-carotene in promoting growth of deficient rats, but a t least as active in promoting tissue storage of vitamin A (Johnson and Baumann, 1948). I n the chick, With and Wanscher (1939) reported cryptoxanthine to be about twice as active as 0-carotene. Callison et al. (1953) separated p-carotene from other partially active carotene isomers present in yellow corn meal. The latter included significant amounts of neo-@-caroteneB, neo-p-carotene U, and an inactive pigment. The biologically active pigments as they occur in corn meal proved to be 82% available to the rat. Lower availability values given for corn meal result from inclusion of inactive pigments with the p-carotene fraction.

VITAMIN A REQUIREMENTS O F ANIMAL SPECIES

125

Other dietary constituents also influence the utilization of carotene. Kemmerer et al. (1947) reported that xanthophylls and chlorophylls reduced the utilization of carotene for liver storage of vitamin A in the ra t by about 20%. Johnson and Baumann (1948) found a similar depression of storage t o be caused by lutein, although it did not interfere with the absorption of 0-carotene. 5. V i t a m i n B l z

High and Wilson (1953) have shown that rats receiving a supplement of vitamin Blz in addition to vitamin A, when fed a diet low in vitamin A and Blz,will gain more weight than those receiving vitamin A alone. The vitamin BIZhad no effect, however, on liver stores of vitamin A. On the other hand, when the rats were given vitamin Blz in addition to p-carotene, not only was there a weight increase but also a n increase in liver stores of vitamin A over those animals receiving only ,&carotene. 6. Antibiotics

Burgess et aZ. (1951) noted an increase in the liver storage of vitamin A in chicks fed a ration containing penicillin. The authors believed this to be a specific effect not depending on body weight. Coates et al. (1952) demonstrated that penicillin a t a level of 25 mg. per kilogram of ration increased stores of vitamin A in the Iivers of chicks only in those cases where growth stimulation occurred. They concluded that the effect is nonspecific. Hartsook et a2. (1953) have shown that aureomycin does not affect the requirement of the rat for vitamin A for growth nor does it affect liver storage of vitamin A.

7. Other Dietary Constituents

a. Mineral Oil. The effect of relatively large amounts of mineral oil in decreasing the utilization of carotene and vitamin A has been demonstrated by various workers (Burrows and Farr, 1927; Dutcher et al., 1927, 1934; Mitchell, 1933; Jackson, 1934; Curtis and Horton, 1940). At lower levels in the diet of the rat, Burns et al. (1951) found that as little as 0.08 % mineral oil significantly diminished the utilization of 6-carotene. The effect on vitamin A was not so great but was significant a t 0.16%. Steigmann et al. (1952) studied this interference in human beings and demonstrated that it is of practical significance only when appreciable quantities of mineral oil are taken with meals. Doses of 2.5 ml. or less taken three times daily with meals caused no significant interference with vitamin A or carotene absorption, nor did the usual 30 ml. therapeutic dose taken a t bedtime.

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SAUL H. RUBIN AND ELMER DE RITTER

b. Other Fats and Oils. A great many publications have appeared on the subject of the effects of fats and oils on vitamin A and carotene utilization. A few typical examples are cited below to illustrate the general trend of such experiments. In chickens, carotene absorption has been correlated with the dietary level of fat, whereas vitamin A absorption was not affected (Russell et al. 1942). As previously noted, Deuel et al. (1951) observed augmented biological activity of carotene when fed to human beings in margarine. Mayer and Krehl (1948) found that the isocaloric replacement of sucrose by fats in a vitamin A-deficient diet fed to rats afforded increased protection against development of the vitamin A deficiency syndrome. This was particularly noticeable in the case of lard. Kaunitz and Slanetz (1950a) obtained a similar protective effect with a molecularly distilled forerun fraction from lard. Subsequently Kaunitz and Slanetz (1950b) demonstrated the activity of this so-called “lard factor” as well as of vitamin A in protecting against deficiency symptoms caused by rancid lard. More recently, these workers have reported (Herb et al. 1953) that this biological activity of lard is largely attributable to the presence of hitherto unrecognized typical vitamin A. The difficulty of ascertaining the presence of this low level of vitamin A in lard also raises a reasonable doubt as to whether other biological materials claimed to have vitamin A activity actually contain unrecognized vitamin A. Oxidized fats have been reported to affect the utilization of vitamin A. Fridericia (1924) found that lard heated in thin layers and then fed to rats along with butter fat would destroy the vitamin A in the butter. Lease et al. (1938), using rat liver storage as a criterion, found that the rate of destruction of vitamin A by rancid fats increases in general with increasing peroxide number of the fat. A similar decrease in the biological action of vitamin A has been reported by Halpern and Biely (1948) for chicks, when an oxidized vegetable oil was fed together with a fish oil. Reviews of the literature on the effect of oils and fats on absorption of vitamin A and also on the effect of rancidity of fats on vitamin A utilization have been made by Burr and Barnes (1943), Quackenbush (1945), and Barnes et al. (1948). c. Protein. Mayer and Krehl (1948) reported that in vitamin A-deficient rats, increased levels of dietary protein resulted in a generally increased severity of the symptoms of vitamin A deficiency. Evidence was presented by these authors suggesting that “ a t least part of the effects of an increased protein level on vitamin A deficiency is mediated through the concomitant decrease in vitamin C reserve.” James and ElGindi (1953) compared the effects of casein, lactalbumin, gluten, and zein in isocaloric, isonitrogenous, and essentially isophosphoric rations on the nutritional utilization of 0-carotene by growing albino rats. Liver storage of vitamin

VITAMIN A REQUIREMENTS OF ANIMAL SPECIES

127

A was two to five times higher with the casein diet than with the other

three proteins. Rats which received casein or lactalbumin had blood levels of vitamin A about 17% higher than those of rats r,eceiving gluten or zein. d. Choline. Bentley and Morgan (1946) investigated the effect of choline on liver storage of vitamin A by rats. Storage by depleted rats fed vitamin A with a high-fat-low-protein diet was not affected by choline deficiency. With a low-fat-low-protein basal diet, the addition of choline increased the liver vitamin A. When carotene was fed with the high-fat diets, the vitamin A deposits were very small and somewhat less in the fatty than in the normal livers. With the low-fat diet carotene caused better vitamin A deposition, which was little affected by choline. 8. Stress Factors

a. Cold. Ershoff (1950) fed a vitamin A-deficient diet to immature rats kept at 2" C. or 23" C. The rats kept in the cold room showed more rapid depletion, less body weight a t the time of depletion, and shorter survival time than the rats kept a t room temperature. b. Thyroxine. Greaves and Schmidt (1936) and Kelly and Day (1948) have shown that thyroxine accelerates the release of vitamin A reserves from the livers of rats on vitamin A-deficient diets. Also in rats, Johnson and Baumann (1947b) have shown that thyroxine increased hepatic storage of vitamin A from dietary carotene but not from preformed dietary vitamin A. In a study of cows and goats, Chanda and Owen (1952) found thyroxine to produce increased yields of vitamin A in milk when the diets contained carotene. When no carotene was fed, thyroxine caused a large increase in the proportion of vitamin A present in the milk as alcohol; this arises as a result of the mobilization of hepatic reserves.

V. UTILIZATION OF OTHER FORMS OF VITAMIN A 1. Vitamin A z The isolation of pure vitamin A2 was reported by Shantz (1948), and its biological activity was determined by Shantz and Brinkman (1950). By the U.S.P. rat growth assay, these workers found pure vitamin A2 alcohol to have a potency of 1,300,000 units per gram, which is about 40% of the activity of vitamin A1 alcohol. Rat liver storages of vitamin A2 alcohol and acetate were reported as 56 % and 50 %, respectively, of the values for the corresponding forms of vitamin Al. The vitamin A, appeared to be stored as such rather than converted in vivo to vitamin A1. 6. Neovitamin A

The isolation of this isomer of vitamin A was reported by Robeson and Baxter (1945, 1947). It was suggested that neovitamin A was a geometric

128

SAUL € RUBIN I. AND ELMER DE RITTER

isomer of vitamin A having the cis-configuration of the double bond nearest the hydroxyl group (5-cis.). A quantitative evaluation of the biological activity of neovitamin A has been reported by Harris et al. (1951). I n both rat growth and liver storage tests, these authors found neovitamin A t o have about 85% of the potency of all-trans vitamin A Regardless of whether neo- or all-trans vitamin A were fed t o the rats, an equilibrium mixture containing neovitamin A and all-trans vitamin A in the ratio of 1/1 t o 2/1 is deposited in the rat liver. 3. Other Isomers and Role in Vision

Ordinary crystalline vitamin A, as well as the bulk of commercial, synthetic vitamin A, is the all-trans isomer. The 5-cis stereoisomer has RHOOOPSIN

/ \

NEORETINENE b

+ SCOTOPSIN

NEOVITAMIN A b

--

ALL-TRANS

RETINENE

, ALL-TRANS

+ SCOTOPSIN

VITAMIN A

FIG.8. Formation and bleaching of the visual pigment, rhodopsin (Wald, 1953).

been isolated and called neovitamin A as noted above. Two other stereoisomers are expected, namely the 3-cis and the 3,5-di-cis. The stereoisomers of vitamin A are of particular interest in regard t o the role of vitamin A in vision, which has been investigated intensively by Wald and his co-workers (Wald, 1953; Hubbard and Wald, 1952). The system wherein vitamin A has an important role is summarized in Fig. 8. Briefly, vitamin A is oxidized to retinene by the alcohol dehydrogenase, cozymase system. Retinene and scotopsin combine t o form the visual pigment, rhodopsin. I n the presence of light, rhodopsin is bleached to form scotopsin and retinene, which is in turn reduced t o vitamin A. A specific isomer is necessary for the formation of rhodopsin. Retinene enters rhodopsin in the form of neoretinene b, a 3-cis isomer, the synthesis of which was reported by Graham et al. (1949). On bleaching of rhodopsin, retinene comes out primarily as all-trans retinene, which must be re-isomerized t o the active form before it can regenerate rhodopsin. Before this process is completed much of the retinene is reduced t o all-trans vitamin A.

129

VITAMIN A REQUIREMENTS O F ANIMAL SPECIES

Hubbard and Wald (1952) studied the formation of rhodopsin in the dark in a four-substance system containing vitamin A, crystalline alcohol dehydrogenase from horse liver, cozymase from yeast, and scotopsin from rods. When fish liver oil was used as a source of vitamin A, rhodopsin formation occurred; but with synthetic, all-trans vitamin A or the 5-cis neovitamin A, only a trace of rhodopsin was formed. Both of the latter forms of vitamin A, however, are readily converted t o equilibrium mixtures of stereoisomers by exposure to light in the presence of a trace of iodine. After this treatment, both preparations form rhodopsin as efficiently as liver oil vitamin A (Hubbard and Wald, 1952). These authors consider it likely t ha t any geometrical isomer can fulfill all the nutritional needs for vitamin A because of the isomerization of vitamin A which occurs in the body. Support for this view is provided by the recent experiments reported from Morton’s laboratory (Collins et al., 1953), in which synthetic (alltrans) vitamin A was used in rhodopsin regeneration tests. These workers excised the retina from the eyes of frogs, rats, and cows and made rhodopsin preparations. After bleaching the rhodopsin with light, it mas incubated with potassium succinate, phosphate buffer, magnesium sulfate, nicotinamide, cytochrome c, DPN, ATP, and crystalline, all-trans vitamin A. Almost complete regeneration of rhodopsin from frog retinas was obtained, as shown in Table XIV. When either A T P or vitamin A alcohol TABLE XIV

REGENERATION OF RHODOPSIN USING FROGRETINASA N D CHOROIDS* Yield per eye (fiducial limits No. of Percentage for P = 0.05) samples yield Unbleached Bleached Regenerated with complete medium ATP not added Vitamin A not added

0.036 0.008 0.033 0.016 0.018

k 0.0037 k 0.0022 rt 0.0033

rt 0.0053

f 0.0076

8 8 8 3 5

100 22 92 44 50

Final concentrations. of reactants were: potassium succinate, 0.024 M ; phosphate buffer, pH 7.4, 0.034 h l ; magnesium sulfate, 0.005 M ; nicotinamide, 0.03 M ; cytoM ; ATP, M ; vitamin A alcohol, 2.8 X 10-4 M ; DPN, 5 X chrome C 1 X 1.3 x 10-3 M.

* Collins et al., 1953. was omitted from the medium, only half the rhodopsin was regenerated. Using r a t retinas as a source of rhodopsin and a simple medium containing only phosphate buffer, magnesium ions, and all-trans vitamin A alcohol,

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SAUL 11. RUBIN AND ELMER DE RITTER

these authors obtained 76 % regeneration of bleached rhodopsin as shown in Table XV. These results would appear to be in contradiction to those of Wald (1953), who found all-trans vitamin A ineffective for regeneration of rhodopsin in a relatively pure system. It appears that the cruder system of Morton may be closer to the in vivo system occurring in the eye. In TABLE XV REGENERATION OF RHODOPSIN FROM RAT RETINAUSINGCRYSTALLINE SYNTHETIC VITAMINA (ALL TRANS)*

Unbleached Bleached Regenerated with complete mediumt

* Collins el al.,

1953.

t Phosphate buffer, magnesium

Yield per eye

Percentage yield

0.0236 f 0.006 0.0075 f 0.0006 0.0179 rt 0.0008

100

32 76

ions and crystalline synthetic vitamin A alcohol.

view of these results, the demonstrated isomerization of vitamin A in the body, and the fact that no eye dysfunction has been reported in animals fed synthetic vitamin A, it appears that a dietary source of the 3-cis isomer is not essential for proper vision and that all-trans vitamin A when ingested is sufficient for all physiological functions of vitamin A. VI. SUMMARY Comparative data are presented on the requirements for vitamin A of laboratory animals, poultry, farm animals, and human beings. I n general, the minimum requirement of a species has been considered to be that level of vitamin A which gives good growth and prevents outward signs of deficiency. When expressed on the basis of a unit of body weight, the minimum requirements for all mammalian species fall in a similar range of approximately 20 to 100 International Units of vitamin A per kilogram per day. For poultry the minimum requirement for vitamin A is approximately five times greater. When other criteria are applied, such as maintenance of liver stores or blood levels, longevity, maximum growth, or normal teeth and eyes, considerably higher vitamin A levels are required for optimum response. Most of the studies in this regard have been carried out in the rat, but ample evidence is available to show that similar considerations apply to other species. Recommended allowances for various species have been designed not only to meet growth requirements but also to provide for moderate storage and for the increased demands for reproduction and lactation.

VITAMIN A REQUIREMENTS OF ANIMAL SPECIES

13 1

Many factors have been demonstrated to influence vitamin A requirements. These include dietary level of tocopherol, types of fats and proteins fed, antibiotics, vitamin Biz, and stress factors such as cold or thyroxine dosage. The greatly increased absorption of vitamin A when given orally as an aqueous dispersion as compared to an oil solution has a beneficial effect on vitamin A utilization, which is of particular importance in disease conditions where absorption mechanisms are impaired. Since dietary carotene is a major source of vitamin A supply, the factors affecting its utilization are of primary interest. On the basis of rat assays the International Unit was established as equivalent to 0.6 microgram of p-carotene or 0.3 microgram of vitamin A alcohol; but by adjustment of diet of the rat and provision of adequate tocopherol, it is possible to obtain equal activity of vitamin A and p-carotene on a weight basis. However, the rat is one of the more efficient utilizers of carotene. Other animal species such as cattle, sheep, horses, and swine require five to six times as much carotene as vitamin A. Similarly high ratios have been reported for human beings. The factors denoted above, which influence vitamin A utilization, apply also to carotene, and generally t o a greater degree. The isomers of vitamin A are of particular interest by virtue of their role in the process of vision. Although a cis-isomer of vitamin A is essential for rhodopsin formation, recent experiments with excised frog and rat retinas have demonstrated efficient regeneration of rhodopsin after addition of synthetic, all-trans vitamin A. Apparently the eye tissues contain an enzyme which catalyzes the isomerisation of vitamin A. Mixtures of cis-and trans-isomers of vitamin A have been found in rat livers after feeding crystalline, all-trans vitamin A, and no report of eye dysfunction has been cited in animals fed synthetic vitamin A. It appears that a dietary source of the cis-isomer is not essential for proper vision and that all-trans vitamin A when ingested provides for all the physiological functions of vitamin A.

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