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The biological assay of fish oils for vitamin A Miller, John Peat 1937-12-31

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THE BIOLOGICAL ASSAY 03* FISH OILS FOR VITAMIN A by John peat M i l l e r A Thesis Submitted i n P a r t i a l Fulfillment the Requirements for the Degree of MASTER OF SCIENCE IN AGRICULTURE i n the Department of Poultry Husbandry The University of B r i t i s h Columbia A p r i l 1937. AMOwXEDGEMMT The writer wishes to acknowledge his indebtedness to Professor E. A. Lloyd and Mr. J* B i e l y of the Department of Poultry Husbandry for t h e i r keen interest and invaluable c r i t i c i s m i n the preparation of thi s manuscript; also to Messrs. J . B. O'Heil and C. W. Wood for their untiring assistance during the experimental work. THE BIOLOGICAL ASSAY OE FISH OILS FOR VITAMIN A TABLE OF CONTENTS I. Introduction--Vitamins i n Nu t r i t i o n I I . Discovery of Vitamin A .Page 1 I I I . Vitamin A in the Plant Kingdom " 4 1 . Vitamin A Content of Plants . .. " 4 2. Synthesis of Vitamin A i n Plants ..... " 9 3. PIaxit Carotinoids and Vitamin A ...... " 12 4. E x t r a c t a b i l i t y of Carotinoids from plants 14 [V. Vitamin A in the Animal Kingdom n 15 1 . Synthesis of Vitamin A in Fish... " 15 2. The Vitamin A Content of Animal » 19 Products 3. Storage of Vitamin A i n the Body " 25 4. Transmission of Vitamin A from Parents to Young " 27 5. Conversion of Carotinoids into Vitamin A " 28 r. Vitamin A Therapy « " 32 1# _Cn GTo3.cl s • • • • • • • • • o « » » * « * a « « • • • « • « > * • « • ^ 32 2. In the Dressing of Wounds " 34 3. Antagonism between Vitamins A and C, and Vitamin A and Thyroxin " 38 TABLE OF C.OFIBNT S ( Cont' d ) VI. B i o l o g i c a l Assay of Vitamin A Using the Rat as the Test Animal ............Page 41 1. Feeding Technique " 41 2 . Results of Vitamin A Deficiency xn Kelts ••»«*>**«»4*«*>«»>««*««««*o 45 3. Assimilation of Vitamin A i n the Presence of Mineral Oils . " 49 4. Influence of Vitamin A on Fat VII. Chemical and Physical Properties of Vitamin A 53 1. The Chemical Structure of Vitamin A.. " 53 2 . The Effect of Chemical and Physical Agents on Vitamin A c " 57 3. Oolorimetric Reaction .. " 63 4. Absorption Spectrum " 69 5. Solvents for Carotene " 73 6. Vitamin A. and Anti 7. Concentrati on of Vitamin A ........ „ u 76 8. The Measurement of Vitamin A {International Units) . .. . " 78 VII I . Vitamin A i n Poultry .. " 83 1. Symptoms and Lesions due to Lack Gi* \Pxtctii2X.Q .A. e t * > « « « « * . > « « « « « > « f t 6 * > 0 o " 83 2. Requirements of Chicks for Growth " 89 3. Requirements of Hens for Egg !Px*o^.liGtxon « » « « « o e * « » * * » » » » » e * » * « ^ X03 TABLE PIT CONTENTS (Cont'd) IX. Experimental . .Page No, 1. Experiment No. 1 (a) Introduction...... .. " 109 (t>) Material and Methods ........ " 109 (c) Results .. u 111 (d) Discussion u 117 2. Experiment No. 2 (a) Introduction. " 121 (t>) Material and Methods .... « 122 (c) Results . ... " 124 (d) Discussion .. . .. " 149 3. Experiment No. 3. (a) Introduction ... .. . . " 154 ("b) Material and Methods ........ " 154 (c) Results . " 155 (d) Discussion .. " 159 X. Summary " 165 168 XI. References ........... - - - - 11 I. TEE. BIOLOGICAL ASSAY OF FISH OILS FOR VITAMIN A. Introduction. Domestic animals must be furnished with a ration that i s complete i n carbohydrates, f a t s , proteins, vitamins, a,nd minerals i f normal health, reproduction, and growth are to be maintained. A deficiency i n any nutrient or vitamin may cause retardation of growth i n young animals, or decreased output i n mature animals of such products as milk or eggs. Furthermore deficiencies i n diet may r e s u l t i n diminished health or vigor as evidenced by such diseases as xerophthalmia* n u t r i t i o n a l roup, r i c k e t s , pellagra, a,naimia, or goiter. The causes of the most common of these deficiency diseases have been tra.ced, in recent years, to a lack of some sp e c i f i c vitamin. Of the many different vitamins known to exist today, Vitamin A has received considerable attention. Many investigators have shown that this vitamin i s indispensable for the normal growth of animals although the actual amount needed i s very small. Of t h i s small t o t a l r e l a t i v e l y moderate quantities are required for maintenance, somewhat larger quantities for growth, and r e l a t i v e l y large quantities for the production of eggs or milk. Since Beach (12) in 19 23 showed that Vitamin A was essential for normal poultry n u t r i t i o n , a great deal of work has be.n done by other investigators to determine the I I . quantitative requirements of chicks for this vitamin. Ordinarilys Vitamin A i s supplied through the feeding of yellow corn, dehydrated a l f a l f a , or green feed. These feeds .as a r u l e , must be supplied in comparatively large amounts to meet the Vitamin A requirements of growing chicks and p a r t i c  u l a r l y of laying b i r d s . In recent years f i s h o i l s have become widely u t i l i z e d as sources of Vitamin A for poultry because they are very potent sources of t h i s vitamin. According to Praps and Treichler (83) a good grade of Cod Liver O i l contains 600-1000 units of Vitamin A per gram, while a good grade of yellow corn contains only 5. units, of Vitamin A per gram and dry a l f a l f a 12 units per gram. It w i l l be readily seen, there fore, that the Vitamin A content of a rat i o n can be readily f o r t i f i e d and controlled by•the mere addition of a small quantity of a Vitamin A potent f i s h o i l . Amongst the f i s h o i l s , Cod Liver O i l has hitherto occupied a pre-eminent position a.s a source of Vitamin A i n both human and animal n u t r i t i o n . In recent years i t has been shown, however, that many other f i s h o i l s a.re just a.s r i c h sources of Vitamin A. Amongst the various f i s h -oils, BRITISH COLUMBIA  PILCHARD OIL i s already extensively used as a source of Vitamin D. On account of t h i s , i t was deemed advisable to carry out at The University of B r i t i s h Columbia investigations regarding the Vitamin A potency of this o i l . The purpose of these investigations were'not only to throw l i g h t on the Vitamin A content of Pilchard O i l , but to I I I . determine the Vitamin A requirements of growing chicks as w e l l . At the same time, i t appeared that a satisfactory method should he developed, i f possible, for the b i o l o g i c a l and chemical assay of Vitamin A i n f i s h o i l s . The present investigation consists of three d i s t i n c t experiments, (a) Estimating the Vitamin A content of B r i t i s h Columbia pilchard O i l i n terms of International Units. (b) Developing a suitable basal ration free from Vitamin A for the b i o l o g i c a l assay of Vitamin A i n f i s h o i l s , using the chick as the test animal, (c-^) Comparing the b i o l o g i c a l and chemical methods of assaying B r i t i s h Columbia Pilchard O i l . ( c 9 ) Determining the Vitamin A content of Pilchard Oils produced at different periods of the f i s h i n g season. l o reference i s made i n this investigation to the storage of Vitamin A in the l i v e r s of chicks fed varying amounts of Vitamin A. Work on t h i s phase of the problem i s in progress. DISCOVERY Off VITAMIN A. The s p e c i f i c growth-promo ting property which i s now "associated withihe occurrence of Vitamin A was f i r s t observed by McCollum and Davis (142) i n 1913, who found that an ether extract of butter or egg yolk i n a synthetic ration had a stim ulating action on growth which v/as not possessed by other fats such as lard or o l i v e o i l . These workers pointed out that certain mixtures of fats of animal o r i g i n , as butter f a t , egg f a t , and the fats ex tracted from the internal organs, eg. the kidney, l i v e r , etc., contain something which i s absolutely indispensable for either maintenance or growth, and that t h i s substance i s not found i n vegetable o i l s or fats and i n but very small and inadequate amounts i n the bodyfats of animals. They showed that when the diet i s inadequate i n i t s content of this substance which they designated as fa,t-soluble A, the animals become emaciated and suffer from edema, of the • eyes. . Blindness results i f the animals are permitted to go without this dietary essential or with an inadequate supply for a s u f f i c i e n t time. Very shortly thereafter Osborne and Mendel (171) pub lished experiments i n which i t v/as pointed out that th e i r milk rations had special dietary properties not found i n their milk- free rations and that t h i s c h a r a c t e r i s t i c seemed to be true of rations carrying an equivalent amount of butter as w e l l . Later 2. (172) they obtained uniform success by substituting cod l i v e r o i l for a portion of the. lard i n their standard d i e t s . Stephenson (238) i n 1920 found that a crude alcohol- light-petroleum extract of dried carrot when added to a fat not containing vitamin confers upon i t the growth promoting property and the power of protecting the animal from or...curing i t of keratomalacia. His work showed that the substance of substances i n the extract, responsible for these properties was not carotene. Other experiments that he conducted demon strated that the colouring matter of butter fat may be complet ely removed or destroyed by f i l t r a t i o n through charcoal with out i n the least affecting the vitamin content of the butter. Z i l v a (267) i n 1919 found that the fat soluble A fact or i n butter became inactivated when the butter was exposed to u l t r a v i o l e t l i g h t for eight hours. Drummond and Coward (60) i n 1920 decided that no hard and fast l i n e could be drawn between the animal and the veget able o i l s and fats when their value as a source of Vitamin A was being considered. These investigators also found that the animal f a t s , taken as a class, possessed a growth-promoting power superior to that of the vegetable o i l s . This superior growth promoting power of the animal o i l or fat appeared to be influenced considerably by the diet of the animal. In 1919, Haring, Bea.ch and J a f f a (26) reported a, study of several outbreaks of a disease occuring i n flocks of pullets i n C a l i f o r n i a , which, so far as could be determined by a search 3. of the l i t e r a t u r e , had not been previously described. The symp toms resembled those of "roup" more than of any other disease of fowls known at that time, but differed enough so that a d i f f e r  e n t i a l diagnosis was re a d i l y made. Later on Beach (12) defin i t e l y showed that the disease with which he was dealing i n poul try was due to a lack of Vitamin A. 4. VITAMIN A IN THE PLANT KINGDOM  Vitamin A Content of Plants* Steenbock, Kent and Gross (226) i n 1918, discovered that barley contained an abundance of the wa.ter soluble v i t a - •min but was deficient i n the fat soluble vitamin. Osborne and Mendel (173) i n 1919, were the f i r s t i n  vestigators to note that green vegetables supplied an import ant addition to the diet of man because the staples such a.s cereals, meats, potatoes, fats and sugar furnished too small an amount of the vitamin to meet f u l l y the requirements of an adequate dietary. As early as 1919 Steenbock and Gross (228) demonstrat ed the high fat-soluble vitamin content of carrots and yellow sweet potatoes as compared with other roots. A year l a t e r , Steenbock and Boutwell (229) showed that yellow corn contained enough of the fat-soluble vitamin to allow growth at the norm a l rate to take place i n the r a t . White corn on the other hand did not contain any demonstrated amount of the fat-soluble vitamin. In 1920 Steenbock and Gross (230) found that 5 % of clover or a l f a l f a as the sole source of fat-soluble vitamin i n a r a t i o n , when other dietary requirements were met, allowed normal growth and the rearing of some young. Steenbock, S e l l and Boutwell (234) worked with peas to f i n d their fat-soluble vitamin content i n r e l a t i o n to their pigmentation. The results of this investigation demonstrated that i n ri p e peas, those of a green colour, also carrying con siderable yellow pigment, were far richer i n their fat-soluble vitamin content than yellow peas which contained much less yellow pigment. About the same time as thes.e investigations were going on Coward and Drummond (35) were investigating nuts as a source of Vitamin A. They found that the various nuts contained a large percentage of fat but possessed l i t t l e or no vitamin A. Their results further substantiate the theory that Vitamin A i s formed i n the green part of the l i v i n g plant and i s not stored to any appreciable extent as such i n the seed and other resting tissues. Willimont (259) showed that 5 c c . of navel orange juice contained s u f f i c i e n t Vitamin A for growth and well-being i n the ra.t. The Vitamin A content of barley was intensively studied by Hughes (124). He noted that when growth was used as a measure of the Vitamin A content of barley yellow corn, white corn and barley, the indications were that the Vitamin A content of barley lay somewhere between that of yellow corn and vtfiite corn. When growth and vaginal smears were used as a measure of the Vitamin A content of barley, i t was shown that the quantity i n barley was low, i n f a c t , much lower than that of yellow corn but higher than that of white corn. The experiment d e f i n i t e l y showed that barley, as the only source of Vitamin A i n the diet did not produce normal growth i n ra t s . In conclusion he stated that barley contained less than one six t h as much Vitamin A as 6. did yellow corn. Extensive tests of the Vitamin A potency of a l f a l f a were carried out by M i l l e r and Bearse (155a.). They found that a sample of commercial dehydrated a l f a l f a contained twice as much Vitamin A as a. sample of commercial sun-cured a l f a l f a . After a year's storage the potency of the two samples was s t i l l the same. They also found that the dehydrated a l f a l f a s they used were t h i r t y times more potent than samples of yellow corn. Hauge (106) found that the Vitamin A a c t i v i t y of fresh l y cut young a l f a l f a was preserved when the enzymes were des troyed immediately before drying. A direct correlation was found between the effect of temperature on the Vitamin A and on the enzyme a c t i v i t y . The influence of the sun's rays on the destruction of the Vitamin A was thought to be due to the prod uction of temperature which accelerated enzyme a c t i v i t y . Guilbert (98) observed no loss on drying but consider able loss on autoclaving and during sun-drying, most of which was due to the photochemical action of the sun, though i n slow drying, enzyme and b a c t e r i a l action also played a part. Temp erature was the most important factor influencing loss i n stor- o age; below 5 c. no loss was detected i n 6 months but the rate of loss increased very rapidly with r i s e of temperature. Recent work on a l f a l f a , hay by V a i l , Tobisha and Doug lass (252) showed how Vitamin A may be l o s t or conserved acc ording to methods of handling. 1. Loss or inactivation of Vitamin A i n a l f a l f a hay 7. results from: (a) Usual practice of curing and stocking. (b) Exposure to u l t r a - v i o l e t l i g h t i n presence of moisture. (c) Storage of a l f a l f a meal in cloth sacks. 2. Conservation of Vitamin A i n alfalfa- hay results f r om: (a) Curing indoors. (b) Curing by rapid a r t i f i c i a l drying. (c) Curing by crushing and rapid drying. (d) Storage i n the bale. (e) Storage of a l f a l f a meal i n paper sacks. The Vitamin A i n a l f a l f a hay tended to become more i n  active the longer i t was stored. Woods et. a l . (266 a ) found that t h i r d cutting a l f a l  f a hay, cock cured and i n fresh condition, contained 308± 13 Sherman units of Vitamin A a c t i v i t y per gram (leaves 48 3-t 34, stems 121 dr. 7 u n i t s ) . A sample of the same hay ground and stored i n diffused l i g h t for 4 months, contained only 233 _ 20 unit s . Hay cured i n the swathe for 3 days, then cock cured and sweated i n the stack contained 116dr 9 units per gram com pared with 144±10 units for si m i l a r hay which had been exposed i n the swathe only one day before cocking. Sweating i n the stack caused a si g n i f i c a n t reduction i n the Vitamin A a c t i v i t y from 233+ 20 Sherman rat units for cock cured, 2-g- months old unsta.cked hay to 1 4 4 i l 0 units for similar* hay sweated i n the stack 49 days. 8, Praps, Treichler and Kemmerer (84 a ) showed that a l  f a l f a products containing from 7.3 to 63.5 micrograms of caro tene had a Vitamin A potency of from 13 to 77 Sherman-Munsell units (15.6 to 92.4 international units) per gram, with an average value of 1.4 units (1.6 international units) per micro gram of carotene. They also observed that one microgram of carotene i n the international standard had a value of 1.4 Sherman-Munsell units, and 1 microgram of p u r i f i e d carotene had the same value. 9. Synthesis of Vitamin A i n Plants. Wilson (260) i n 1922 found that either etiolated or green wheat sprouts furnished an adequate amount of Vitamin A when the dried sprouts made up 5 % of the diet of white r a t s . He drew the conclusion that Vitamin A i s produced i n the grow ing plant with or without any accompanying photosynthesis. Coward (37) i n 1923 pointed out that l i g h t i s nece ssary for the formation of Vitamin A i n plant tissues. This process, however, can "be carried out i n the absence of carbon dioxide and of oxygen i n the surrounding atmosphere. The production i s also independent of the u l t r a - v i o l e t rays of the spectrum (or such of those as are held back by 2 plates of window glass) and can be carried on under the influence of e l e c t r i c l i g h t i n the absence of sunlight. On the other hand the presence of chloroform i n the atmosphere prevents the formation of the Vitamin. In 1925 Coward (39) further showed that Vitamin A i s not used i n any process carried on by l i v i n g plant tissue i n the dark. The Vitamin A appears to increase when a le a f loses i t s green color and becomes yellov/; but i s completely des troyed when the leaf dries up and dies. The same investigator (40) l a t e r presented data to show that the l i g h t from a quartz mercury vapor lamp was ef fective i n accelerating the formation of Vitamin A i n l i v i n g plant tissues. These short u l t r a - v i o l e t rays from such a lamp, used i n conjunction with the v i s i b l e rays, did not have any influence on the ultimate amounts of Vitamin A contained 10. i n the tissues. The investigation also showed that the amount of Vitamin A i n etiolated shoots i s an inverse function of the temperature at which they have been grown, this having i n f l u  enced markedly the rate of growth. Moore (159) corroborated the work of both (260) and (40). He further showed that rats fed for some months on etiolated wheat shoots appeared lean and rough-coated when compared with similar rats receiving a l i b e r a l d i et. The year after the above evidence had been published Moore (160) came out with the opposite idea. He fed et i o  lated wheat shoots to rats under conditions involving the minimum of red l i g h t i l lumination consistent with the feeding and handling of the animals. They were effective as a source of Vitamin A, thus supporting his conclusion that l i g h t i s not essential during any stage of the formation of the V i t a  min from seed. Dye, Mediock and Cr i s t (72) observed that leaf l e t  tuce exceeded head lettuce i n the promotion of growth i n rats that had ceased to gain on a diet deficient i n Vitamin A. The outside green leaves of head lettuce were far superior to the inside yellow leaves i n furnishing the Vitamin. Heller and St. Ju l i a n (113) found that Vitamin A was formed and stored c h i e f l y i n that portion of the plant exposed to l i g h t and that i t did not migrate to other portions of the plant* Luce and MacLean (141) stated that Vitamin A i s syn thesized by the yeast c e l l and the synthesis may be brought 11. about in the absence of sunlight. According to Goode (91) the Vitamin A content of white corn sprouts was increased by i r r a d i a t i o n and the Vitamin was transferred i n part from the sprouts to the grain during the i r r a d i a t i o n of the growing process. 12. Plant Carotinoids And Vitamin A. Palmer and Kennedy (174) i n 1921 showed that carotin oids and fat-soluble Vitamin are not even quantitatively asso ciated i n the plant tissues i n which both are presumably syn thesized. Two years l a t e r Coward (38) stated that the chief point i n the formation of the Vitamin which was apparent i n her experiments was that some lipochrome (generally carotene) was always associated with the Vitamin i n plant tissues; and that where carotene was found, p a r t i c u l a r l y carotene exposed to sunlight, there the Vitamin may be expected to be present also. Smith and Morgan (223) l a t e r found that chlorophyll was not a necessary intermediary i n s i t u for the formation of carotene, lycopene, or any other possible precursors of V i t - min A. Fr u i t s which develop carotene and Vitamin A a c t i v i t y under normal l i g h t conditions do so also under glass and i n the dark although usually i n s l i g h t l y smaller amounts. They also noted that a constant r e l a t i o n exists between the le v e l of daily intake of aarotene by Vitamin A free rats and their growth per unit of t o t a l carotene ingested. This r e l a t i o n i l l u s t r a t e s the "Law of Diminishing Returns." Norris (169) discovered that the exposure of etiolated plants to a r t i f i c i a l l i g h t resulted i n the formation of chlor ophyll and xanthophyll i n constant proportions. Carotene de creased at f i r s t , but after 4—5 houES increased more rapidly than the chlorophyll andxanthophyll. With increasing oxygen 13. i n the atmosphere carotene developed s t i l l more rapidly, hut formation of chlorophyll andxanthophyll reached a maximum with 20% oxygen. The proportion of a l l pigments increased with r i s i n g carbon dioxide i n the atmosphere up to 3-5^, hut sub sequently declined. Indications were obtained by Macv/alter and Drummond (149) that the lipochrome content of young f i s h may be increas ed when they are fed on a diet containing a t y p i c a l green alga. Examination of the lipochromes of Pucus Vesiculosus by Heilbron and Phipers (111) showed that wheras the dead material contained beta carotene a.nd zeaxanthine, the l i v i n g plant con tained beta carotene and fucoxanthine. No trace of xanthophylls usually associated with the higher plants could be found and zeaxanthine was probably a post mortem product of fucoxanthine. Heilbron, Parry and Phipers (112) found that i n the unsaponifiable matter from extracts of cladophora, sauteri, n i t e l l a opaca, oedogonium and rlodymenia palmata, various sterols could be i d e n t i f i e d and also the lipochrome pigments, l u t e i n , taroxanthine and beta carotene. A l f a carotene was observed only i n oedogonium. 14. I n t r a c t a b i l i t y of Garotinoids from Plants. In 1920 Steenbock and Boutwell (232) cound that when carrots were saturated with lard or corn o i l and then extract ed with ether, none or l i t t l e of the fat-soluble vitamin was removed. The l a r d preparation gave no evidence of containing the vitamin, but the corn o i l preparation contained i t i n small but persistent amounts. They also noted that ether had l i t t l e solvent properties for the fat-soluble vitamin as found i n car rots; chloroform and carbon disulphide removed some of i t ; while alcohol and benzene removed considerable amounts of i t . While the vitamin i s not extracted from forn by ether, alcohol removed i t quantitatively and with l i t t l e i f any, destruction. They give their procedure for fractionating an extract from a l f a l f a . These same two investigators (229) l a t e r demonstrated that the fat-soluble vitamin as found in the plant kingdom in a grain,,in l e a f and stem tissue, i n fleshy roots and in a cucurbitous vegetable was comparatively stable at a high temp erature. 15. VITAMIN A IN THE ANIMAL KINGDOM. Synthesis of Vitamin A i n Fish.. In 1922 Jameson, Drummond and Goward.(125) demonstrated that pure cultures of a common marine diatom grown i n Miguels' solution or s t e r i l i s e d sea water synthesised large amounts of Vitamin A. These investigators draw a p a r a l l e l between the de pendence of land animals on fresh green leaves and that of mar ine animals on the synthetic a c t i v i t y of the marine f l o r a for their supplies of Vitamin A. Working with brown trout, Coward and Drummond (36) found that the ova of these trout normally contain r e l a t i v e l y large amounts of Vitamin A. I f i n the po s t - l a r v a l period or even before the yolk sac i s completely absorbed, the f i s h are given food r i c h i n Vitamin A the i r growth and development are satisfactory and they appear able to store that factor i n their tissues. Drummond and Z i l v a (63) concluded from their work that the ultimate o r i g i n of the Vitamin A found in the o i l s derived from f i s h , and p a r t i c u l a r l y f i s h l i v e r o i l s , to be chiefly the un i c e l l u l a r marine plants. Except very occasionally these org anisms are not consumed, d i r e c t l y by the f i s h . These investigators also noted that the extraordinary r i s e i n the number of marine plants which begins as soon as the intensity and duration of sunlight increase, early i n the year, i s followed by a rapid r i s e i n organisms largely copepods and l a r v a l decopods and molluscs, whose growth and development are dependent on their food supply which consists of minute plants. These minute animals, which form a la,rge proportion of plankton contain r e l a t i v e l y large quantities of Vitamin A presumably de rived from the deatoms on which they have thriven. The plankton forms the staple food of innumerable species of marine animals from small f i s h to some whales. This no doubt accounts for the presence of Vitamin A in the tissues or fat depots of these animals. Two years la.ter Z i l v a , Drummond and Graham (268) dem onstrated that the sexual condition and age of the cod did not influence the Vitamin A potency of the l i v e r o i l . Bailey (6) l a t e r corroborated t h i s work while working with Ling Cod of the p a c i f i c Coast. Finn (82) i n 1931 published data to show that pilchard o i l contained a substance which promoted the growth of rats which had f a i l e d to grow on a Vitamin A deficient d i e t . B i e l y and Chalmers (16) working with chicks further demonstrated the Vitamin A potency of pilchard o i l . Truesdail and Boynton (246) undertook a study of the Vitamin A content of body o i l s of 5 species of P a c i f i c Coast Salmon. They found a l l 5 were decidedly i n f e r i o r to the sample of high-grade medicinal cod l i v e r o i l with which they were compared. The Vitamin A potency was found to be proportional to the intensity of the natural yellow colour of the o i l . While working with halibut l i v e r o i l , Lavern, Edisbury and Morton (139) obtained very interesting r e s u l t s , ilo p a r a l l -ism could be traced between the Vitamin A and Vitamin D pot encies. In certain species, the Vitamin A content of the l i v e r o i l was found to increase with the age and or size of the f i s h , the t o t a l Vitamin A reserve increasing more rapidly than the o i l potency. They believe that halibut l i v e r o i l i s by far the richest known natural source of Vitamin A available i n quantity. It has been found, however, that the o i l varies i n potency over a wider range than any other source. Eo correlation has emerged between the immediate diet of the halibut and the o i l potency. Their work also showed that halibut l i v e r o i l s exhibit well- marked seasonal fluctuations in Vitamin A concentration which cannot be attributed to changes in the o i l content of the l i v e r occasioned by spawning. The best o i l s from the standpoint of Vitamin A content are obtained from large halibut caught in northern waters i n the late spring or early summer, and i n the autumn. Very r i c h o i l s at other times of the year are except ional . Working along similar- lines Lavern and Sharp (140) found that the diet of the halibut was of a general nature, with no outstanding r i c h source of Vitamin A to account for the high potency of halibut l i v e r o i l . Taking the glycogen content of the l i v e r as a c r i t e r i o n of intensity of feeding, they found that no correlation could be established between intensity of feeding and the Vitamin A potency of the o i l . They obtained further evidence that i n general the l i v e r s of older f i s h aff orded a more potent o i l than those of younger f i s h . 18O Weekly records were taken of the f a t , Vitamin A and Vitamin D contents of the l i v e r s of halibuts(caught near Seattle by B i l l s , Imboden and Wallenmeyer (18). The o i l content i n  creased slowly from January (12%) to June, there was then a sudden r i s e to a majcimum value [25%) i n August, followed by a slow decline during the rest of the year. The Vitamin A content (January 240,000, August 35,000 international units per g.) and Vitamin D content (January 1,400, August 900 international units per g.) moved inversely with the fat content although in the case of Vitamin A the fluctuation was wider than i n the case of Vitamin I). B i l l s et. al.(19) conducted a toxonomic-study of the di s t r i b u t i o n of the Vitamins A and D i n many species of f i s h . They found that for a given species, no relationship could be established between the Vitamin A and D potencies of the l i v e r or body o i l s . In general, but with many exceptions, l i v e r o i l s r i c h i n the other, and the potency tended to vary inversely with the o i l content of the l i v e r . 19. The Vitamin A Content of Animal Products. In 1917 McCollum, Simmonds and Steeribock (143) stated that fat free milk, when included i n a diet consisting other wise of p u r i f i e d food substances, promoted growth and prevented decline of animals i n a manner which indicated i t s t i l l contain ed the fat-soluble essential in appreciable amounts. This led them to suspect that the factor was appreciably soluble i n water. They demonstrated that when butter fat was melted and thoroughly agitated with twenty successive portions of water i t no longer contained the fat-soluble factor. Continuing along this l i n e of thought, Steenbock, Boutwell and Kent (227) found that while the vitamin was remov ed from the washed butter f a t the washing s did not contain i t . They were convinced that somewhere i n the course of the man ipulations to which the butter fat had been subjected, fa.ctors had been introduced which were responsible for the vitamin de stru c t i o n . They then r e a l i z e d that heat alone i n the absence of water or i n the absence of conditions designed to bring about intimate contact with air was responsible for the vitamin de struction observed i n the early experiment. Hopkins (122) found that the fat-soluble A substance i n butter, which displaying marked resistance to heat alone at o temperatures up to 120 was readily destroyed by simultaneous aeration of the f a t , presumably because i t i s a substance prone to oxidation by atmospheric nitrogen. Working along the same line s as Hopkins (122), were 20. Drummond and Coward (61). They noted that the destruction of the Vitamin present i n "butter fat occured on heating i n the presence of a i r . They concluded that the loss was due to chang es' of an oxidative nature. They also noted that the destruct ion took place rapidly at high temperatures hut provided the exposure to a i r i s extensive, considerable loss of n u t r i t i v e value takes pla.ce at temperatures as low as 37o. Steenbock, S e l l and Buell (233) found that butter fat showed a seasonal v a r i a t i o n i n the fat-soluble vitamin content when obtained from' s t a l l fed cows during the winter and pastur ed i n the summer. The note that the fat-soluble vitamin con tent of butter fat does not run closely p a r a l l e l to the yellow pigment; yet in general, due to determination by their content in thle feed, butter highly pigmented are r i c h i n the vitamin; butter low i n pigment should be looked upon with suspicion. In beef fats the relations are somewhat simi l a r ; those most pigmented are also generally richest i n their fat-soluble vit° amin content. These investigators demonstrated that the fa t - soluble vitamin withstood severe methods of saponification. This indicated that i t was not a fat and probably not an ester. The inactivating action of some fats on Vitamin -A i n other fats was studied by F r i d e r i c i a (85) i n •1925. He reported that rats did not grow on an apparently adequate diet when the butter fat y i e l d i n g the Vitamin A of the diet was mixed (after melting) with a brand of hydrogenated whale o i l . This hydro- genated whale o i l , had no toxic action on the oh ; the: growth, of 21. rats but had an inactivating action on the Vitamin A of butter fat when the two fats were mixed after melting at a low temp- erature. Two hydrogenated vegetable oils(hydrogenerated coc- panut o i l , s-nd hydrogenated hemp-seed o i l ) and a non hydrogen- ated vegetable o i l (cocoanut o i l ) showed neither any toxic effect on the growth of rats nor any inactivating action on the Vitamin A of butter f a t , t h i s action accordingly not being reg u l a r l y connected with the process of hydrogenating. Untreated abdominal pig fat did not inactivate the Vitamin A of butter f a t . o After being heated i n thin layers to 1G2 to 105 6. for 24 hours on exposure to a i r , the same pig fat acquired an inactivating action on the Vitamin A of butter fat when the two fats were mixed after melting© This inactivating action i s supposed to depend on the generation of peroxides i n the aerated heated f a t , the Vitamin A of butter fat being destroyed by these per oxides through oxidation© Sjorslen (219) showed that butter fat gave a reaction with sulphuric acid but not in such low concentrations as cod l i v e r o i l and colour indices do not attain so high a value© Lard did not give a reaction with sulphuric acid© A mixture of equal parts butter fat and non-heated l a r d gave a reaction with sulphuric acid. When butter fat Y/as heated 4 hours i n the a i r i t s a b i l i t y for a typ i c a l sulphuric a,cid reaction disappeared. Gillam (88) found that on the average, the r a t i o of carotene to xanthophyll in butter i s 14 to 1 by weight. Many investigators have studied the vitamin content of 21 Q Ci cow's milk. Kennedy and Butcher (131) noted that the presence of Vitamin A i n cow's milk i s e n t i r e l y dependent upon i t s occ ur ance i n the rat i o n . S t a l l fed cows w i l l produce a milk r i c h in vitamins provided their ration consists of a proper combin ation of grains and leafy foods. Bussel et. a l . , (195) found that i n fresh milk the vitamin content may vary a.s a result of change i n l a c t a t i o n period and of variations of provitamin A and the Vitamin A content of the ration when the assay extends over a period of months. They show that the percentage output . of vitamin expresses the relationship between intake and output but does not take into consideration the p o s s i b i l i t y that some of the Vitamin A in the milk may come from the body stores. The percentage of the factor which appears in.the milk decreases as the amount of i t in the r a t i o n i s increased and that the i n  crease i n Vitamin A content of the milk is not proportional to the increased consumption. In a comparison of Ayshire and Guernsey butters, Wilbur* Hilton and Hauge (255) found that, although the Ayshire butter contained 1.8mg carotin per 100 gr. of butter and the Guernsey 4.0mg. per lOOgr. of butter, they were i d e n t i c a l i n Vitamin A a c t i v i t y , Hathaway and Davis (103) demonstrated that the Vitamin A content of milk i s closely ass ociated with the butterfat content. They show that skim milk or separated milk containing only a small quantity of butterf at contains also only a small amount of Vitamin A. They also noted that Holstein cream i s mire potent i n Vitamin A than i s Jersey cream. As the percentage of fat i n the Holstein and Jersey 22. milk approach each other the difference i n Vitamin A content of the cream separated from them w i l l disappear. In further tests Hathaway and Davis (104) shov/ed that there i s l i t t l e difference i f any i n sour and sweet cream butter. Margarines on the other hand are poor sources of Vitamin A. Investigating the Vitamin A content of butter, Praps, Copeland and Hreichler (84) demonstrated that cows receiving feeds which were low i n Vitamin A content produced butter low in Vitamin A potency. The Vitamin A content of butter depends both upon the Vitamin A potency of the feed and the length of time the cow has been fed upon i t . When a cow i s on a. feed supplying i n s u f f i c i e n t quantities of Vitamin A, the Vitamin. A in the butter fat decreases with the period of time the cow has been on the feed or the stage of la c t a t i o n on account of the depletion of the reserve of Vitamin A stored by the cow at the beginning of lactation,, In the cow i t requires approximately eleven units of.Vitamin A to give one unit of Vitamin A in the butter. The cow uses Vitamin A much less e f f i c i e n t l y than poultry. According to Moore (163) carotene when supplied to cows undergoes conversion to Vitamin A. Guilbert and Hart (96) found that carotene being the pr i n c i p l e pigment of beef f a t , could be withdrawn from the adipose tissue during Vitamin A privat i o n without a coincident reduction of fat reserve. They showed that the carotene in the fat of c a t t l e constituted a s i g n i f i c a n t part of the tota l V i t -23. amin A reserve. These same investigators noted that the storage of Vitamin A i n the l i v e r of newborn calves i s r e l a t i v e l y low re gardless of the storage i n the dam. The Vitamin A content of the l i v e r s of normally fed oxen, guineapigs, rabbits, rats and dogs was tested biologic- a l l y by Simonnet, Busson and Asselin (218) and found to vary considerably. The l i v e r of the ox was the most potent while that of the guinea pig was the least potent. Using a dog. these same investigators (217) fed l i b e r a l d a i l y doses of carotene for a, month. I t wa.s then k i l l e d and the lungs, l i v e r , kidneys, brain and part of the body fat were tested for Vitamin A. They found that only the l i v e r and kidneys contained Vitamin A and these in approximately equal amounts. Ahmad and Malik (5) showed that animals d i f f e r i n their a b i l i t y to synthesize Vitamin A from carotene, as judged by the Vitamin A content of the l i v e r . With the a b i l i t y of rats to synthesize Vitamin A rated at 100, chickens give a rating of only 24, while rabbits give a rating of only 16 and cats zero. They conclude that the Vitamin A potency of feeds may vary for different animals according to the r e l a t i v e proportion of the Vitamin A and carotene present. Localisation of Vitamin A in tissues by a fluorescent microscope was noted by Querner (178). In the c e l l s of the l i v e r and other organs a fluorescent material was present which was ra,pidly destroyed by exposure to u l t r a v i o l e t l i g h t • This 24. "Leuchtstoff" was found to correspond to the amount of Vitamin A present. The n u t r i t i v e value of lard was extensively studied by Drummond et. a l . (62) i n 1920. They concluded that the pig was able to store up supplies of Vitamin A i n the body fat when fed a diet containing ample supplies of the factor. When the diet was deficient i n Vitamin A no appreciable amounts of the dietary factor could be found i n the body fat. The process employed i n the manufacture of lard causes a very marked des truction of the vitamin present in pig f a t . Investigating t h i s problem of the Vitamin A content of l a r d further, Mallon and Clark (151) concluded that lard made from leaves and back fat of hogs whose diet contained Vitamin A, did not contain an adequate supply of this vitamin to prevent xerophthalmia even when fed in large amounts. 25. Storage of Vitamin A i n the Body. The storage of Vitamin A i n the body was reported by Goldblatt and Soames (90) i n England and Steenbock, S e l l and Nelson (235) i n America i n 1923. Both groups of investigators cagie to the same conclusion that Vitamin A i s stored i n the l i v e r and that i t is stored in rough proportion to the amount contained i n the d i e t . Continuing this work further, Sherman and Cammack(207) showed that by feeding diets graded i n their content of Vitamin A, the rich e r the diet i n t h i s vitamin, the greater i s the am ount stored i n the body. The attainment of the maximum store of Vitamin A i s a process of gradual accumulation which i s re l a t i v e l y rapid i n i t s e a r l i e r stages and becomes slower as the maximum i s approached. A rapid storage of the entire maximum amount i s not possible. McGoord and Clausen (145) demonstrated that when 1 drop of halibut l i v e r o i l was added to the diet of the rat and the animal k i l l e d 24 hours l a t e r , there was a definite increase of Vitamin A i n the l i v e r and body f a t . When 4 drops were so added, marked increase was found i n a number of other organs and tissues, p a r t i c u l a r l y the adrenals. Working on the problem quantitatively Baumann, R i i s i n g and Steenbock (10) found that 95 % of the Vitamin A i n the body was stored i n the l i v e r . The remainder was located i n lung and kidney tissue. The minimum daily dose of Vitamin A nece ssary to produce storage i n the l i v e r was between 25 and 50 blue units. When Vitamin A was fed i n the form of halibut 26. l i v e r o i l , the amount stored i n the l i v e r was found to p a r a l l e l the amount administered, hut only 10 to 20 % of the Vitamin could be accounted f o r . When equal amounts of Vitamin A were fed to normal and to Vitamin A depleted r a t s , the l i v e r stor age was greatest i n the normal animals. When equal amounts of Vitamin A were fed to animals i n various stages of deple t i o n , the amount stored was inversely proportional to the state of depletion. The absorption and storage of Vitamin A to a large extent takes place within 6 hours after ingestion of the Vitamin. The elimination of Vitamin A from the l i v e r s of rats was studied by Davies and Moore (49). Female rats 18 months old, which had finished breeding and had been fed a diet well supplied with Vitamin A, received for 12 weeks, i n addition, a large amount of a Vitamin A concentrate. The mean Vitamin A concentration i n the l i v e r at the end of th i s time was 18,000 blue units per gram. The surviving animals then re ceived a diet deficient i n Vitamin A and the concentration i n the l i v e r f e l l r a pidly, being reduced i n 4 weeks to an average of 2,700 blue.units per gram; after 12 weeks i t f e l l further to about 400 units and v<ras s t i l l at that l e v e l after 24 weeks. The rate of depletion of these enormous reserves of Vitamin A was far more rapid than a rate representing the rats' daily requirement. 27. Transmission of Vitamin A Prom Parents to Young. The problem of Vitamin A transmission from the mother to the foetus has "been extensively studied by Dann (44) and '(46). He showed that Vitamin A i s normally found i n the l i v e r of the rat and rabbit at b i r t h , but only i n small amounts which cannot be increased by giving the mother a diet r i c h i n carotene during gestation. The store of Vitamin A i n the l i v e r of the young rat increases 2 or 3 f o l d during suckling, but the increase may be greater when the lactating parent receives a diet r i c h i n the factor. There i s a l i m i t to the amount of t h i s increase, due i n turn to a l i m i t a t i o n of the amount of Vitamin A which can pass into the milk. Working with chicks, however, Bearse and M i l l e r (13) have shown that the hen's diet markedly controls the amount of Vitamin A found in baby chicks. The work of Hale (101) i s interesting i n that he produced 3 l i t t e r s of pigs born without eyeballs or with very serious eye defects when the mother had received a diet de f i c i e n t i n Vitamin A before mating and during the f i r s t 30 days of gestation. 28 © Conversion of Carotinoids Into Vitamin A. The transformation of carotene into Vitamin A was studied by Copper (30) i n 1930. He used the absorption spec- u u t r a of rat l i v e r o i l s . He found that the band at 325 generally attributed to Vitamin A was absent from the absorp tion spectra of the l i v e r o i l s of rats suffering from Vitamin A deficiency, but was shown by the l i v e r o i l s of similar rats which had been subsequently cured by massive doses of carotene, u u The 325 / / band was absent from the absorption spectrum of carotene, therefore, he concluded, the substance responsible for the exhibition of this band by the l i v e r o i l s of carotene- treated r a t s , had been synthesised i n vivo from the carotene, Moore (161) noted that the conversion of carotene into Vitamin A took place i n the l i v e r , but the efficiency of the conversion was by no means quantitative. The absorption of carotene from the i n t e s t i n a l tract was investigated by Ahmad (3) v/ho observed that i t may be con siderably affected by the composition of the diet. Studying the isomers of carotene Karrer, Euler and Hell stroza (128) showed that the blue values given by alpha and beta isomerides i n the Sb.Clg reaction were i d e n t i c a l . Rat growth tests for Vitamin A a c t i v i t y however, demonstrated that the beta i s more active than the alpha isomeride. The curative effects of 5 to 10 y of pure carotene on rats suffering from symptoms of Vitamin A deficiency was d e f i n i t e l y established by Glanzmann (89). He found that the blood p l a t e l e t s of the rats f a l l i n numbers rapidly when 29.. diets deficient i n Vitamin A are given, and r i s e rapidly when carotene i s supplied. The Vitamin A a c t i v i t y of palm o i l s was found to be "in accord with their carotene content by Ahmad (4). After hydrogenation, however, a l l the a c t i v i t y was l o s t . Sea and Drummond (180) investigated the formation of Vitamin A from carotene i n the animal organism. In the incu bation of the l i v e r of rats and cats with solutions of caro tene, they f a i l e d to demonstrate the formation of Vitamin A. The intravenous i n j e c t i o n of c o l l o i d a l suspensions of carotene into the l i v e r s of cats which had received a Vitamin A d e f i  cient diet, produced no detectable increase i n the Vitamin A content of the l i v e r s as shown by spectrographic examination of the non-saponifiable fractions before i n j e c t i o n , and 4 to 72 hours after i n j e c t i o n . Conversion of carotene into Vitamin A i n the animal body was studied by Skarzynski (220). He made spectroscopic and colorimetric examinations of extracts from the caecum, spleen, l i v e r and serum of young rats which had developed xerophthalmia on ©• Vitamin A deficient diet and which had died or been cured with carotene ( 4 mg. i n 36 days). In the extracts from the deficient group which had died, no trace of Vitamin A or carotene could be detected. In the group which received carotene, Vitamin A was found i n the l i v e r and serum, and carotene i n the caecum and probably i n the l i v e r ; the spleen contained neither. The author sug gests that transformation of carotene does not take place i n 5 0 . the gut as supposed "by Ahmad (4), but in the l i v e r from which i t passes into the blood. The Vitamin A content of rat l i v e r after feediing .alpha, beta and gamma carotene was reported by Brockmann and Tecklenburg (22). They examined the unsaponifiable fraction of the l i v e r o i l with SbCl 3 and found that beta carotene produced 2 or 3 times as much Vitamin A as the other two. Moore (164) reported i n 1933 that carotene i s u t i l i s e d i n the body as e f f i c i e n t l y as preformed Vitamin A at levels approaching the minimum dose. Using a spectrophotometer method, Drummond and MacWa.lter (68) observed that after i n j e c t i n g carotene into the portal c i r c u l a t i o n of rabbits no increase i n the Vitamin A i n the l i v e r takes place u n t i l eight days after the experiment. They suggest the p o s s i b i l i t y that formation of the vitamin occurred within much shorter periods but either i t was u t i l  ised and converted into another substance or that the amounts were too small to be detected by their technique. The fats of carotene introduced into the circ u l a t i o n was investigated by Drummond, Gilding and MacWalter (69). These investigators injected intravenously into anaesthetised cats a c o l l o i d a l suspension of carotene i n isotonic glucose solution. Blood samples were taken i n the ensuing 15 minutes and after death, the remaining blood and organs were examined for carotene Wo carotene was. found i n any of the blood samples and, of the organs, only i n the lungs, l i v e r and spleen. In the lungs the 31. carotene appeared to be trapped in the c a p i l l a r i e s . The greater part was absorbed s e l e c t i v e l y by the l i v e r and was chi e f l y located i n the Kupffer c e l l s . Working with r a t s , Drummond and MacWalter (70) re ported that Vitamin A and carotene were evenly distributed through the three lobes of the animals l i v e r , Pure c r y s t a l l i n e xanthophyll was shown not to be i d  en t i c a l with Vitamin A by Willimott and Moore (257) i n 1927. A few years l a t e r Kuhn, et. a l . (134) showed that the xantho- p h y l l s , l u t e i n , zeaxanthin and violaxanthin were negative as well as azafrin methylester, dihydrocrocetin dimethylester and chlorophyll. Certain microorganisms were found by Baumann, Steen- bock and Ingraham (9) to synthesize carotene. Whenever an organism showed Vitamin A a c t i v i t y as determined by feeding experiments, enough carotene was found to be present to acc ount for that a c t i v i t y . The Vitamin A a c t i v i t y of microorgan isms did not appear to be affected by the presence of yellow pigments other than carotene. They concluded that since spectrographic determination f a i l e d to reveal any absorption band at 328 M i t i s exceedingly improbable that Vitamin A as such i s generally present i n bacteria. Attempts, however, to effect the transformation of carotene into Vitamin A by microorganisms f a i l e d . 32» VITAMIN A 'THERAPY III Ool&B I t i s generally recognized that a lack or deficiency of Vitamin A i n the Ration results i n the greater s u s c e p t i b i l i t y of animals as well as man to various Respiratory infections. In view of the wide prevalence of simple colds i n man, con siderable emphasis has been l a i d on the inclusion of optimum amounts of Vitamin A i n the diet. As a matter of fact, direct evidence that Vitamin A plays the role of an an t i - i n f e c t i v e agent, as has been commonly accepted, Is lacking. In t h i s connection i t i s interesting to note Cameron's work (27), (28) and (29), on Vitamin A Therapy i n colds. In an experiment (27) conducted at the University of West V i r g i n i a , he used f i v e groups of women students. The members of one group took a tablespoonful of cod l i v e r o i l daily from November to March 1932-33, and the other groups an equivalent dose of a Vitamin A concentrate, an inert t a blet, a cod l i v e r o i l residue i n tablet, form, and no medication. Records were made twice a week during the experimental period on the presence and duration of colds. At the end of the experiment i t was found that the number of days with colds reported from the group taking no medication was considerably higher than from any of the other groups. The Vitamin A concentrate and the eod l i v e r o i l residue tablets appeared to give s l i g h t l y better results than the cod l i v e r o i l . The following i s a quotation from the result of the above experiment. "Ho conclusion i s as yet j u s t i f i e d from these results, but the volunteers p a r t i c i p a t i n g i n the experiment were unanimous i n reporting subjective improvement from cod l i v e r o i l and i t s concentrates. This improvement took the form of increased appetite, weight, or endurance, or r e l i e f from skin eruptions or from sinus trouble, as well as i n reduction i n number and duration of colds. These changes can probably be attributed to the improved n u t f i t i o n r e s u l t i n g from an abundance of Yitamin A, and t h i s improved n u t r i t i o n i s worth while i n i t s e l f whatever f i n a l conclusions are reached with reference to Yitamin A Therapy against common Golds". In his second experiment (28) the effect of increasing the Yitamin A intake of men and women students, either by increasing the amount of foods r i c h i n Yitamin A, of by direct dosing with cod l i v e r o i l , haliver o i l , cod l i v e r o i l tablets or carotene solution, was studied i n regard to the number and severity of colds during a period of two years. The control groups received no medication or a lactose supplement. S t a t i s t i c a l examination of the results indicated that increase i n the Vitamin A intake did not decrease the number of colds, but did tend to lessen the duration and severity of the infection. Approximately f i f t y percent of the subjects of the test reported improvement i n powers of endurance, i n appetite and i n decrease of skin eruptions. He concludes, however, 34. that Vitamin A Therapy should not be considered as a specific against oolds. From his t h i r d investigation (29) he gave the follow ing conclusion: "Whether the effect of Vitamin A upon colds, therefore, rests upon i t s a b i l i t y to maintain intact a barrier of healthy mucous membrane to act as a l o c a l defense against invasion by virus or bacteria, or whether i t s effect i s due also to i t s influence on c i r c u l a t i o n and nervous mechanisms which weather and dietary imbalance undoubtedly play a part, i t has been shown to be a factor i n the duration of colds i n adults and worth a t r i a l i n a l l susceptible individuals." In the Dressing of Wounds. The l o o a l use of Vitamin A i n the treatment of wounds has been observed by many workers. Horn and Sandor (123) observed that dressings of a concentrate of Vitamin A, containing two thousand international units i n 1 ml. of an o i l y medium, influenced favorably the healing of f l e s h wounds and of infected tissues. Zoltan (269) obtained good results when purulent wounds were treated with an ointment containing cod l i v e r o i l , carotene and irradiated ergosterol. Direct application of crude cod l i v e r o i l was found by Steel (225) to be most successful i n the treatment of severe burns and ulcers. The use of the cod l i v e r o i l prevented the formation of scar tissue and pigmentation following healing. Drigalski (55) made wounds of a measured size with scissors i n the skin of guinea pigs and dressed them with a cod l i v e r o i l ointment "Norguentolan1', or with the same ointment through which a i r had been bubbled at 150° c. for t h i r t y - s i x hours. B i o l o g i c a l tests on rats showed the destruction of Yitamin A by th i s procedure to be complete. Healing was considered to be more favorable and more rapid i n wounds treated with unoxidized "Unguentolan", whether on the same or on different animals, even when the wounds were experimentally infected with stophylococci or streptococci. Shortly after the above observations had been made "Strauss (259) reported that over one hundred twenty cases of wounds, burns, etc., were s a t i s f a c t o r i l y treated with "Unguentolan". He stated that pain was reduced since the wound seldom needed redressing and drains were unnecessary. Using t h i s same ointment "Unguentolan", Dreyfus ( 5 3 ) observed that i s applic ation to burns of the second and t h i r d degrees resulted i n rapid and complete healing. Similar results were obtained i n purulent and gangrenous ulcers, but l i t t l e improvement was obtained i n erythrodermia ichthyosiforma. He suggests that the healing might be due to Vitamin A or D, or the Phosphorus or Iodine content, or the protective effects of the preparation or a combination of these factors. An extensive study of the effect of ointments contain ing Vitamins on the healing of wounds was conducted by Lauber and Rooholl (135). Surface wounds on white mice were treated with various ointments containing Vitamins "H", "B", "C", "A", "D", and with control ointments without Vitamins. The wounds treated with vogan (Vitamin A) i n a base containing Cholesterol healed best; vogan i n a vaseline-lanoline base was considerably less e f f e c t i v e , and i n large doses even tended to i n h i b i t healing. Vitamin "H" had some ben e f i c i a l effect and Vitamin "B", very l i t t l e , while Vitamins "C" and "D" appeared to hinder the healing process. Dri g a l s k i (56) disagreed with the above results and stated.that they would go no further than the authors" own findings. The effect of Vitamins on the growth of tissues and transplanted tumors was studied by G-ordonoff and Ludwig (92). They noted that the growth of fibroblasts from embryo chicken heart, or of small transplants of mouse tumors, was very much l e s s ' i n plasma from rats which had been kept on diets deficient i n Vitamin A, than i n plasma from rats which had received large doses of vogan. The growth of normal and path ological tissues seemed also to be inhibited i n plasma from rats deprived of the Vitamin "B" complex, but not i n plasma from rats receiving doses of yeast. There was no difference i n plasma from rats on MoCollum's rachitogenic diet 3143, whether the animals received Vitamin "D" or not. The presence of Vitamin "0" seemed to have an i n h i b i t i n g e f f e c t , as shown by cultures i n plasma from scorbutic and normal guinea pigs. 57. The work of Turner and Loew (249), shows that carotene i s protective against "bacterial invasion of the upper respirat ory tract and the development of the pathologic systemic conditions accompanying Vitamin A deficiency i n rats. Carotene therapy reduces the number of spontaneous supperative lesions i n the upper respiratory tract occuring i n animals deprived of Vitamin A; Xerophthalmia i s cured i n one hundred percent, and normal health regained i n seventy-four percent. The percentage incidence of bacteria i n the nasal cavities and middle ear i n animals fed active carotene i s noticeably less than that encountered i n animals given faded carotene or that i n animals receiving no source of Vitamin A. In another investigation (247), they noted that the withdrawl of Vitamin A from the monkey does not develop a characteristic suscept i b i l i t y toward infection of the upper respiratory tract. They also observed while working with rats (248), that the age of the animal, the previous storage of Vitamin A, the season, had no marked effect on the bacterial f l o r a . In reviewing the researches on the value of ood l i v e r o i l plastus i n the healing of burns and wounds, Gortzen (92), points out that ood l i v e r o i l does not i n h i b i t the growth of bacteria i n cultures. He claims i t s . healing effect cannot, therefore, be explained by any bactericidol action on infected tissue. Hayaski (107) suggests that i t i s the l i p o i d content of cod l i v e r o i l that has the b e n e f i c i a l effect on infected 38. wounds and not Vitamins "A" and "D". Antagonism Between Vitamins "A" and "C" and Vitamin "A" and  Thyroxin Wendt and Schroeder (253) studied the antagonism between Vitamins "A" and "C". When guinea pigs were given large simultaneous, daily doses of Vitamin "A" (1.5 ml. vogan) and Vitamin "C" (60 mg, Cebain Merck), they showed no signs of hypervitaminosis "A". I f only 0.25 to 0.5 ml. daily of vogan was given together with 10 mg. Cebain Merck, there was not even retardation of growth. The Vitamin "A" content of the l i v e r s of the guinea pigs receiving both Vitamins was lower than that of others having Vitamin "A" only, but the Vitamin "G" content of the organs was not affected by the presence or absence of Vitamin "A", While studying the problem of hyperthyroidism, Schneider and Windmann (201) noted that when rats were f u l l y depleted of Vitamin "A", oral administration of carotene for ten days did not cause deposition of Vitamin "A" i n their l i v e r s j although administration of the Vitamin i t s e l f caused storage i n large amounts. When the l i v e r s were depleted of glycogen by i n j e c t i n g the animals with the thyrotropic principle of the pi t u i t a r y , f a i l u r e of deposition of Vitamin "A" after administration of carotene also occured. This f a i l u r e i s attributed to an increased demand for Vitamin "A" rather than to i n a b i l i t y of the l i v e r t-o convert carotene into the 39. Vitamin. They suggest that the metabolism of glycogen i s closely bound up with that of Vitamin "A", Wendt (254) obseryed that an excess of thyroxine increases the rate at which Vitamin "A" i s metabolised. In hypothyroidism the capacity to convert carotene to Vitamin "A" and to store carotene and Vitamin "A" i n the l i v e r are reduced. Further studies by Sherwood and Luchner (216) dealt with the h i s t o l o g i c a l effect of ood l i v e r o i l on the thyroid gland. They found that excessive daily doses of carotene, "haliver" o i l or cod l i v e r o i l , over a period of several weeks, resulted i n a;depletion of the o o l l o i d and an increase i n the height of the e p i t h e l l o l c e l l s i n the thyroid glands of young albino rats. Carotene and "haliver" o i l produced i n addition a marked increase i n stroma. Administration of potassium iodide, i n amount approximately equivalent to the iodine i n 0.3 ml. daily of ood l i v e r o i l , had no effect up to the eighteenth day. There was then an increase i n c o l l o i d and the a c i n i were distended, with extremely low epitheluim. They concluded that the iodine of ood l i v e r o i l was less available for metabolic a c t i v i t y than that of potassium iodide, possibly being l o s t by excretion of the o i l , and that such changes i n the gland as were produced by cod l i v e r o i l were due to Vitamin "A", rather than to iodine* The work of Abelin ( l ) noted that the ingestion of a Vitamin "A" preparation by r a t s , diminished the r i s e i n basal 40. metabolism r e s u l t i n g from thyroid administration. The action of excess of doses of thyroid i n lowering the growth rate was also p a r t i a l l y counteracted by administration of Yitamin "A", which was shown to increase the glycogen content of muscles. He suggests that Thyroxine and Vitamin "A" are antagonistic because of t h e i r opposite influence on l i p o i d and carbohydrate metabolism. In spite of the w e l l recognized importance of Vitamins i n n u t r i t i o n , p r a c t i c a l l y no work has been done on the associative action of the various Vitamins. While i n the case of poultry and other domestic animals the minimum and optimum amounts have been f a i r l y accurately determined, no information i s available as to the most satisfactory combin ation of any two or more Vitamins to promote normal growth, health and reproduction. BIOLOGICAL ASSAY OF YITAMIN A USING .THE RAT AS THE TEST ANIMAL. .  Feeding Technique. The technique used in carrying out the Vitamin A assays i s described i n great d e t a i l by the United States Pharmacopoeia (see appendix 1). These recommendations have arisen as the result of very careful work conducted during the past decade by several investigators of whom Coward of Great B r i t a i n has been the leading investigator. In 1920 Drummond and Coward (59) stated that i n test ing foodstuffs for the presence of the fa,t-solub"le vitamin the greatest care- should be devoted to ensuring that the basal dietary i s rendered as free from that vitamin as possible. They give d e t a i l s for the preparation of a highly p u r i f i e d r a t i o n . They conclude that the f a i l u r e to work with a suff i c i e n t l y pure diet may lead to c o n f l i c t i n g and misleading r e s u l t s . St eenbock,;: Nelson, and; Black.-(236) noted that the ab sence of growth on suitably constituted rations cannot be taken as an indication of the absence of Vitamin A unless the an t i r a c h i t i c factor i s supplied. Steenbock and Coward (237) advocated the use of the incidence of ophthalmia as a, sign of exhaustion of the animals' store of Vitamin A i n preference to cessation of growth. The two are often simultaneous, but the use of the former c r i t e r i o n prevents loss of animals through the very sudden and rapid de cl i n e that may ensue while waiting to become certain that growth 42. has r e a l l y ceased. Growth ceases during the worst stages of ophthalmia and i s only resumed when definite improvement i n the animals' condition i s observable. In the b i o l o g i c a l assay for Vitamin A according to J a v i l l i e r and Emerique (126) i t i s necessary to observe cert ain precautions with regard to: an adequate supply of Vitamin D i n the diet of the experimental animals; 2. The period of the test proper which should begin only after there has been a loss in weight of 10%; 3. The large number of animals (10, 20, 30) to be used i n the tests of each dose; 4. The similar sex of a l l animals compared; 5. The manner of administration of the sub stance tested. The J a v i l l i e r unit of Vitamin A is defined as "the quality of the vitamin which, when added to the minimal dose of maintenance (per lOOg. of rat's body weight under the specified conditions) w i l l induce a growthresponse l a s t i n g at o least 30 days, the angle of growth being 30 and the gain in weight about 30%." Sherman and Batchelder (208) claim on s t a t i s t i c a l grounds that with levels of Vitamin A feeding which induce a gain i n weight of about 3 grams a week, a decrease of about 25% or an increase of 33% i n the value of the daily intake of Vitamin A can undoubtedly be measured by this method. According to Honeywell, Butcher and Ely (121) the response to administration of Vitamin A of rats depleted of this factor i s influenced by the type of yeast used i n the diet. This influence i s thought to be a function of the Vitamin B complex. The authors Suggest that Vitamin A' consists of two factors, one possessing anti-xerophthalmic properties, concerned i n maintaining the normality of the cornea and other e p i t h e l i a l tissues, and another with a purely growth-promoting function. Nelson, Walker and Jones (167) noted that when single doses of material containing Vitamin A were given to rats de prived of the factor, the growth response and subsequent dur ation of l i f e of the animal were d i r e c t l y proportional to the amount of Yitamin A given. The investigators Polak and Stokvis (175) found that daily doses of 0.5 to 1.0 gamma of carotene were sufficient to prevent and cure xerophthalmia i n rats on a diet deficient i n Yitamin A. Working along si m i l a r lines too(1.75) were Baumann, R i i s i n g and Steenbock (11). A solution of carotene i n cotton seed o i l in which no deterioration could be detected spectr o- scopically throughout the experiment, was administered for 8 weeks to rats on d i e t s deficient i n Yitamin A. A weekly dosage of 3 to 5 gamma of carotene did not cure or prevent xerophthal mia but 10 gamma did so and also promoted moderate growth; amounts larger than 20 gamma weekly did not enhance growth. They found i t impossible to maintain rats at a growth rate of 3 grams per week, and also emphasise the danger of using a standard of animal response rather than the international stan dard substance for reference. Coward and Key (42) found that the mean weight i n  crease of groups of rats receiving 7 mg. of cod l i v e r o i l 44. weekly, after depletion of Vitamin A, was the same whether 1 mg. was administered da i l y , or 4 mg. on Thursday and 3 mg. pn Monday. The high degree of s t a t i s t i c a l probability that the result i s v a l i d i s worked out. The determination of Vitamin A values by a method of single feedings was reported by Sherman and Todhunter (209). V/hen male or female rats, depleted of Vitamin A received single large doses of 28, 56 or 112 gamma of carotene, the weight i n  crease and survival time were proportional to the dose for each sex. The value best expressing this relationship was obtained by p l o t t i n g weight increase and subsequent decline against the -time i n days and estimating the area, bounded on the upper side by this curve and on the lower side by a base l i n e drawn para l l e l to the abscissae through the point representing the av erage weight loss of a group of untreated controls, at the end of the f i r s t week from beginning of dosage. This method i s specially recommended for perishable or unstable materials. 45. Results of Vitamin A Deficiency i n Bats. In studying avitaminosis A of r a t s , McCollum, Simmonds and Becker (144) found that the feeding of excessive amounts of any one element or ion did not induce ophthalmia. The h i s t o l o g i c a l changes are very well described by Tyson and Smith (250) who say "The characteristic h i s t o l o g i c a l changes found i n rats fed a diet lacking Vitamin A are the substitution of s t r a t i f i e d heratinizing epithelium for normal epithelium in various parts of the respiratory t r a c t , aliment ary t r a c t , eyes, paraocular glands and the genito-urinary tract i " Replacement of the normal epithelium of . the body organs with keratinized c e l l s was observed i n mice maintained on V i t  amin A free diet by Wolfe and Salter (265). Xerophthalmia dev eloped i n from 25 to 120 days although metaplastic changes i n the respiratory tract were found previously to t h i s . Sure and Smith (242) noted that Vitamin A deficiency i n rats did not alter the true blood sugar, the alkaline reserve of the blood or the glycogen content of the l i v e r . The reducing non-sugars, however were frequently increased, thus r a i s i n g the apparent blood sugars. Pathologic changes i n the tissues during early stages of Vitamin A deficiency were investigated by Thatcher and Sure(244) Their results showed metaplasia i n the posterior portion of the tongue, i n the salivary glands, and i n the respiratory and urinary tracts i n the majority of cases of early Vitamin A def iciency, a l t ho ugh i n many individuals there were as yet no ex ternal symptoms such as cessation of growth, persistence of the cornified c e l l stage of the oestrous cycle or incipient ophth almia. The effect of Vitamin A on metabolism was recorded by Chevallier and Baert (33). Young rats with t y p i c a l signs of Vitamin A depletion showed a basal metabolic rate 13 per cent higher than that of similar normal rat s . Rats receiving daily 2G00 U.S.P. units of Vitamin A and a guineapig receiving daily 10,000 such units, showed a rate lowered by 10 to 15 percent. According to Malmberg (152) increase i n weight and i n t a i l length siakens simultaneously i n young rats in process of depletion of Vitamin A. Prom Tornblom's (245) experiments no sign i f i c a n t d i f f  erence could be detected i n the oxygen consumption of rats dev eloping well maxked symptoms of deficiency, on a diet deprived of Vitamin A, and rats maintained i n health on the same diet, by the daily addition of 10 gamma of carotene. Hematopo&ietic function i n deficiency diseases was i n  vestigated by Sure, et. a l . (241). Prom the i r work they con cluded that after Vitamin A deficiency has progressed to t he ophthalmic stage inanition complicates the blood picture, so that the high figures of hemoglobin and erythrocytes may be an expression of anhydremia, indicated by the concentration of t o t a l blood solids of the pathological animals as compared with normal animals of the same age and weight. In the pre- ophthalmic stage they found a suggestion of an anemia charact erized by reduction i n either hemoglobin or erythroytes. Ho 47. connection could toe established between aisritaminosis and pern icious anemia. Batchelder (7) found that urinary c a l c u l i were present in rats receiving small quantities of Yitamin A hut were not present i n rats receiving no Yitamin A. He also noted that the general "breakdown resulting in death, though accompanied "by sim i l a r symptoms, was postponed to successively l a t e r stages as the Yitamin A content was increased. Sutton, S u t t e r f i e l d and Krauss (243) demonstrated that nerve degeneration occurred about the same time as ophthalmia and became extensive before external symptoms of paralysis appear. Working with a polarizing microscope on formalinr.fixed frozen peripheral tissues, they showed that after external symp toms of paralysis appear, adequate amounts of Vitamin A do not relieve them but nerve degeneration i s arrested. The data published by Evans (80), demonstrated that i n  adequate Vitamin A injures the female reproductive system so that f e r t i l i z a t i o n and implantation often f a i l . In this respect i t d i f f e r s r a d i c a l l y from the reproductive impairment due to low Vitamin E content, where, t y p i c a l l y the eggs are always healthy and implantation takes place but resorption follows. A study of the effects on the male genital organs was carried out by Sampson and Korenchevsky (196). They observed that in most rats on a Vitamin A deficient diet the weights of the testes and especially of the penis and of the prostate with seminal vesicles were greater than those of rats kept on a 48. paired complete d i e t . The testes of rats on the deficient diet were oedematous. The decreased food intake produced no not iceable effect on the penis, decreased the weight of the pros tate with seminal vesicles and in some cases s l i g h t l y increased the weight of the testes. 49. Assimilation of Vitamin A in the Presence of_Mineral O i l . Rowntree (191) i n 1931 undertook a study of the effect of the use of mineral o i l upon the absorption of Vitamin A. V(hen' rats previously deprived of Vitamin A were given small doses of cod l i v e r o i l s u f f i c i e n t only to allow of slow growths the addition of mineral o i l to the diet i n amounts equivalent to the human therapeutic dose cause the anijjials to lose weight and die. When, however, the dose of cod was an adequate one, t the ingestion of the mineral o i l did not appear to interfere with the absorption or u t i l i z a t i o n of the vitamin. The assimilation of carotene and Vitamin A i n the pres ence of mineral o i l was studied by Dutcher et. a l . (71) i n 1934 Rats depleted of Vitamin A recovered when receiving 40 mg. of butter"daily, but-not when they also received 100 mg. of min eral o i l , with or without hydroquinone. A comparable result was obtained with carotene but the adverse effect was overcome i f the carotene was considerably increased r e l a t i v e to the mineral o i l . Mineral o i l exercised scarecly any unfavorable effect when administered with cod l i v e r o i l or a Vitamin A con centrate. When carotene was fed with mineral o i l , i t could be recovered from the faeces i n an am&unt proportional to that fed, indicating that the action of the o i l was to i n h i b i t the absorp tion of carotene from the gut. The influence of Vitamin A on fat metabdlism was f i r s t investigated by Drummond (58) i n 1919. He found that rats were able to absorb large amounts of fa t t y acids, and presumably synthesise these into f a t s , i n the absence of Vitamin A i n the 50-. di e t . Several years l a t e r , Green (95) worked along similar 1 l i n e s . From his work no evidence was obtained that a diet • very r i c h i n fat accelerates the u t i l i z a t i o n of Vitamin A i n the body. The diminuation i n the t o t a l crude fat of the whole body i s not significantly, different i n . the Vitamin A deficient rat from that in the rat on a. complete diet whose food intake is, r e s t r i c t e d to that of the deficient animal. A r i s e i n the iodine value of the f a t t y acids of the l i v e r , coincident with a f a l l i n the percentage amount of fat i n the l i v e r , occurs i n the end stages of Vitamin A deficiency. The r i s e i s of a sim i l a r loss of l i v e r fat has been produced by a r e s t r i c t i o n of food intake or by i n f e c t i o n . Working on the esterase content of the blood-serum Green (94) reported that Vitamin A deficiency i n the rat"produced a large and progressive decrease of this enzyme. Relatively large amounts of cod l i v e r o i l i n the diet, however, produce a r i s e i n the serum esterase content well above normal. 51. Influence of Vitamin'.A on Fat Metabolism. The toxic effects due to large doses of Vitamin A was studied by Dri g a l s k i (54) i n 1935. He noted that rats given bread, oats and water together with 20,000 or 40,000 rat units of Vitamin A daily in the form of a concentrated commercial preparation died i n 5 to 19 days with loss of weight and the development of conjunctivitis and hemorragic r h i n i t i s . Control rats, given the same concentrates after the destruction of the Vitamin A by u l t r a v i o l e t i r r a d i a t i o n , remained i n normal health. Tn<the same year Bomagk and Dobeneck (52) made a detailed h i s t o l o g i c a l examination of rats of 80 gram weight, which had received 100,000 "units" of Vitamin A i n I c e . of sesame o i l da i l y for a week. Kupffer's star c e l l s of the l i v e r , the pulp c e l l s of the spleen, endothelial c e l l s i n the. kidney and epith e l i a l c e l l s of the stomach were f i l l e d with f a t , as compared with those of controls, receiving sesame o i l only. The c e l l s of the gastric epithelium showed abnormally rapid multiplies, t i o n . A year later Davies and Moore (48) fed massive doses of a Vitamin A d i s t i l l a t e to young rat s . They observed that their doses proved t o x i c , causing emaciation, haemorrhagic r h i n i t i s , loss of hair round the mouth and in one rat gross lung lesions. Their attempts to induce hypervitaminosis A by feeding carotene f a i l e d on account of i t s limited s o l u b i l i t y i n fats. Lewis and Reti (136) studied the effects, of large doses of carotene when fed to ra t s . They noted that when young rats were; given large amounts of a Vitamin A concentrate (vogan) they declined i n weight, and died within 10-14 days, with the 52 a usual symptoms of hypervitaminosis A (emaciation, conjunctivitis skin lesions on the face and paresis of the hind legs). Yi/hen - they fed 40 mg. of pure carotene dissolved in an innocuous o i l there were no i l l e ffects. P r a c t i c a l l y no work has been reported on the toxic effect i f any, of Vitamin A i n domestic animals. The majority of i n  vestigations to date have been concerned with determining the minimum and optimum requirements of domestic animals for Vitamin A. ;CHEMICAL AND PHYSICAL PROPERTIES Off VITAMIN A. The Chemical Structure of Vitamin A. A great deal of work has "been done on the Struct - and Chemical composition of Vitamin A and carotene* contained 9 easily saturated double bonds in a conjugated series, further conjugated with 2 other unsaturated linkage either double bonds or cyclopropane linkages. He suggested that this system was responsible for the colour of the carotene molecule. Bruins, Overhoff and Wolff (23) calculated the ratio of the molecular weights of carotene and Vitamin A and from this they deduced a molecular weight of about 330 for Vitam: A. Erom this value they concluded that the assumption of a simple chemical r e l a t i o n between Vitamin A and carotene to appear improbable. In 1931 Karrer, Morf and Schopp(129) suggested the following formula for Vitamin A,(C20H30O)» which i s now the "provisional formula." Smith (222) interpreted his observations that caroten - CH- OH- Cr CH-. CH= CH-Ci CH- CHgOH :-CH* A year l a t e r , Heillbron, Morton and Webster ( 1 1 0 ) 5 4 . '• suggested the previous formula, and also the followi C H H 2 C H 2q H GH G - C H N C C Hg •^3^' 3 9 H3 C'H=dH-C=CH-CH2QH The next year Karrer, Morf and Schopp (130) synthesised perhydro-vitamin A, (C ? QH 0) with the following structure: H, H R / \ C ( G H 3 ) 2 C H <? C H - C H - C H 2 - C H - C H P - C H P - C H Q - C H - G H 0 » C H o 0 H H C H ^ (L<, d 2 2 The i d e n t i t y of the substance thus synthesised with that prepared by the reduction of the natural Vitamin A was estab lished by comparing the b o i l i n g point, density and molecular ref r a c t i o n . According to Drummond (67) the formulae for a l f a and beta carotene are as follows: 55. © j c\j . o O . - o Oi o t o o W o i - o . t o X o — o W CM O — O , 0 CD CD +> O n3 +» CD pq t o w o — o - o - 0 = t 1 w t o 0 W 11 0 w 0 8 - O H K CD 0 > 1 •H W tio O K 0 w -*-> < 0 9 CD a O fH •rt II CD '3 Cd 0 4-» 1 CQ •H w . -M i> •H rH w (—4 0 0 «• CO K © 0) O c rH 11 CD 3 •O O I O CD K r4 rH 0 o3 O SI O g CES 0 1 j p M CD O w a 0 56. He also summarized and compared the properties of Vitamin A and carotene. * Carotene , Synthesised i n plant Orange red Antimony t r i c h l o r i d e gives steel blue colour, o hand at 5900 A. Vitamin A.s Stored in animal Almost colourless o Band i n U.V. at 3280 A Royal blue colour with antimony t r i c h l o r i d e , o band at 5720 and 6060 A. 57 The Effect of Ghemioal and Physical Agents on Yitamin A Observations upon the chemical nature and properties of Yitamin A were reported by Drummond (57) i n 1919. He noted that the fat soluble accessory food factor A was readily destroyed by short exposures (one hour to a'tempature of 100°), Destruction was less rapid at temperatures of 50° to 100°# He believed at this time that the destruction was not due to oxidation or hydrolysis. The factor was not extracted from o i l s by water or d i l u t e acid but was soluble i n alcohol. The fat soluble A faotor could not be i d e n t i f i e d with any of the recognized components of f a t s , such as glycerol, saturated or unsaturated f a t t y acids, cholesterol, l e c i t h i n , phosphatides or the lipochromes. In conclusion he suggested that i n view of the low temperature at which destruction occurs, fat soluble A may be a l i a b l e substance of i l l - d e f i n e d constitution. Working with whale o i l s Delf (50) found that the temper ature used for extracting the o i l played an important part i n the potency of the o i l . In his work he also found that the sperm whale gave from i t s head an o i l richer i n Yitamin A than from the blubber. Emmett and Luros (77) showed d e f i n i t e l y that benzine or acetone did not extract from the pancreas, thymus and suprarenal glands a f a t that contained the fat soluble A Yitamin. 58 Brummond, Channon and Coward (64) prepared a concentrate of Yitamin A from Cod Liver O i l and examined i t a chemical properties. They found i t contained no detectable traces of iodine or nitrogen, so that these elements are not related to the physiological action of the o i l i n promoting growth. Their results showed that approximately 50% of the unsaponifiable matter from Cod Liver O i l i s cholesterol, which may be removed quantitatively without loss of vitamin a c t i v i t y . Yitamin A was found on d i s t i l l a t i o n to pass over at 180-220° at 2-3 m.m. A chemical examination of the active d i s t i l l a t e indicated the presence of (a) a saturated s o l i d alcohol, (b) the unsaturated hydrocarbon spinaeene, (c) one or more than one unsaturated alcohol, b o i l i n g about 200° at 2-3 m.m. Spinaeene and the s o l i d alcohol were without Yitamin A action. Cody and Luck (25) studied the effect of different gases upon Yitamin A. They discovered that SOg rapidly destroyed the active p r i n c i p l e of Cod Liver O i l . A l f a l f a and spinach, when sulphured i n the dry and green conditions, experienced no loss of Vitamin A a c t i v i t y . Phosphorous penta- chloride, chlorine, acetyl chloride, nitrous fumes and Benedicts' alk a l i n e copper reagent destroyed the active principle i n Cod Liver O i l . Prolonged treatment with sodium bisulphite had the same effect. Hydrogen sulphide, ethylene, ammonia and Benedicts' reagent after neutralization exhibited no destructive action. Formaldehyde had l i t t l e effect. 59 Hydrogen peroxide brought about a p a r t i a l loss. They concluded that Vitamin A a c t i v i t y i s the property of a specific atomic grouping rather than of a specific molecule, and that the active p r i n c i p l e of Cod Liver O i l possesses aldehyde properties, The destruction of Vitamin A by u l t r a - v i o l e t radiations was studied by Norris (168), He found that the curve of destruction of Vitamin A i n Cod Liver O i l by u l t r a - v i o l e t i r r a d i a t i o n , determined by means of b i o l o g i c a l experiments, differed i n several particulars from that obtained by use of the color t e s t . The f i r s t showed no disappearance of the Vitamin up to one hour's exposure, thereafter there was a rapid destruction, only 1.5$ of the o r i g i n a l content remaining after four hours i r r a d i a t i o n . The color test, on the other hand, indicated that destruction started at once and proceeded slowly, the curve following that of a bimolecular reaction. Forty-four percent of the o r i g i n a l ohromogenio value s t i l l remained after sixteen hours and t h i r t y percent after t h i r t y - two hours i r r a d i a t i o n s . This suggested that Vitamin A and the ohromogenio substance were separate e n t i t i e s . Marcus' (153) found the storage of the ether-soluble portion of the unsaponifiable f r a c t i o n of Cod Liver O i l with f i n e l y divided s o l i d s , such as lactose granulate, f e r r i c sulphate etc., i n the presence of either a i r or carbon dioxide caused a progressive destruction of Vitamin A, often complete i n f i f t e e n days. Destruction also occured when the concentrate 60 was stored with "nuchar" charcoal from which a l l traces of a i r had "been removed, showing that i n this instance oxidation was not the destructive agent* The presence of hydroquinone or water as ten percent of the t o t a l material delayed, but did not prevent, the destruction of the Yitamin. - From the work of Monaghan and Schmitt (158), carotene, the precussor of Yitamin A i n the animal body, greatly i n h i b i t s the oxygen uptake of l i n o l e i c acid. Oxidized carotene, on the other hand, s l i g h t l y accelerates the oxygen uptake of this acid. Yitamin A i n small concentration may completely i n h i b i t the oxygen uptake of l i n o l e i c acid for some hours. This i n h i b i t i o n wears off, as i n the oase of carotene, when the vitamin i s destroyed by oxidation. They suggest the p o s s i b i l i t y that Yitamin A may be concerned with phospholipid metabolism. Measurements made i n a Warburg apparatus by Euler and Ahlstrom (79) showed that the rate of oxygen uptake of a series of f i s h - l i v e r o i l s and Yitamin A concentrates increased with increasing "blue value" and growth-promoting power. Liver sections from rats showed a higher rate of oxygen uptake i n blood than i n Ringer's solution. They suggest that Yitamin A plays an important part i n oxidation processes i n the body. Davies (47) i n his work with the absorption spectrum observed that at room temperature, Yitamin A deteriorated less rapidly i n l i v e r specimens treated with potash than i n untreated 61 tissues kept without preservative treatment. In the case of specimens of l i v e r transferred immediately post mortem to potash solution and then stored at room temperature, no ser ious decrease i n Vitamin A content was.to be anticipated i f the assay was carried out within fourteen days after death. The production of high grade feeding o i l from pilohards and similar f i s h was investigated by Brocklesby and Bailey ( 2 1 ) . A dry, acid-free pilchard o i l stored i n a cool dark place and having a minimum access to a i r w i l l remain unoxidized for a very long period. Samples kept i n t i g h t l y stoppered glass and t i n containers and stored i n a dark cup board for f i v e years showed but a trace of oxidation. An o i l with 0 . 0 0 1 $ maleio acid dissolved i n i t w i l l r e s i s t oxidatine rancid i t y twice as long as an untreated o i l . G-utteridge ( 1 0 0 ) at the Central Experimental Farm, Ottawa, showed from an examination of the free fatty acid and nitrogen content of several v a r i e t i e s of Cod Liver O i l , that, i n general, o i l s with a high fatty acid content also had a high nitrogen content. Such o i l s abnormally high i n these constituents were either manufaoture.d from stale l i v e r s or improperly processed. One sample of Cod Liver O i l was deaminised and compared with the untreated o i l by feeding experiments. The deaminised o i l was superior to the untreated o i l i n that i t had the effect of equalising the rate of - growth and lowering the mortality i n growing chicks, while 62 also rendering more e f f i o i e n t u t i l i s a t i o n of food for egg production. E e t i (184) proved the invariable existence of Yitamin A i n e s t e r i f l e d form i n the l i v e r . Aocording to M i l l e r (156) the Yitamin A potency of Cod Liver O i l i n a feed mixture was preserved by mixing the o i l with cottonseed meal "before incorporating i t i n the feed. The chemical and physical constants of God Liver O i l was extensively studied by Lindholm (138), Fifteen samples of Cod Liver O i l showed remarkable constancy i n color, v i s c o s i t y , a c i d i t y , saponification value, iodine value, unsaponifiable matter, sulphuric acid test of the U, S, Pharmacopoeia, Carr and Price test, i n a few samples, i n the Yitamin A value, generally as determined spectrographically but i n a few samples b i o l o g i c a l l y . The Yitamin A content was easily diminished by oxidation without affecting any of the other constants, but i f any of these became seriously altered,; the Yitamin A was also found to be completely destroyed. 63 Oolorimetrlo Reaction Among the f i r s t to use the colorlmetric method of assaying the Vitamin A content of God Liver O i l were Rosenheim and Drummond (186). They used arsenic t r i c h l o r i d e , 1 o.c. to one drop of o i l . They found that the o i l dissolved rapidly and gave a blue solution which i n a few seconds changed to purple and gradually faded. Complete agreement was found to occur between the color intensity and growth-promoting a c t i v i t y as tested b i o l o g i c a l l y . Drummond, Coward and Hardy (65) demonstrated the s e n s i t i v i t y of color reactions with trichloroacetic acid or dimethyl sulphate, and found that they seemed to be of about the same order as the animal feeding test. They stated, how ever that the reaction with arsenic t r i c h l o r i d e was decidely more delicate. Fearon (81) found that phosphorus pentoxide formed a deep v i o l e t color on addition to o i l s containing Vitamin A, Using a 12% solution of trichl o r o a c e t i c acid i n dry l i g h t petroleum as a condensing agent, pyrogallol and other poly phenols intereacted with o i l s containing Vitamin A to give stable pigments which were suitable for colorimetry. Continuing this work were Willimott, Moore and Wokes (256). They noted that concentrated sulphuric acid and phosphorous pentoxide were less sensitive tests for Vitamin A than were arsenic t r i c h l o r i d e or antimony t r i c h l o r i d e . They 64 concluded, "In view of the transient nature of the colors obtained with both these reagents, i t i s suggested that read ings be taken not more than t h i r t y seconds after mixing. Using the antimony t r i c h l o r i d e test Wilson (261) observed that the fatty extract from the human l i v e r gave the same color reaction as Vitamin A found i n Cod Liver O i l , In a study of effect of heat and oxidation on Cod Liver O i l , Wokes and Willimott (263) used four color tests--coneen- trated sulphuric acid and phosphorus pentoxide being qualitat ive only and arsenic and antimony t r i c h l o r i d e s being quantitat ive as well as qua l i t a t i v e . They obtained results i n agreement with those of workers using animals, Drummond and Morton (66) measured the color reactions with a Lovibond tintometer and found their results were in agreement with those obtained by Rosenheim and Drummond (186) and also those obtained by the absorption bands. They found that colorimetric analysis was i n agreement with the spectr oscopic methods and recommended either for giving a rapid, r e l i a b l e quantitative measurement of the Vitamin A content of God Liver O i l s . Wokes (264) conducted a spectroscopic study of the colors produced by the"Vitamin Reagents" (arsenic and antimony tri c h l o r i d e s ) on a series of Cod Liver Oils and concentrates whose Vitamin A content had been ascertained by feeding tests. In each case he found two absorption bands which appeared to 65 be characteristic of the chromogen. Arsenic t r i c h l o r i d e gave bands at about 587 and 475 J1 ja and antimony t r i c h l o r i d e gave hands at about 614 and 530/1 He observed that the chromogen on standing i n contact with either reagent gradually passed from the stage giving the i n i t i a l band (at about 587 or 615?" J1) to the stage giving the second band (at about 475 or 530 z1 J 1). This change was accompanied by a gradual loss i n blue color and gain i n red color, which can be measured by means of the t i n t  ometer."-? He also stressed the time effect i n reading the results as (256) had done previously. According to Smith and Hazley (221) the unsaponifiable fra c t i o n of God Liver O i l gives with antimony t r i c h l o r i d e i n chloroform a blue color proportional to i t s concentration. The l i n e representing the d i l l u t i o n effect for the t o t a l unsaponifiable f r a c t i o n i s tangential at the origin to the d i l u t i o n curve for the corresponding God Liver O i l . They describe a method for oarrying out the color test on the unsaponifiable f r a c t i o n extracted with chloroform, Emmerie, Eekelen, and Wolff (76) found that by treating a Yitamin A preparation from Cod Liver O i l with drops of furan, methylfuran, p y r r o l , indol or skatol prior to use of the antimony t r i c h l o r i d e reagent, a purple color i n place of the usual blue was given. The 610' J absorption band was found to be suppressed, but the 572//u band was unaltered. They conclude that the v a r i a t i o n i n different specimens of l i v e r 66 o i l may perhaps be due to variations i n th e i r content of indol- l i k e substances, Gillam and Morton (87) observed from t h e i r work that l i v e r o i l s contained two chromogens which with antimony t r i  chloride gave colored substances with absorption maxima at 606 u f and 57 2f F respectively. In concentrates these maxima were displaced to 620vu F and 583/U F , A comparison of u l t r a  v i o l e t absorption spectra with a spectroscopic data on the color test disclosed, " ( l ) that the parallelism between the intensity of the 6G6)1 Ju band and the intensity of the 328,u u band breaks down so seriously i n extreme cases or to render i t improbable that the 606 J1 chromogen i s Vitamin A; (11) that u u the 572; / chromogen and the substance responsible for the 328 f- P- band are probably i d e n t i c a l ; ( i l l ) that the blue color for r i c h o i l and concentrates i s often much deeper than would be expected on the basis of correlation between blue color and u l t r a - v i o l e t absorption, "Hence they concluded that the matching of blue colors with Lovibond glasses, though i t may act as a rough guide to Vitamin A potency was theoretically unsound. The ideal conditions for accurate colorimetric deter minations are stated and reviewed by Heilbron, Gillam and Morton (109). They observed that i n a considerable number of o i l s characterised i n the color test by predominance of the 572f J1 band over the 606 f F band, a large increase i n the 67 intensity of the l a t t e r band could be obtained by treating the o i l beforehand with ozonised oxygen, hydrogen peroxide or benzoyl peroxide. O i l s which i n i t i a l l y showed an excess of the 572 /u J3- chromogen over the 606 7 U J3- chromogen underwent a slow spontaneous ageing which resulted i n a marked increase i n the intensity of the 606 JP-. J3- band. Increases i n the 606 ,u u absorption were not at the expense of the 572 J3- J 1 chromogen and were not accompanied by similar increases i n the l a t t e r , and the absorption at 328 m /u remained p r a c t i c a l l y constant throughout. Rosenthal and Erdelyi (187) (188) found that a modif i c a t i o n of the antimony t r i c h l o r i d e color reaction for Yitamin A by the addition of pyrocatechol distinguished between Yitamin A and the known carotenoids. The viol e t - r e d color resembling that of a dilute solution of potassium permanganate was more stable than the blue of the Carr-Price reaction. A short time l a t e r these same two Investigators (189) noted that a bfo guaiaool solution produced with Yitamin A the same red v i o l e t color as &fo catechol. The s t a b i l i t y of the color rendered the guaiaool test suitable for quantitative purposes. Continuing t h i s work Rosenthol and Weltner (190) found that the purplish red color of the antimony tr i o h l o r i d e - catechol test for Yitamin A, when examined speotroscopically 68 within the f i r s t ten minutes, showed maxima at 545 and 475 /u /U on longer standing two other maxima appeared. When catechol was replaced by guaiacol the purple color remained unchanged for hours, with maxima at 545 and 478 f- /U, Anderson and Levine (5 a) observed that on heating the reaction mixture to 60° C. the blue color given by Vitamin A with the antimony t r i c h l o r i d e reagent was changed to red, whereas the color given by carotene remained blue. They con cluded that the use of pyrocutechol, as recommended by (187), was not necessary to bring about the change of color i n the case of Vitamin A* According to Przeydziecka (177) i f the mixture i s heated i n the presence of guaiacol for two minutes on a water bath at 60° 0, a rose or red color i s produced which i s stable and may be compared with a standard solution of suelan three. Holmes and Bromund (119) found that the orthodox method of matching against potassium diohrornate was satisfactory for estimating carotene when dissolved i n petroleumether. Solvents of higher refractive index, such as benzene and chloroform, however, caused a change i n the color towards the red end of the spectrum, and accurate matching against a dichrornate solution was impossible. Accurate results could be obtained by using solutions of o r y s t a l l i n e bixen as a standard. 69' Absorption Spectrum The absorption spectra of Yitamin A was observed f i r s t by Schultz and Morse (199) and Schultz and Zeigler (200). Shortly after these investigators were Heibron, Kamm and Morton (108). In 1928 Morton and Heilbron (165) claimed that Vitamin A was characterised by an absorption band with a maximum at 328 u u . They also suggested that one of the decomposition products of Vitamin A had an absorption band near 275 to 285/u Morton, Heilbron and Thomson (166). found that a crude God Liver O i l of high potency gave additional selective absorption between 565 and 585 J1 J1 i n the blue solution. Dann (45) showed that Vitamin A was more rapidly o destroyed than carotene by radiation of wave length 2650 A. He d e f i n i t e l y denied the p o s s i b i l i t y that Vitamin A was the end product of the photochemical reaction of carotene with t h i s radiation, Chevallier and Ghabre (32) applied a spectrophotometrie method, which permitted a very accurate measurement of the u l t r a - v i o l e t absorption given by a substance to the measurement of intensity of absorption at 3280 1 of different samples of Cod Liver O i l . The examination of a great number of o i l s of different origins showed, that, besides the presence of Vitamin A, the free a c i d i t y of the o i l and i n certain cases i t s pigment ( i f very concentrated) must be taken into account 70 i n consideration of the absorption i n the neighborhood of o 3280 A. They concluded that when the free acid content of the o i l was low, the results of the physical and b i o l o g i c a l tests agreed f a i r l y w e l l , the differences not exceeding the usual experimental errors inherent i n b i o l o g i c a l methods. B i l l s (17) demonstrated that the spectrograph gave two adjaoent spectrograms, and an opti c a l wedge i n front of the collimator provides means of revealing d i r e c t l y the intensity of the banded absorption due to Vitamin A, Macwalter (150) found that the aeration of God Liver O i l for a short time reduced i t s absorption at 328 /u y u but aeration for a long time increased i t s absorption greatly. He devised a method which uses a curve r e l a t i n g absorption at 328 u u to duration of aeration, whereby an accurate measure of the absorption due to Vitamin A may be obtained. When this u u method was impracticable, the absorption of 328 7 / of the unsaponifiable f r a c t i o n of an o i l was a truer measure of Vitamin A than the absorption of the o i l i t s e l f . In Hotevarp's (170) experiments the Hilyer Vitameter A was equipped with a simple photographic device which made i t possible to determine E. 328 J1 J1 with an accuracy approaching that attained by conventional spectrophotometry methods. The values thus obtained for pure and crude God Liver O i l s , halibut l i v e r o i l s , concentrates of herring o i l , etc., agreed 71 w e l l with the blue values obtained by the antimony t r i c h l o r i d e method, when the l a t t e r were calculated according to a special formula. With Cod Liver Oils the absorption of the unsapon i f iable matter was 85 to 90$ of that of the o r i g i n a l o i l , the reduction being attributed chiefly to losses i n the preparation of the concentrate. He also noted that the value for E 1 cm, 328 U u w a s reduced from 0,52 to 0.027 i n a God Liver O i l exposed for s i x months on a roof i n a white glass bottle. Shrum and How (210) found that the results of bio- l o g i o a l estimations of Yitamin A i n a Cod Liver O i l and i n a concentrate, agreed well with the value found by multiplying the extinction coefficients (S 1 / G m at 328/ J ) by 1600. In two samples of pilchard o i l , however, the physical method gave values more than three times the b i o l o g i c a l values for the o i l s and 2,5 times those for the unsaponifiable fractions. Removal of the pigment by absorption on diatomaceous larch reduced the absorption only s l i g h t l y . To measure the absorption due to substances other than Vitamin A, the l a t t e r was destroyed.by oxidation, and the absorption of the treated material measured. After subtraction of this value from the t o t a l for the untreated material, the remainder, due presumably to Vitamin A, yielded a value only 30% greater than the bio l o g i c a l value. They believed that the high values obtained for pilchard o i l were caused by other substances absorbing i n the region of 328 yu >u. They concluded that the spectroscopic method can he applied to pilchard o i l only when the Vitamin A i s removed completely from the o i l without otherwise modifying or affec t i n g i t . Sample Vitamin A Content Bi o l o g i c a l Physical Standard Solution Cod 1 2 , 5 0 0 1 1 7 0 0 Liver O i l ' 6 5 0 ' 6 2 Q Pilchard O i l No. 1 1 2 0 380 Pilchard O i l No. 2 175 5 9 0 The findings of Shrum and How. According to McFarlane and Rudolph ( 1 4 8 ) , at the University of Alberta, there i s need for further investigation of pilchard o i l to bring into closer agreement the results of the b i o l o g i c a l assay and physical measurements of t h i s o i l for Vitamin A. Milne ( 1 5 6 ) found that spectrophotometrie measurements of the Vitamin A content of pilchard o i l did not equal the bio l o g i c a l value of the o i l . I t was also found that the carotenoid pigment of this o i l was too small to account for the high b i o l o g i c a l value. 73 Solvents for Carotene Dann (43) reported that Yitamin A was rapidly oxidized by a i r when dissolved i n some solvents, and slowly or not at a l l i n others. He found i t to be p a r t i c u l a r l y stable i n ethyl alcohol, alcoholic potash and ethyl acetate. In ethyl alcohol i t was stable toward hydrogen peroxide. The solvent (or' impurities associated with the vitamin or solvent) appears to play a leading part i n determining the s t a b i l i t y of Vitamin A. McDonald (146) found that carotene dissolved i n ethyl butyrate, laurate, or palmitate, or i n peanut o i l , and stored with access of a i r , was destroyed to the extent of 80% i n four weeks, even at 5°C., while with the same conditions, only 8% was destroyed i n Cod Liv e r , Wesson or Maize o i l . Higher temperatures accelerated, and storage i n vacuo retarded destruction. Baumann and Steenbock (8) examined the s t a b i l i t y of carotene i n a number of solvents, quantitative estimation being made speotrophotometrically by observing the intensity of absorption at 485 and 460 m J1. Refined cottonseed o i l (Wesson o i l brand) was most satisfactory; cocoanut o i l , among others, was less good. The effect of various conditions of storage was studied. I t was calculated that when the solutions were kept i n the conditions exacted by th e i r use i n a feeding test, 95% of carotene was lo s t after a month when dissolved i n olive or 74 coooanut o i l , "but only 26$ when cottonseed o i l was used. The addition of hydroquione increased the s t a b i l i t y of carotene i n certain solvents (ethyl laurate, ethyl sebosate) but not i n others. An elaborate study of the-;,-stability of carotene i n ethyl esters of fa t t y acids and i n l i v e r and vegetable o i l s was conducted by McDonald (147). A 0.05$ solution of mixed a l f a and beta carotene i n ethyl butyrate, laurate, and palmitate, i n peanut o i l , ood l i v e r o i l , maize o i l and Wesson o i l was stored i n partly f i l l e d stoppered bottles at 27°, 24°, and 5° C and i n evacuated sealed tubes at 37°C. and the carotene content observed spectrographically (462 mj1 l i n e ) from time to time. The oarotene was conserved best i n the Wesson and maize o i l s and worst i n the esters. Dyer and Key (73) found the b i o l o g i c a l value of the carotene, used as international standard for Yitamin A, was greater i n solution of oocoanut or arachis o i l than i n hard ened cottonseed o i l or ethyl laurate. Three samples of cocoa- nut o i l , with and without the addition of quinol, gave approximately the same r e s u l t . God l i v e r o i l gave a higher b i o l o g i c a l value for Yitamin A, when diluted with coooanut o i l , than with hardened cottonseed or olive o i l . "Vitamin A and Antioxidants Jones and Christiansen (127) found that hydroquione i n a concentration of 0.03% retarded the absorption of oxygen and the deterioration of the "blue value" of two samples of refined halibut l i v e r o i l . I t also retarded without wholly preventing the deterioration of the Yitamin A value of one sample as estimated b i o l o g i c a l l y . Maleic acid did not act as an a n t i  oxidant i n halibut l i v e r o i l . L e c i t hin and hydroquinone as antioxidants for Yitamin A was studied by Holmes, Corbet and Hartzler (120). The effect of l e c i t h i n and of hydroquinone i n delaying the oxidation of Yitamin A, as measured by the SbCl g reaction, i n halibut l i v e r o i l and cod l i v e r o i l was studied both at room temperature and i n an atmosphere of a i r , and with the aid of specially designed apparatus, at 96° C., i n an atmosphere of oxygen. The a n t i  oxidant action of both substances was confirmed under a l l conditions and at a l l concentrations. The, action of l e c i t h i n i n supplementing the action of hydroquinone was much greater than would have been expected from i t s action alone, and the best results were obtained with a combination of both substances. In some instances, and notably with hydroquinone i n cod l i v e r o i l , there appears to be an optimum concentration for the a n t i  oxidant, and less satisfactory results are obtained i f t h i s concentration i s exceeded. At 96° C, the presence of water s l i g h t l y retarded the rate of oxidation. Concentration of Vitamin A W i l l i m i t and Wokes (258), working with spinach, pre pared an extract that was two hundred times as concentrated as fresh leaves. This extract, they found, was potent source of Vitamin A, i n that 25 mg. daily gave complete freedom from xerophthalmia and induced satisfactory growth. Moore (163) prepared concentrates from the l i v e r o i l s of rats and pigs which had received diets containing large amounts of carotene as red palm o i l and compared them with con centrates derived from turbot and s o l - l i v e r o i l s . Although the i n i t i a l "blue values of the o i l s varied widely, l i t t l e difference could he detected i n the a c t i v i t i e s of the f i n a l concentrates, which approached an average value of 2400 B.TJ. per mg. (Pharmacopoeia color value 45,000) i n the Shcl, test, corres ponding to a minimal dose of ahout 0.001 mg. i n rat growth experiments. He believed that the concentrates must have been consisted substantially of actual Vitamin A, or alternatively that the vitamin must have been associated with other substances of similar s o l u b i l i t y properties i n remarkably constant pro portion. The preparation of a potent Vitamin A concentrate was described i n 1935 by Holmes et a l . (118). They prepared concentrates of halibut l i v e r o i l having blue values up to 140,000, and claimed them to be 40$ more potent than any other previously prepared samples. 77 Karrer et a l (129) suggest the following process for concentrating Vitamin A, HALIBUT LIVER OIL saponify UN" SAPON IE I ABLE RESIDUE \ ' cool i n MeOH to -15° to -60° (to remove sterols) SOLUTION \ Fractionally adsorb on Fuller's l a r t h . VISCOUS OIL CONTAINING OVER 50$ OF VITAMIN A • ' " V • E s t e r i f y VITAMIN A ACETATE (Crude) \ saponify and d i s t i l i n vacuo. NEARLY PURE VITAMIN A (•10,00.0 times as active as o r i g i n a l material) The Vitamin A concentrate obtained from the above process represents about one part i n two thousand of the ori g i n a l o i l . The dose needed to cure a rat i s as l i t t l e as l/lOOO of a m i l l  igram ( s 1/30,000,000 of an ounce). 78 The Measurement of Yitamin A (International Units) The need for a unit measurement of Yitamin A has been recognized by investigators throughout the world. To avoid confusion the so ca l l e d "International Unit" has been evolved after several years of intensive investigation. U n t i l recently the Sherman and Munsell unit (206) has been extensively used i n the Yitamin A determinations of foods, Eexelen, Emmerie and Wolff (73 a ) i n 1934 converted the various "units" used to express the results of Vitamin A estimations by the antimony t r i c h l o r i d e method, into Inter national units. Their findings were as follows: 1 Cod Liver O i l unit (Rosenheim and Webster) equals 208 international units, 1 blue value (Drummond and Hilditch) equals 20.8 international units, 1 lovibond unit (Wolff) equals 4.2 international units, 1 blue unit (Moore) equals 0.39 international units* They also showed that by similar calculations an o i l having Q j £ c e n ^ 328 u u equals 1 i s assumed to contain 5 Cod Liver O i l units, 50 blue values, 250 lovibond units (Wolff), 2750 blue units (Moore) and 625 gamma Vitamin A or 1042 international units per gram. The f i r s t International Conference for the Standardis ation of Vitamins was held i n 1931 (183.) i n London England. A provisional unit of Vitamin A was established i n terms of the 79 bi o l o g i c a l a c t i v i t y of a sample of carotene. The second Confer ence was held i n 1934 at which a solution of pure beta carotene was adopted as the permanent standard i n terras of which units of Vitamin A were i n future to be expressed (154). The Pharmacopoeia of the United States has evolved a standard procedure for the b i o l o g i c a l testing of Vitamins A and D. Their standards are Reference Cod Liver Oils of known standard potency. See Appendix No. 1 for the procedure. The above two standards are for the use of rats. Up to the present there i s no standard procedure for Vitamin A determinations with chicks. Many investigators have used chicks as a test animal but a l l express t h e i r results differently and therefore i t i s very d i f f i c u l t to compare the results of one investigator with another. A standard procedure using chicks as the test animal i s needed i n order that the findings of different investigators may be comparable. The d e f i n i t i o n of a unit of Yitamin A as outlined by the "Permanent Standards Commission of the Health Organization of the League of Nations" i s as follows: (a) International Standard The Conference recommends that pure B—carotene be adopted as the International Standard for Vitamin A, The Standard Preparation s h a l l conform to the requirements stated in Note 1 i n regard to i t s chemical and physical constants. 8D (b) Definition of Unit The Conference reoommends that the unit for Yitamin A provisionally adopted at the 1931 Conference s h a l l be maintained. I t has been established that one such unit i s contained i n 0 a6 microgram (0.6 gamma) of pure B--carotene. The International Unit for Yitamin A recommended for adoption s h a l l be defined as the Yitamin A a c t i v i t y of 0.6 microgram (0.6 gamma) of the International Standard Preparation. Daily doses of two to four International Units of Yitamin A, when administered to young rats suitably prepared on a Yitamin A deficient diet, have been found adequate to restore growth; somewhat larger doses are required for the cure of xerophthalmia. (c) Mode of Preparation I t i s recommended that the Health Organization of the League of Nations s h a l l be requested to obtain a sample of B—carotene as defined by the Conference (see Note 1), and that the National Institute for Medical Research, London, acting for this purpose as central laboratory on behalf of the Health Organization of the League of Nations, s h a l l undertake the care, storage and di s t r i b u t i o n of the International Vitamin A Standard so obtained. (d) Mode of Distribution The Conference recommends that the International Standard 81 Preparation s h a l l be issued i n the form of a standard solution i n o i l , the strength of the solution being such that 1 gramme .contains 500 International Units, or 300 micrograms (300 gamma) of B--carotene. (e) Adoption of Subsidiary Standard of Reference The Conference recommends that a sample of God Liver O i l , the potency of which has been accurately determined i n terms of the International Standard Preparation of B—carotene, shall be provided as a Subsidiary Standard of Reference, In view of the fact that the Reference Cod Liver O i l of the United States Pharmacopoeia, which has been accurately assayed i n terms of the provisional International Standard adopted i n 1931, has been i n effective use i n the United States of America for some time, the Conference recommends that the Board of Trustees of the United States Pharmacopoeia be approached and invited to place a quantity of their Reference God Liver O i l at the disposal of the Health Organization of the League of Nations, with a view to i t s adoption for inter national use as a Subsidiary Standard for Vitamin A. In the event of the Reference God Liver O i l of the United States Pharmacopoeia not being available for international adoption, the Conference recommends that another sample of God Liver O i l be selected, i t s potency i n terms of the Inter national Standard Preparation of B—carotene accurately determined by b i o l o g i c a l comparison and independently by 82 spectrophotometries measurements, and that this selected sample be then adopted as a Subsidiary International Standard for Yitamin A. 83 YITAMIH A.IN POULTRY  Symptoms and Lesions Due to Lack of Vitamin A . Beach (12) i n 1924, was the f i r s t investigator to des cribe avitaminosis A i n poultry. He noted that the character i s t i c symptoms of t h i s disease were confined to the head L . > involving the nasal passages, the mouth, pharynx, esophagus and the eyes. According to his early work avitaminosis A was associated with the following symptopathology: "A discharge from the n o s t r i l s , of a watery or v i s c i d f l u i d i s nearly always present. Later t h i s may coll e c t i n the i n f r a o r b i t a l sinuses, become transformed into a G a s e o u s mass and cause a swelling of the face. "The lesions i n the mouth, pharynx, esophagus and crop consist of collections of white caseous material i n the mucous glands. "The lesions i n the eye consist of an ophthalnia which produces puffiness of the eyelids, reddening of the con junctiva, a profuse watery secretion which soon becomes v i s c i d and may glue the eyelids together. "The kidneys are usually pale and marked by a network of fine white l i n e s which are u r a t e - f i l l e d tubules. Occasion a l l y there i s a deposit of urates on the heart, pericardium, l i v e r , omentum and intestines. In some cases the uraters are greatly distended with urates. "The p o s s i b i l i t y that i t i s Vitamin D rather than 84 Vitamin A i n the ood l i v e r o i l that prevents the development of th i s disease would seem to "be controverted by the following: 1. "The s i m i l a r i t y of the ophthalmia to that occuring i n rats fed a ration deficient i n Vitamin A and the t o t a l lack of any symptom of rickets suggestive of Vitamin D deficiency. 2. "Exposure of the fowls to an abundance of direct sun l i g h t did not prevent the development of this disease, although i t did prevent r i c k e t s . 3. " I t would seem, therefore, that t h i s disease which the writer has previously designated as a n u t r i t i o n a l disease resembling roup should now be designated a n u t r i t i o n a l disease caused by Vitamin A deficiency although the name n u t r i t i o n a l roup might be more suitable for use by the poultrymen". In 1930 S e i f r i e d (203) described the essential histo- pathological changes i n the respiratory tract of chickens caused by avitaminosis A. He said, "There i s f i r s t an atrophy and degeneration of the l i n i n g mucous membrane epithelium as well as of the epithelium of the mucous membrane glands. This process i s followed or accompanied by a replacement or substit ution of the degenerating o r i g i n a l epithelium of these parts by a squamous s t r a t i f i e d k e r a t i n i z i n g epithelium. This newly formed epithelium develops from the primitive columnas epithelium and divides and grows very rapidly. The process appears to be one of substitution rather than a metaplosia, and resembles the normal keratinization of the skin or even more 85 closely the incomplete keratinization of the mucous membranes. A l l parts of the respiratory tract are about equally involved i n the process, and the olfactory region as we l l , so that the sense of smell may be l o s t . The lesions, which f i r s t take place on the surface epithelium and then i n the glands, show only minor differences ". "The protective mechanism inherent i n the mucous membranes of the entire respiratory tract i s seriously damaged or even enti r e l y destroyed by the degeneration of the c i l i a t e d c e l l s at the surface and the lack of secretion with bacter i c i d a l properties. Secondary infections are frequently found, and nasal discharge and various kinds of inflammatory processes are common, including purulent ones, especially i n the upper respiratory t r a c t , communicating sinuses, eyes and trachea. The development of the characteristic h i s t o l o g i c a l process i s not dependent upon the presence of these infections, since i t also takes place i n the absence of infection. The specific h i s t o l o g i c a l lesions make i t possible to differentiate between A avitaminosis and some infectious diseases of the respiratory t r a c t " . Continuing t h i s work further S e i f r i e d (204) said "When fowls are placed on a diet lacking i n Vitamin A lesions appear i n the upper alimentary tract which are confined largely to the mucous glands and the i r ducts. H i s t o l o g i c a l l y i t i s shown that the o r i g i n a l epithelium becomes replaced by a s t r a t i f i e d 86 squamous k e r a t i n i z i n g epithelium and that secondary infections are r e l a t i v e l y common. The ducts of the glands may be blocked leading .to distention with secretions and nurotic materials. These lesions macroscopically resemble very closely certain stages of fowl pox and the two conditions can be separated only by h i s t o l o g i c a l examination. These lesions produced by a lack of Vitamin A may enable bacteria and other viruses to enter the body". Ackert, Mcllvaine and Crawford (2) found that the resist ance of growing chickens to the i n t e s t i n a l roundworm, Ascaridia Lineata, was lowered when the fowls, four to seven weeks of age, were kept on a diet deficient i n Vitamin A. The larger number of the roundworms remaining i n the chickens on the Vitamin A deficient diet were attributed i n part to evidences of weakened p e r i s t a l s i s , thus making i t less d i f f i c u l t for the young worms to withstand the rigors of p e r i s t a l s i s . According to investigations conducted at the University of Idaho, (251) the symptoms and lesions of chicks fed a Vitamin A free ration are as follows: "A characteristic wobbly gait, sore eyes, swelling under the throat, excessive mucous i n the mouth, r u f f l e d feathers, general i n a c t i v i t y and i n a b i l i t y to maintain normal equilibrium, and extreme paleness of skin, beak, and shanks. The internal lesions observed consisted of extreme paleness of the kidneys, followed by a characteristic white network of urate deposit i n 8 7 the kidney tissue, enlargment of the ureters with an accumulat ion of urates, and enlargment of the g a l l bladder and provent- .riculus. A white deposit often appeared over the surface of the internal organs and walls of the body cavity. A gelatinous substance- was frequently found around the heart or over the breast muscles. In those chicks which survived to or beyond s i x Yi/eeks of age, t y p i c a l white abscesses (pustules) developed on the mucous membrane of the throat, and cankerous growths appear ed i n the mouth. In some instances the small intestines exhibited extreme inflammation, with definite hemorrhagic spots. As previously stated, these lesions did not appear uniformly i n a l l the chicks. The urate deposits i n the kidneys and the enlargement of the ureters, g a l l bladder, and proventriulus, were the lesions which appeared more consistently. The above symptoms occur i n chicks at about four weeks of age and become more severe before the chicks f i n a l l y die". Hinshaw and Lloyd (114) report that poults fed a Vitamin A deficient ration from the time of hatching developed symptoms of avitaminosis A i n twenty-five days. Chicks kept as penmates to the poults showed similar symptoms i n twenty- seven days. The disease was much more acute among the poults than among the chicks. Lesions i n the poults were confined to mucous membranes of the head, the upper digestive t r a c t , the respiratory tract and the Bursa of Fabricus. Deposits of urates i n the kidneys and the ureters seldom occured i n the 88 poults. They also found that turkeys required a ration contain ing eight percent of dehydrated a l f a l f a leaf meal (containing approximately 130 gamma of carotene per gram), for normal growth to t h i r t y weeks of age. Chicks kept as penmates to the turkeys made normal gains and showed no evidence of avitamin osis A on a l e v e l of four percent a l f a l f a meal. Prom their investigation they prepared tables showing the d i s t r i b u t i o n of lesions i n the turkeys and the chickens. S e i f r i e d (205) i n l a t e r work showed that chicks developed the f i r s t macroscopic e p i t h e l i a l changes after thirty-three days on a diet deficient i n Yitamin A. M i l l e r and Bearse (155 a ) noted that the f i r s t symptom observed i n chicks i n a Yitamin A deficient diet was a stagger- ing gait. This condition became progressively worse u n t i l death occurred. Staggering g a i t , unthrifty appearance, and soiled f l u f f were the only external symptoms of deficiency i n chicks dying early i n the t r i a l . Those dying l a t e r frequently showed a watery eye condition which was often accompanied by pus. They also found that the post mortem examination of chicks on a Yitamin A deficient diet revealed enlarged g a l l bladders, enlarged gray kidneys (due to accumulation of urates), ureters distended with urates, enlarged proventriculi and gray hearts. Gullet lesions were observed i n only a few birds* 89 Requirements of Chioks For Growth Sugiura and Benedict (240) i n 1923 studied synthetic diets for the n u t r i t i o n of pigeons. In th e i r work they used two different rations and obtained normal growth and reprod uction. Ho. 1 diet consisted of casein 22, cane sugar 10, starch 27, agar 2, s a l t mixture 3, butter fat 30 and yeast 6$. Ho. 2 diet had the starch increased to 37$ and the butter fat replaced by l a r d 20$. They concluded from the i r work, "Pigeons on.a diet of s u f f i c i e n t c a l o r i c value, even though the diet lacks fat and fat soluble vitamins, may maintain excellent condition, and may produce f e r t i l e eggs and rear healthy squabs. Hence fat-soluble vitamin i s not essential i n any stage of avain n u t r i t i o n " . A few months l a t e r Emmett and Peacock (78) showed that young chicks require the fat soluble Vitamin A. They noted that i n the absence of Vitamin A, the onset of the symptoms of ophthalmia appeared and unless the diet was properly reinforced, or an oral treatment r i c h i n the Vitamin was given, death eventually ensued. They claimed that t h i s eye condition resulting from the lack of Vitamin A was the same as the poultry n u t r i t i o n a l roup described by Beach (12.), The presence of urates i n the ureters, kidneys and at times on the surface of the heart, l i v e r and spleen were observed and believed related to the deficiency of the fat soluble Vitamin A. They concluded that, "Young mature pigeons require very l i t t l e i f 90 any, fat soluble A. Yitamin A does however play a role i n the n u t r i t i o n of some species of avian". The following year, Hart, Steenbock, Lephovsky and Haplin, (102) agreed with Emrnett and Peacock (78) and disagreed with Suyiura and Benedict (240), Hauge, Canick and Prange (105) found that when they used 25$ of yellow corn i n their basal ration, the fat soluble A requirements of growing chicks were met for the f i r s t ten weeks of t h e i r l i v e s . On the other hand 50$ of yellow corn met the requirements for the development of p u l l e t s , up to the laying age. Chicks fed on rations deficient i n fat soluble A usually reflected such a deficiency i n t h e i r growth responses at about four weeks of age. Russell and Weber (192) investigated the role played by plant pigments i n the n u t r i t i o n of chickens, Pour groups of chicks were placed on a Yitamin A deficient diet. After four weeks on t h i s diet alone, daily supplements of 0.03 mg, of carotene, xanthophyll and chlorophyll were fed to the chicks i n each of three of the four groups respectively, the fourth serving as controls. The group receiving the carotene throve, but the chicks i n the control group and i n the two groups receiving the other pigments f a i l e d and died i n two weeks. The uric acid i n the blood of the Yitamin A deficient chicks was much increased but starving chicks yielded similar values. The conversion of carotene into Vitamin A was definitely proven by Capper, McKibben and Prentice (31). They also observed that the beaks and shanks of chickens, which had become colorless through the absence of carotenoids from the d i e t , did not become more yellow when carotene was added to the ratio n . They showed that the Vitamin A content of hen l i v e r o i l s was very high and that the Vitamin A requirements of fowl were large. Elvenjem and New (75) used chicks instead of rats i n the i r study of avitaminosis A. They evolved a basal diet consisting of : 58 parts ground white corn, 25 parts wheat middlings, 12 parts crude casein, 1 part common s a l t , 1 part precipitated calcium carbonate, 1 part precipitated calcium phosphate, 2 parts dried yeast. They observed that the chicks grew normally to three weeks when they began to exhibit general incoordination, became drowsy and crouched on t h e i r haunches. The feathers were very r u f f l e d and there was some soreness around the eyes, but there was no t y p i c a l ophtholmia. The beaks and shanks became colorless and most of the birds were dead by the end of the f i f t h week. They also found that the uric acid content of the blood of normal chicks was approximately 5 mg. per 100 c c . of whole blood, while that of Vitamin A deficient chicks went as high as 44 mg. per 100 c c . of blood. The amount of uric acid i n the blood was independent of the protein intake. Lsra Yitamin A deficiency did not disturb the uric acid metaboils but injured the structure of the kidney s u f f i c i e n t l y to prevent normal elimination of ur i c acid. The amount of uric acid i n the blood was dependent upon the degree of kidney damage. The degree of incoordination, however, was independent of the uric acid content of the blood. Kline, Schultze and Hart (132) found that Xanthophyll, m.p. 174^ prepared from spinach did not serve as a source of Yitamin A for the chick. There were, however no toxic effects, even when i t was fed at levels of 0.25 mg. daily per chick. Carotene, m.p, 172,5° prepared from spinach, when fed i n adequate amounts served as a source of Vitamin A for chicks. When the chicks reached the age of seven to eight weeks, 0.03 mg. of carotene daily were not su f f i c i e n t when i t was the sole source of Yitamin A. Chicks that had been depleted of Yitamin A required more than 0.05 mg. of carotene daily i n order to grow to maturity. In determining the minimum amount of yellow corn necessary i n a growing rat i o n Smith {224) used a basal ration which contained: 35 pounds ground corn meal, 5 pounds oat meal, 20 pounds wheat middlings, 7 pounds meat scrap, 3 pounds f i s h meal, 5 pounds dried skim milk, 1^ pounds oyster s h e l l , •g- pound s a l t . When white corn was used i n the basal ration one hundred percent mortality resulted by the end of the eighth week. Normal growth and v i a b i l i t y resulted when twenty-five percent and f o r t y - f i v e and one half percent of the t o t a l r a t i o n ,was made up of yellow corn. He concluded that the minimum amount of yellow corn required i n a growing ration lay between twelve percent and twenty-five percent when other sources of Vitamin A were lacking. The Idaho A g r i c u l t u r a l Experiment Station (251) used the following basal r a t i o n for t h e i r work on avitaminosis A: 43 pounds ground-wheat, 15 pounds ground oats, 20 pounds bran, 10 pounds dried milk, 7 pounds meat and bone meal, 4 pounds oyster s h e l l , 1 pound s a l t . Vitamin D supplied by i r r a d i a t i o n . They observed that the f i r s t indication of Vitamin A deficiency occured at about three weeks. A high death rate resulted after four weeks up to one hundred percent by eight weeks. Studying avitaminosis A i n turkeys Hinshaw and Lloyd (114) fed the following rat i o n : 25 pounds white corn, 25 pounds barley, 25 pounds ground wheat, 10 pounds f i s h scrap, 10 pounds dried milk, 3 pounds bone meal, 2 pounds ground limestone and •§- pound s a l t . They kept chicks as penmates to the poults and their observations are discussed l a t e r under pathology. Gutteridge (99) found that the addition of pilchard 011 or God Liver O i l to a rat i o n otherwise deficient i n Vitamin A, increased growth and prevented the development of deficiency synptoms i n chicks. He also observed that neither pilchard o i l 9 4 nor God Liver O i l , when fed with a Yitamin A deficient ration at levels of one percent and two percent of the t o t a l feed consumed "brought about as rapid growth as was attained by feeding a well balanced ration. In conclusion he claimed that pilchard o i l and God Liver O i l were of equal value i n so far as Yitamin A content i s concerned, and that pilchard o i l was s l i g h t l y more e f f i c i e n t i n this respect. In a study of the carotene and Vitamin A requirements for white leghorn.chicks Irohring and Wyeno (86) used the following ration: 52-§- pounds ground white corn, 10 pounds wheat bran, 15 pounds wheat middlings, 10 pounds meat scrap, 10 pounds skimmed milk powder, 2 pounds calcium carbonate, •J- pound sodium chloride. 100 A.D.M.A. units of Vitamin D per chick per day. They found that the minimum requirements of Yitamin A for a chick at the age of about eight weeks was approximately 65 A.D.M.A. units per day. P r a c t i c a l l y a l l chicks depleted i n Vitamin A showed marked ataxia three to fourteen days before complete depletion and death, even though given an adequate amount of Vitamin D. There was a wide varia t i o n i n the number of days chicks l i v e d on the Vitamin A free diet, no doubt due to variation i n storage from the egg from which the chicks hatched, which i n turn was dependent on the storage or ration of the parent fowl. The addition of carotene or Vitamin A to the carotene, and Vitamin A free ration delayed the appearance 96 of deficiency symptoms and prolonged l i f e i n a rather definite r e l a t i o n to the amount of carotene or Vitamin A added to the diet. Sherwood and Praps (214) fed rations containing 50, 100, 150 and 300 Sherman-Munsell (206) units per 100 grams of feed to chicks hatched from eggs produced by hens receiving rations containing 310, 440 and 560 units of Vitamin A per 100 grams of feed. During the f i r s t few weeks of the experiment the mortality of the chicks from the hens on the lowest Vitamin A l e v e l was so high that the different levels of Vitamin A i n the chick feeds studied did not show the results on chick mortality that they did on the chicks from the hens on the higher levels. The mortality was lower i n the case of the chicks from the hens receiving 440 and 560 units of Vitamin A per 100 grams of feed as the amount of Vitamin A i n the ration increased. Ho advantage was shown i n the weight of the chicks for the 300 units over the 150 units per 100 gms., i n the chick rations. In another study with chicks hatched from hens receiv ing an adequate supply of Vitamin A these same two invest igators found that the mortality i n twelve weeks was as follows: i n s i g n i f i c a n t amount of Vitamin A, 100$ mortality; 42 units per 100 gms. 41%; 84 units per 100 gms, 15%; and 126 units per 100 gms. 12%. The percentage of healthy chicks remaining at the close of the experiment was 0, 24, 72, 83 for the respected l o t s . Record, Bethe and Wilder (181) used a ration consisting of: 58 pounds white corn, 25 pounds wheat middlings, 12 pounds domestic casein, l i - pounds dried yeast, % pound Irradiated yeast (200 D), 1 pound steamed bone meal, 1 pound ground lime stone, 1 pound s a l t . They found In prophylactic t r i a l s that from f i f t y to one hundred gamma of carotene per 100 gms. of feed were required to produce normal growth and prevent symptoms of Yitamin A deficiency during the f i r s t eight weeks of a chicks 8 l i f e . In curative t r i a l s chicks were fed the basal ration for twenty-six days, at which time a large percentage of the birds showed Yitamin A symptoms. They then divided the chicks into groups and fed different amounts of carotene or God Liver O i l and found that i t required approximately f i f t y gamma of carotene or 60 to 100 international units daily of God Liver O i l to produce normal chicks for seven or nine weeks of supplemental feeding. In 1935, Schroeder, Higgins and Wilson (202) reported that chicks up to nine weeks of age required 6000 international units of Vitamin A per pound of feed (about 1320 units per 100 gm) to prevent c l i n i c a l and pathological symptoms of hypovitaminosis A. They also reported that 1200 units of Vitamin A per pound of feed appeared to be adequate to promote f a i r growth; but that there was a tendency for body weights to increase as the Yitamin A l e v e l was increased. The following .on 9 7 year these same investigators (262) used the same basal r a t i i and found 1200 international units of Vitamin A per pound of feed were adequate to promote satisfactory growth. They suggested that t h e i r previous high requirements were due to the severe mixed infe c t i o n that occured amongst the experimental birds They further noted that the storage of Vitamin A was cumulative and bore a marked relationship to the amount of the factor i n the diet. Unit for unit, carotene and Vitamin A obtained from a f i s h o i l concentrate were found to be u t i l i z e d by the chick with equal e f f i c i e n c y . In a study of the Vitamin A storage of growing chicks, Holmes, Tripp and Campbell (116) used the following ration: 52 pounds corn-meal a t t r i t i o n , 15 pounds wheat bran, 15 pounds wheat flou r middlings, 12 pounds ground oat groats, 8 pounds dry skim milk, 5 pounds a l f a l f a leaf meal, 5 pounds meat scraps, 5 pounds f i s h meal, •§• pound d i calcium phosphate, 1 pound oyster s h e l l meal, 1 pound s a l t . When 0.5% sardine o i l was added to the above basal rati o n and fed to young chicks the l i v e r s of these chicks contained s i g n i f i c a n t l y more Vitamin A than when 0.25% was added. The amount stored was approximately four times more for the higher l e v e l . The Vitamin A reserve i n the l i v e r increased i n the period from eight to twelve weeks of age. Older birds showed wide variations both i n egg producing power and i n the Vitamin A content of t h e i r l i v e r s . 9'8 Working at Cornell, Ringrose and IJTorris (185) used the following ration: 55 pounds white corn-meal, 25 pounds wheat flour middlings, 10 pounds commercial casein, 5 pounds dried yeast, 2£ pounds pulverized limestone, 1 pound steamed bone meal, 1 pound cottonseed o i l , -g pound iodized s a l t , Yitamin D was supplied by i r r a d i a t i o n . They established the growth response curve for the chick to Yitamin A feeding, a b i o l o g i c a l assay expressing results i n terms of Yitamin A per gram of material thus became possible. The assay was conducted by feeding the test material at such a percentage of the ration as to induce a growth response which was s l i g h t l y , but d e f i n i t e l y , subnormal. The growth results obtained at eight weeks of age were then applied to the. growth response curve and the units of Yitamin A per 100 gms. of feed determined. By dividing the number of units per 100 gms. of feed by the percent of the test material i n the ration, the potency of the test material i n units per gm, was obtained. They found that the minimum Vitamin A requirement of the chick during the f i r s t eight weeks of l i f e was about 150 U.S.P.X. Revised 1954 units per 100 gms. of feed. Holmes, Tripp and Campbell (117) obtained embryos and young chicks from different sources and determined the Vitamin A stores i n t h e i r l i v e r s and unabsorbed egg yolks by means of the antimony t r i c h l o r i d e reaction. From the eighteenth day of Incubation to the f i f t h day after hatching the Vitamin A 99 content of the l i v e r rose ana that of the unabsorbed yolk f e l l , the combined t o t a l from both sources f a l l i n g slowly. At the University of B r i t i s h Columbia Biely and Chalmers (16) fed chicks the diet of Elvehjem and Neu (76) to which irradiated yeast was added. Once a week a dose of the U.S. Pharmacopoeia Reference Cod Liver O i l was fed directly into the crop of each b i r d i n an amount to supply 0, 25, 50, 75 and 100 international units of Vitamin A daily to the various groups. The group reoeiving no o i l showed symptoms of Vitamin A deficiency after three weeks; these became marked at fi v e weeks and after eight weeks a l l the birds were dead. Of the remainder those receiving 25 international units daily, began to l a g i n growth and to show some symptoms after four weeks. The remainder grew well and showed no symptoms. They concluded that 75 international units daily were suffi c i e n t to ensure normal growth and protect chicks up to eight weeks against any symptoms of Vitamin A deficiency, 100 units appeared to be above the immediate normal requirements of the chicks up to t h i s age. When yellow corn was substituted for the white corn (59$) i n the ration with the omission of Cod L i v e r . O i l , they found approximately the same performance as the dose of 75 international units daily had given. Record, Bethke and Wilder (182) used the basal diet of Elvehjem and Neu (75) i n which irradiated yeast supplied 4,000 international units of Vitamin D per 100 grams of feed. They IQO conducted both prophylactic type and curative type of feeding. The prophylactic t r i a l s showed that, under the experimental conditions employed, i t required a minimum of approximately 50 to 100 micrograms of carotene or 80 to 160 international units of Yitamin A from Ood Liver O i l per 100 gms. of ration for normal growth and the prevention of external and internal symptoms of Yitamin A deficiency i n chicks to about eight weeks of age. The curative experiments showed that about 100 micro grams of carotene or 120 to 200 international units of Yitamin A from Ood Liver O i l were required every other day to cure and prevent symptoms of Yitamin A deficiency and restore good growth i n chicks depleted of t h e i r Yitamin A reserves to ten to twelve weeks of age. Both types of experiments showed that the chick u t i l i z e d carotene as a source of Yitamin A, The response of the chicks to carotene or Vitamin A was approx imately similar when equivalent rat units were fed; indicating that the chick and rat u t i l i z e carotene as a source of Yitamin A i n the same order. They presented data to show that the Yitamin A requirements of chicks inoreased with age. In conclusion they pointed out that these experiments showed no sig n i f i c a n t storage of Vitamin A i n the l i v e r s of the chicks u n t i l several times the minimum l e v e l , as determined by growth and external and internal symptoms was fed. At the Western Washington Experiment Station Bearse and M i l l e r (13) used a basal ration consisting of: 44i pounds 101 ground white corn, 15 pounds ground wheat, 15 pounds ground oat 10 pounds millrun, 7 pounds meat scrap, 7 pounds powdered skim milk, 1 pound oyster s h e l l f l o u r , £ pound s a l t . Yitamin D was supplied by i r r a d i a t i o n . They supplemented this ration with varying levels of dehydrated a l f a l f a for a twenty-four week growing period. These levels furnished approximately 87.5, 175, 350, 700 and 1400 Sherman-Munsell (206) Yitamin A units per 100 gms. of ration. The results based on avitaminosis A mortality, growth and l i v e r storage of Yitamin A showed that 175 Sherman-Munsell Yitamin A units per 100 gm. of ration met the Yitamin A requirements of the chicks. Feeding efficiency-was greater i n the l o t s reoeiving the highest levels of Yitamin A during the f i r s t eight weeks of the t r i a l . This difference was not so great for the entire twenty-four week period. There was a close correlation between mature body weight and t o t a l feed consumption. In summarizing the findings of many investigators Dr. Parkhurst (174 a) stated: "When feeds are mixed previous to the feeding period, the minimum Yitamin A requirements of chickens to eight weeks of age would appear to be i n the v i c i n i t y of 150 U.S.P. Yitamin A units per 100 grams of feed (680 units per pound) and the p r a c t i c a l requirement for s a t i s  factory growth and l i v e r storage about 300 units per 100 grams s 102 of feed (1362 units per pound. For chickens to t h i r t y weeks age, the requirement may be double. Turkeys require at least double the amount of Vitamin A i n the i r ration as do chickens 103 Requirements of Hens For Egg Production Bethke, Kennard and Sassaman (15) reported i n 1927 that -the fat soluble vitamin content of hen's yolk was greatly influenced by the amount of these substances present i n the ration and by environment of the laying hen. The yolks of eggs l a i d by hens which had access to a blue-grass range were approximately f i v e times as potent i n Yitamin kt and ten times as active .antirachitfeally as the yolks of eggs l a i d by hens which received the same basal diet but were confined indoors. When two parts of Ood Liver O i l were added to the mash there was a f i v e f o l d increase i n the a n t i r a c h i t i c and fat soluble A vitamin content of the egg yolks. Holmes, D o o l i t t l e and Moore (115) found that the addition of fat soluble vitamins to the poultry rations d e f i n i  t e l y stimulated egg production. The average weight of eggs, produced by the o i l fed birds was s l i g h t l y greater than that of the eggs produced by the control birds. In t h i s experiment they observed that the percentage of eggs containing blood clots decreased consistently with the increase of Ood Liver O i l i n the experimental ration. The number of eggs discarded during incubation either from being i n f e r t i l e or on account of containing a weak germ was less for the o i l fed group. The number of chicks obtained and the v i a b i l i t y of these chicks was greater i n the birds fed the supplement. Contrary to 104 previously observed conditions, the body weight of the high producing birds did not decrease but their weight at the end of experiment exceeded that of the controls. As the experiment progressed the fat soluble vitamin potency of the eggs from the experimental birds increased while those from the controls decreased. No detectable flavor was imparted to either the egg or the f l e s h of the birds fed the God Liver O i l . They d e f i n i t e l y concluded that supplementary fat soluble vitamin feeding increased the reproductive performance of domestic fowl. From the work of Sherwood and Fraps (211) i n 1932, i t was found that yellow corn alone was not s u f f i c i e n t l y high to supply enough Vitamin A to pullets for egg production. They estimated that pullets required for maintenance alone, about 105 units of Vitamin A potency per day, or 33 units per pound per day, (t h i s i s eight times the estimate of 4 units per day per pound for maintenance of growing r a t s ) . Ordinary a l f a l f a was found not to supply enough Vitamin A to produce eggs of high potency i n this vitamin, even i f fed at Q$ of the mash. They calculated that one unit of Vitamin A i n the egg required 6.3 units i n the feed at 270 units, 5,7 units at 336 units, and 4,0 units at 44-4 units i n the feed. The following year Sherwood and Fraps (212) published further data on the subject. In th i s experiment three rations were used: 105 No. 1 HO*: 2 20 pounds yellow corn 50 pounds yellow oorn 20 " wheat shorts 50 " white com 20 " wheat bran 20 " ground oats 20 " meat and bone scrap No. 3 A l l White Corn These rations were fed to groups of pullets that had been normally fed. At the close of the experiment the yellow corn group (No, 1) averaged 25% more i n weight than the white corn group (No. 3) and the mixed oorn group (No. 2) weighed 17% more than No. 3. These rations had a definite effect on the number of eggs l a i d after the experiment had been i n pro gress two months. During the l a s t four months of the experiment the yellow corn group l a i d approximately 65% more eggs than did the white corn group and the mixed corn group l a i d 55% more eggs than did the white corn group. The Vitamin A content of the eggs showed d e f i n i t e l y , as the experiment advanced, that none of the rations contained enough Vitamin A for high egg production. The Vitamin A content of the eggs from a l l of the groups became loiver as the experiment progressed. They con cluded that i t required approximately 1,365 units of Vitamin A (Sherman-Munsell units) per day for a laying p u l l e t for main tenance and production of eggs at the rate of twenty eggs per month.. • 106 Russell (193) also observed that the Yitamin A content of eggs from hens receiving yellow corn was greater than those .from hens receiving white corn. He found that 47 to 48% of the Yitamin A fed was contained i n the eggs. In 1934 Sherwood and Fraps (213) stated, "The percent age of vitamin recovered i n the eggs i s greatest at the lowest l e v e l and least at the highest l e v e l of feeding. This apparent percentage recovery i s not correct because the Vitamin A stored by the hen i s not taken into account and some of the Yitamin A or carotene fed i s used for maintenance while some of i t i s probably not digested". They found that on the average 1 unit of Vitamin A i n the egg required 4.7 units i n the feed. The units of Vitamin A required i n the feed for 1 unit i n the eggs decreased as the average number of units fed increases. Bishey, Appleby, V/eis and Cover (20) conducted an . investigation to correlate the Vitamin A a c t i v i t y of egg yolks with the amounts of carotinoid pigments they contained. They observed that there was a d i s t i n c t gradation of the color i n the egg yolks from the hens on the different rations and that the color of the yolks on a given ration was uniform. They concluded that while there seemed to be some relationship between color and growth, the Vitamin A a c t i v i t y of egg yolks could not be explained on the basis of the carotinoid pigments they contained. They also noted that the Vitamin A 107 a c t i v i t y of the egg yolks was d i r e c t l y dependent upon the rations of the hens, Mohler (157) claimed that the optimto l e v e l of Cod Liver O i l feeding i n the diet of chickens was between 1 and 2%. I f this l e v e l was exceeded, impairment i n egg production and hatchability was l i k e l y to occur. The work of DeVaney, Litus and Nestler (51) showed that extra Vitamin D, as 0.5$ vi o s t e r o l (160 D), i n the diet of chickens receiving graded amounts of Cod Liver O i l had no effect on the transfer of Vitamin A to the egg. Eggs from pullets receiving 8$ of Cod Liver O i l were several times richer i n Vitamin A than those from pullets receiving lower doses. Russell and Taylor (194) determined the Vitamin A con tent of the egg yolk and the diet "by the method of Sherman and Munsell with a reference o i l as a standard. The results were expressed i n U.S.P. 1934 (international) units by refer ence to a curve of response. They found that with varying egg production and intake of Vitamin A, the output of Vitamin A i n the eggs, expressed as a percentage of intake, varied from 11 to 32$. In 1935 Sherwood ana Praps (215) estimated that hens laying per year one hundred and f i f t y eggs, high i n Vitamin A potency required approximately 600 Sherman-Munsell biological units of Vitamin A per day, or 7.5 units per gram of feed. 108 Koeriig, Kramer and Payne (133) found that the eggs of pul l e t s l a i d i n the t winter about the fourth month of egg production, and tested by the method of Sherman and Munsell, contained about 25 Sherman-Munsell units of Vitamin A per gram, whether the birds were of high or low productivity. Autumn eggs produced near the end of the f i r s t year contained 20 and 33 Sherman-Munsell units per gram, when l a i d by birds of high and low productivity, respectively. They conclude that egg production made pronounced n u t r i t i v e demands upon the hen and that the recognized demand for Vitamin A was more pronounced the greater the number of eggs produced and the longer the laying period. Working at the Western Washington Experiment Station, Bearse and M i l l e r (14) found that eggs from hens receiving different quantities of Vitamin A i n th e i r rations contained different quantities of Vitamin A i n t h e i r egg yolks i n proportion to the amount of Vitamin i n the ration. Chicks hatched from such eggs l i v e d and grew on Vitamin A deficient ration i n proportion to the amount of Vitamin A i n the breed ing hen r a t i o n . They concluded that 500 Sherman-Munsell units of Vitamin A per 100 gm. of feed i n the breeding hen ration supplied s u f f i c i e n t Vitamin A for maximum hatchability. 109. EXPERIMENTAL Experiment No. 1. Preliminary experiments conducted at The University of B r i t i s h Columbia by Wood (266) indicated that Pilchard O i l , as produced i n B r i t i s h Columbia, contained an appreciable amount of Vitamin A. In fact, Wood's experiments showed that one-half on one percent of a sample of commercial Pilchard O i l when fed i n combination with a basal ration free from Vitamin A, promoted normal growth and development of chicks to eight weeks of age. The purpose of the present experiment was t o deter mine the Vitamin A potency of a sample of commercial Pilchard O i l i n terms of International Units, using the chick as the test animal. The results of t h i s test were' compared with those of B i e l y and Chalmers (16), who determined the Vitamin A re quirements of growing chicks up to eight weeks of age by feed ing them gradecd doses of Reference Cod Liver Oil(251a). The method followed by B i e l y and Chalmers was to feed ©, 25, 50, 75, and 100 units of the Reference Cod Liver O i l once a week with a pipette per orem. The chicks in the experiment described herein, were fed their Vitamin A supp lement i n the mash, but were brooded under similar conditions. Consequently, the data may be considered as comparable. Experimental Methods: Day-old single comb White Leghorn cockerels were 110. placed i n battery brooders under the same conditions as des cribed by B i e l y and Chalmers (16). These cockerels had been separated-from the p u l l e t s by the Japanese method of "chick sexing". The basal ration fed to;the chicks consisted of 59 pounds ground white corn, 25 pounds wheat middlings, 12 pounds crude casein, 1 pound calcium carbonate, 1 pound calcium phos phate, 1 pound s a l t , and 1 pound irradiated yeast (70 D). The . chemical composition of this ra.tion was as follows: Protein ,20.84%, Calcium 1.32%, Phosphorus .54%. This basal r a t i o n i s similar to that used by Elvehjem and Hew (75) i n their vitamin determinations, except that i r r  adiated yeast (70 D) served as the source of Vitamin D i n the present experiment i n place of u l t r a - v i o l e t l i g h t as used by the above investigators. It d i f f e r s from the basal ration used by Hart, Kline, and Keenan (101a) for the production of rickets i n chicks, i n that, the yellow corn i s replaced by white corn, and the yeast i s changed t o irradiated yeast. The basal ration was supplemented with Pilchard O i l a. s follows: .Lot I 1/8% Pilchard O i l Ho. 1 Lot I I 1/4% Pilchard O i l Ho. 1 Lot I I I 1/2% P i l c h a r d - 0 1 1 1 Ho. 1 Lot IV 1/4% Pilchard O i l No. 2 Lot V 1/2% God Liver O i l Lot VI Control Wesson O i l was used to d i l u t e the l / 8 % and 1/4% lots of o i l up to 1/2% by weight while the controls received 1/2% 1 1 1 . Wesson O i l only. Results: The average weekly weights of the various lots of chicks are given i n Table No. 1, and the growth curves are shown i n Figure. 1,. It w i l l he readily seen from Table No. 1 that the control chicks began to lag i n growth between the second and . t h i r d week. From then on the. difference in the weight of the control chicks, and those which received Vitamih A as contained i n Pilchard O i l ( l / 4 % or 1/2%) or God Liver O i l (1/2%), became progressively greater as the experiment neared complet ion. This i s s t r i k i n g l y shown i n Figure No. 1 . Furthermore, the control chicks showed d i s t i n c t symptoms of avitaminosis A at two weeks of age, and characteristic lesions at the time of death which usually followed three to four days after the f i r s t appearance of symptoms. In every instance, the control chicks showed considerable loss of weight before death. The control chicks were a l l dead by the end of the eighth week. From Table No. 1, i t w i l l be seen that the groups of chicks which received various amounts of Pilchard O i l or God Liver O i l , grew at a f a i r l y uniform rate during the f i r s t four weeks of the experiment. . After the fourth week, the group which received 1/2% Pilchard O i l No. 1, and the group which received 1/2% Cod Liver O i l , grew faster than the groups which received either 1/8% or 1/4% Pilchard OilNo. 1. At eight weeks of age, the average weight of the chicks which were fed 1/2% Pilchard O i l No. 1 exceeded the average weight of the chicks 112. co CQ M CD CD | CQ CD CO © l [ © © "I © © O J ©J H i rH O o rH o O J rH o CO 9 « o e CO co o> o> £> o CO I O I O m CC t o CO CO t o *te **. % O J CO rH T j • rH CO 00 .crj © IO t o IO CO Oi O J co rH Oi CO co co t o CO co t o t o '^ o « o . 9 4 LO IO CO OS rH t o CO tO to t o CO CO t o t o O J O CO O J rH co O CO O J ' 0 e © 9 • CO o t o rH C - t> o> co t o O J 02 O J O J O J tO Oi co t o O J CO Oi t o 0 « 0 c CO tO t o ^ < o o o O co O J O J o j O J rH t o t o O J IO o co to CO IO D « « •a o rH Oi to t o '^ Oi rH rH rH rH rH r - CO Oi t o ^ rH O J co t o « « « a t o IO IO Oi o> Oi o> CO to £> o t o rH rH to, 0 « o e o r to rH co CO <o CO CO CO o t o z> t o rH t o O J - 0 • o « co CO CO t o t o t o t o t o « 0 « rH rH H O J 4 J © © rH P- P rH rH rH rH •H • r t •H •H •H O O o O o t s •cs t » . •CS © r4 ?H n > CO Co Co n3 •H & ^ , 3 H o o O o rH rH rH rH nd •H •H •H •H o PM A * rH f 4 O V*. ^ . ^ ^ CQ o . y » o co & t o o O J CO Oi o 00 e CO rH O tO rH tO rH O Oi t o o rH 9 CO CO to o CO « t o CO 0 2 \ rH Oi rH O u +» a o a X X *5 $ 114. which were fed 1/2% Cod Liver O i l . It should be noted that the average w eight of the chicks which received 1/4% of Pilchard O i l Ho. 2 were only 9 grams less than the average v^eight of the chicks which received 1/2% Cod Liver O i l and also, only 20 grams below the average weight of the chicks which were fed 1/2% Pilchard O i l No. 1* With the exception of a few chicks which received 1/8% of Pilchard O i l No. 1, none of the chicks showed any evidence of avitaminosis A. As judged externally, the chicks appeared ' to be quite normal and healthy i n everyrespect. Post-mortem examination of a few. chicks i n each l o t did not reveal any indications of Vitamin A deficiency in the internal organs. The s t a t i s t i c a l analyses of the data are given i n Tables No. 2 and 3. Prom these results, i t w i l l be seen that the difference between the average.weight of the chicks which received the basal ration supplemented with either 1/2% Pilchard O i l No. 1 or 1/2% Cod Liver O i l and the chicks which received 1/8% of Pilchard O i l No. 1, was s t a t i s t i c a l l y s i g n i  f i c a n t . Furthermore, i t i s important to note that the difference between the average weight of the chicks which were fed 1/4% Pilchard O i l No. 2 and. the chicks which were fed 1/8% Pilchard O i l No. 1, was s t a t i s t i c a l l y s i g n i f i c a n t . As previous l y stated, i t i s of considerable importance to note that the average weight of the chicks which received 1/4% of Pilchard O i l No. 2 compared very favorably with the average weight of the chicks which received either 1/2% Pilchard O i l No. 1, or 1/2% Cod Liver O i l . The results of the s t a t i s t i c a l analysis 115. O - p 4Jl 0 r-t" •H O ,Q 0 ra o i> o o •H +> ro •H > CD A .H CQ ro +J to H I m - h i o> t o o CVJ o O J o O J ® 6 * +1 02 - f i O J - I - l O J +1 o O J to • t o o 0 r H OJ OJ 00 o CO OJ t o LO o t—i t o CO o « . . * CO LO r - l rH HI rH rH H 4 1 - I - l - t l t l t l CO O o> LO r-i OV CO » e • 9 « t o CO £> (Jl o> OJ rH o o t o E - o o A o « rH •H to •H H i rH OJ rH rH (CD I"- t l t l + 1 +i + 1 O r H o Oi t o '^ rH o co CO o 9 « © CO co co H I o to LO LO LO LO LO rH r H rH •H •H •H o O o *Si *} (H fH ro ro CO .3 A ' o o O rH rH rH •H «H •H Pi A, OJ rH •H O H CD fc> •H H" •tf O rH •H O i d M co-& o rH •H fH ^ ^ ^ fes. ^ O J ^ \ \ rH rH 116. 4J +>• +» • M •a S3 a S3 CO s CO • ro CD o o o o O •H •H •rH •H •rH C H <M C H <H tH •H •H •H •ri •H EI S3 a S3 0 S O £sQ b O taO •H •rH •H •H •H CO C O C O C O 0 3 4-> 4-» -!-> o ra O o i s ; • S rH -l-> a a o •H « H •H a •H co +» o J25 +•> co o •r+ «H •rH c •(30 •H w +» O 3 c 4J S3 S3 C O cO C o o o o •H •rH •H «H <H <H •H •H ' H £3 S3 (—« bQ EJO •H •ri •H CO C O C O 4-> +i o o o J z j S2i to CM C O » . « 00 o> oo to CM o CO to r H o r H to to CM CQ CM CM o o O © o O CM en CM C O to C M o o> to to co L O o L O e * 9 o £> to to o o CM CM CM CM CM CM " t l t l + 1 t 1 - i l "f 1 O CM to CT> rH CM CO C M C O i> L O * e » * o O rH O o rH o> co £> to L O t o CO C7> r H r H o e to £> t o CM CM, Oi + 1 Hrl- +1 CO c- o> r H t o r H e * ft-o o> H r H - d rH co o rH •H ft HI * d rH CO O • H •H ft CM rH rH rH rH •H rH rH •ri •H •H O "H •rH O O o O O " d •a fH rH 0 - d - d CD rH rH s > rH fH t> ro CO • r i CO cd •H . d 3^ rH .S3 A ' rH o o o O rH rH • d rH rH - d •H •H O •H •iH O ft ft o A ) ft o CM CM < CM CM \ \ \ \ \ H i rH rH rH rH rH e g -=8 «« =3 <=8 H i rH rH rH rH rH •H •H •H •H •H •H o o O O O o ra o r - l •H ft • d m CO 3^ o i H ' H ft • d H co o rH •H ft ^ ^ ^ ^ co \ HI rH T H • r H - d fH CO & o i H •H ft CM rH •H O XS H C O 3^ o rH •H ft r H •t-i o - d fH CO 3^ o rH •H ft r-i r-i •H o •H H i - d o CM r H •ri O • d o rH •ri ft CM CM r H •H O T 3 fH CO 1^ o •H •H ft ^ ^ r H c 3 =8 o • d H CO 4 3 o r H •ri ft ¥ L fe?. ^ CM \ rH "CM ^ rH *H O • d fH CO & o i— ! •ri ft rH •H O fH CD f> •ri t-H • d o © CM 117. (Table No. 3) show ahat the difference i n the weights of the chicks which were fed 1/2% Pilchard O i l No. 1 or 1/4% Pilchard O i l No, 2, or 1/2% God Liver O i l were d e f i n i t e l y of no . s t a t i s t i c a l significance. Discussion: At the end of eight weeks, the average weight of the chicks that received 1/2% Pilchard O i l No. 1 or 1/4% Pilchard O i l No. 2 or 1/2% Cod Liver O i l compared very favorably v/ith those reported by Buckner et. al.(24a), Rlngrose and Norris (185) and Record e t . a l . (182). According to Record e t . a l , (182), at the end of eight weeks the average, w eights of chicks which were fed various amounts of Vitamin A supplement per 100 grams of feed were as follows: Aver. Weight Supplement - 'Qm. 40 units.as C.L.O. 504 9.4 80 11 " C.L.O. 532 8.6 160 «' " C.L.O. - 564 13.8 240 " " C.L.O. 581 13.0 320 " " C.L.O. 542 9.9 1% poultry C.L.O. (700 units) 564 7.7 Records e t . a l . data show that the chicks which receiv ed 160 units of Vitamin A per 100 grams of basal ration grew as well as the. chicks which received 240 and 320 units respect i v e l y . Furthermore, the average weight of the chicks which received 160 units was equal to those which received 1% Cod Liver ©il (700 un i t s ) . Since the chicks which were fed 40 or 118. 80 units of. Vitamin A showed symptoms of avitaminosis A, they concluded that 160 units of Vitamin A per 100 grams of feed may he considered as the minimum requirements of chicks up to eight weeks of age. The average weight of the chicks which were fed 1/2% Pilchard O i l No. 1, or 1/4% Pilchard O i l No. 2, or 1/2% Cod Liver O i l (Table No. 1) were almost identical with the average weights of the chicks of the above mentioned investigators. It may be concluded, therefore, that the chicks which were fed 1/4% Pilchard O i l Np, 2 or 1/2% Pilchard O i l $o. 1 received at least 160 units of Vitamin A per 100 grams of feed from their respective quantities of Pilchard O i l . Since 1/4% Pilchard O i l No. 2 produced a rate of growth equally as good as 1/2% Pilchard O i l No. 1, i t would appear that the Vitamin A. potency of Pilchard O i l No. 2 was greater than that of Pilchard O i l No. 1. Since 160 units of Vitamin A per 100 grams of feed i s considered by several investigators to be the minimum require ment of chicks up to eight weeks of age, i t would appear that l / 4 % of a f i s h o i l containing a minimum of 600 units of Vitamin A per gram of o i l (minimum requirements of U.S. Pharmacopea) would meet the minimum requirements of growing chicks to eight weeks of age. The results of this experiment show that 1/4% of Pilchard O i l No.2, and 1/2% of Pilchard O i l No. 1 amply supplied the above requirements. It may be concluded, there fore, that the Vitamin A content of the two samples of Pilchard O i l used in this investigation ranged between 300 and 600 units 119. of Vitamin A per gram of o i l . Possibly another way of estimating the Vitamin A potency of the Pilchard O i l used i n this experiment i s through estimating the t o t a l feed consumption and the amount of P i l c h  ard O i l contained therein. Unfortunately no record was kept of the amount of feed consumed by each group of chicks. However, the data reported by several investigators (126a), (127a), and (128a) show that normally developing White leghorn chicks w i l l consume 3.6 pounds or 1,634.4 grams feed up to eight weeks of age. This amount of feed would contain 4.086 grams of Pilchard O i l when 1/4% i s added or 8.172 grams when 1/2% i s added. Since the basal ration was pra.ctica.lly free from Vitamin A, the chicks must have of necessity derived their Vitamin A requirements from t he r, bove amounts of o i l i n the 1/4% and 1/2% groups. Since, as has been shown before, a ration must contain 160 units of Vitamin A per 100 grams of feed to promote normal growth of single comb White Leghorn chicks to eight weeks of age, the to t a l number of Vitamin A units supplied i n the ration to this age would have to be approximately 2615.04 units. In view of the normal rate of growth attained by the chicks which received either 1/4% Pilchard O i l No. 2(4.086 gm. o i l ) or l / 2 % Pilchard O i l No. 1 (8,172 gm. o i l ) i t may be concluded that the above number of units were supplied i n the respective amounts of o i l , I f 4.086 grams supplied 2615.04 units of Vitamin A, then 1 gram of Pilchard Oil,.No. 1 would contain 640 units of Vitamin A. Similarly the 1/2% Pilchard O i l No. 1 would contain at least 120. 320 units of Vitamin A per gram of o i l . Biely and Chalmers (16) have shown that the minimum requirements of growing chicks up to eight weeks of age are 50 units of Vitamin A per day. On this basis the chicks i n the present experiment must have received during the entire period(50x56) 2,800 units of Vitamin A. In accordance with the method of calculation shown above,.the sample of Pilchard O i l Ho. 1.would have contained 343 units of Vitamin'A, while the sample No. 2 would have contained 685 units of Vitamin A. Whether the Vitamin A potency of Pilchard O i l i s estimated on the basis of Record e t . a l . (182).minimum requires ment; per 100.grams of feed or B i e l y and Chalmers (16) minimum requirements of 50 units per day, the results are in close agreement, i . e . the Vitamin A content of Pilchard O i l Ho. 1 i s over 300 units and Pilchard O i l Ho. 2 over 600 units. Both methods of calculation show that Pilchard O i l Ho. 2 would meet the minimum requirements for Vitamin A as specified by the United States Pharmacopoeia for Cod l i v e r O i l . 121. Experiment 2- The basal ration used i n the tests? in Experiment 1 was similar to that employed by Elvehjem and Hen (75) i n their studies of 'Vitamin A. I t consisted of 59 pounds of white corn, 25 pounds of wheat middlings, 12 pounds of casein, 1 pound calcium carbonate, 1 pound calcium phosphate, 1 pound s a l t , and 1 pound irradi a t e d yeast. Because of the fact that white corn i s very expensive and not obtainable in Canada, i t was deemed necessary to d evelop a ration consisting of commonly available Vitamin A-free grains. Eor this purpose, a ration similar to that used by Erohring and Wyeno (86) was compounded and a pre liminary test conducted. Unfortunately a large percentage of the experimental chicks developed slipped tendons. Consequent l y , the r a t i o n had to be discarded. At the. time the present experiment was in progress the cause of slipped tendons was not known. I t was generally believed then that an improper balance of calcium and phosphor ous was mainly responsible for the occurrence of slipped tend ons. However, several investigators have since shown that 10 to 20% of ground oats, added to a ration which ordi n a r i l y i n  duced slipped tendons, tends to prevent or diminish their occ urrence. Eor t h i s reason, i t was decided to. include 16% oats i n the rations used i n t h i s experiment and modify the calcium- phosphorous r a t i o by using various amounts of milk and meat. The l a t t e r also provided the animal protein i n the ration, instead of casein which was used i n the ration of the f i r s t experiment. Casein, unfortunately, contains a variable, but appreciable, amount of Yitamin A, and therefore , i s not quite suitable for use i n a basal ration for Yitamin A -experiments. To obtain further information on the residual amount of Vita- .min A i n casein, i t was decided to compare a basal ration containing casein with other rations containing varying amounts of milk and meat. Thus the rations used in the present ex periment varied in their calcium and phosphorus content, and also i n the source of animal protein. The percentage of Calcium and Phosphorus i n the various rations was as follows: Ration Calcium % Phosphorus % No. I 1.426 0.628 No. I I 1.579 0,699 Ho. I l l 1.865 0.853 No. IV 0.978 0.608 A l l rations contained oats for the prevention of slipped tendons, while none contained white corn. TAble No. 4 shows the various rations that were used in this experiment. It w i l l be seen that rations I, I I , and I I I contained 17-g- pounds of animal protein by weight.. Ration No. I contained l2|-% powdered skim milk, and 5% meat scrap. Ration N0.II! contained 10% skim milk, and meat scrap. Ration No. I l l contained 7t% milk, and 10% meat scrap. Ration No. IV contained 12% commercial casein only. Experimental Methods: Day-old single comb White Leghorn cockerels were obtained from a l o c a l hatchery and divided into 16 groups of 25 chicks i n each. 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One compartment in each t i e r contained a heating unit of three carbon filament lamps» The four basal rations used are shown in Table l b . 4. Each basal ration was divided into four l o t s and supplemented with various amounts of Yitamin A„ The amount of supplement added to Lot I was 1/4% of Pilchard O i l ; Lot 2, 1/2% Pilchard O i l ; Lot 3, 1% Pilchard O i l , and Lot 4 served as the control. Wesson O i l was used to dilute the 1/4% pilchard O i l up to 1/2% by weight i n order to assure thorough mixing of the o i l i n the mash. One half percent Wesson O i l was added to the control rations. The respective amounts of o i l were mixed into the mash.and run through a fine-meshed sieve. Mash and wa|er were constantly available to the chicks. A time clock controlled the number of hours of a r t i f i c i a l l i g h t that- the chicks received. I t was set for a twelve-hour day, from 6:00 A.M. to 6:00 P.M. At the end of the experiment, several chickswere examined to determine the percentage of ash i n t he t i b i a f i b u l a . The average ash analysis proved to be over 45%, thus indicating normal bone c a l c i f i c a t i o n . Results: The average weekly weights of the chicks i n the various groups and rations are given in Tables l o . 5 and Ho. 6, and the growth fiurves, i n Figures 2 to 9, inclusive. The mortality of the control groups i s shown i n Table H'o. 7. COS Ml col ©I CO 02 Ml © KM Ml •<D| ©I M to CO ©I © H LO 03 © © CO © ©I 1 to Ml ©1 © OJ M © © •d rH O k e 1 rH CQ 1 * rH l CQ rH I'- .. ° ft S3 « O O •H •H 4-> 4 J CO CO pH £ H 125. 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O fH OJ ^ . a \ \ o rH rH rH* . •& 127, 800 -760 -720 .680 :640 .600 -560 -520 -480 CQ fH J440 -S 4400 i J360 M CO f H © 4320 |j .280 -240 .200 .60 ,20 Figure No. 2 Growth curves of chicks which received Ration #1, plus various supplements of Vitamin A. 1. \$ Pilchard O i l . 2. i% Pilchard O i l . 3. 1% Pilchard O i l . 4. Control. / 3 0 0 . 7 6 0 Figure No, 5. Growth Curves of Chicks which received Ration No, 2, plus various Supplements of Vitamin A. 130. 800 760 |720 680 640 1600 560 C O S 480 440 -S 400 -H © 360 S FH © 320 «{ Figure No. 5 Growth Curves of Chicks which / received Ration No* 4, plus / / various Supplements of Yitamin / / 1 / k A, 1, \% Pilchard O i l . 2, i $ Pilchard O i l . 3. 1$ Pilchard O i l . 4. Control, / V 800 -.760 • 720 .680 -640 .500 .560 .520 4 4 4 0 -5 1st. Figure Ho: 6, Growth Curves of Chicks which received ^ P i l c h a r d O i l , 1, Ration No,'. 1, 2, Ration No. 2, Ration No. 5, 4. Ration No, 4. 1 Week Age i n Weeks — — i — ; i • -•800: .760 .720 -.660 -£40 -.600 .560 1 5 2 0 CQ | 4 8 0 g -1440*5 s HOC" 0 0 1360 k 0 -L320-S; .580 540 -200 • 160 .120 80 Figure No. 7 Growth Curves of Chicks which received %fo Pilchard O i l . 1. Ration No, 1» 2. Ration No. 2. 3. Ration No, 3. I i 1 4. Ration No. 4. / / 1 I / / / / 1 Week Age i n Weeks - — i 800 .760 Figure Ho, 8 Growth Curves of Chicks which received ifa Pilchard O i l . 1. Ration No. 1. 2. Ration No. 2. 3. Ration No. 3. 4. Ration No. 4. 134. 340 X o 5 © o m col o j M M o rH O P3 o o O r H E H H E H a: CQ fH CO a © P H G CQ O U •H © 4-> 4 J •H © rd fH o o G o •H CQ © H-> G •H •d Xi o O i+H : o CQ •M o bO •H G 4 3 -ri o> >» • fH O rH O t>> ©. 4 3 fH o CQ cO G •d © 43 cO © •d © rH O © 42 CQ W © O <H 41 «H CQ O o H-> G « •ri H-» O H P H CO © CO <H 4-> O O ft © CQ rH O X< -H © fH is +> o G 43 © CQ !> I CO ! 43 ft PH © o CQ *d © © o N •H © H 4:) •H H-> -S-> CO fH O © 4d a EJ "d *H © rH ^ © 0 ' 4 3 CQ 43 4-> CQ -H 3 © o 43 CQ 4-> H co © 43 rH « LO • m m rH rH rH •+ -f -+ -1 - + ' + i - + 4 H + > ~|- 43 4-> -t' + + •H S. = • r i = = CM CM CO rH rH -t i - -+ + + - h -f- -f + ~t" "t 43 ^3 +> T ~f* -t-» i - •H — r •H s- r:~ CO LO CM r- co CO rH CM CM CM CQ -W • rH 4 3 O H-> >> ro © •d © 4 3 f H o -d © fH o CQ <H 43 © CO Si G xi © is o 43 02 CQ © rH o •sF CM CQ id rH O >> •d © 4 3 fH o CQ cO G -d © o 43 01 CM 43 T •rH = + 43-+ 1 1- rH <tf .CM rH CO CM rH rH H - ' 43 CT> <vH CM CO CO CM CO rH LO CM o> rH CQ M O A4i o fij ^d G o &\ •& CQ O •H 43 4-> 4-S O CO fH O 4-3 S3 CO o G fH O H-» - H G +>' o ro o P3 o> CM CM CJ> CM CM CO CO CM t.O CM CO 136. The growth rates of the control chicks are in accord with those of Blvehj em and Neu (75), and Bi e l y and Chalmers(16) By the end of the third week avitaminosis A sygiptoms . were observed amongst the control chicks fed Rations No* I, No. IT, and No. I l l * The chicks fed Ration No. IV did not show symptoms u n t i l the fourth week. By the f i f t h week, a l l control chicks showed d e f i n i t e symptoms and were unsteady o n t h e i r feet, and crouched on their haunches. In advanced stages, the chicks were found lying on one side with t h e i r heads f a l l e n • forward.- The feathers were very r u f f l e d , end the beaks and shanks were without any pigmentation. In spite of being i n a weakened condition, the birds made considerable effort to eat and drink, but they a l l l o s t weight, however, before dying. In a l l rations used, a condition was noted i n which feed adhered to the beaks and palate of the control chicks, and the chicks which received 1/4% of o i l . I t was necessary to remove th i s dry, hard accumulation at least two or three times a week. At four weeks of age, some of the chicks showed a l a t e r a l displacement of the mandibles, while i n others, the upper and lower mandibles became permanently curvetf. The cause of th i s condition was, no doubt due to'an impairment of the mucous secreting glands of the mouth, induced by a lack of Vitamin A in the ratio n . No t y p i c a l ophthalmic lesions v/ere observed, yet sev eral chicks shoed slight s.oreness around the eyes. These ob servations are i n agreement with Elvehj em and Neu (75). 137. A l l chicks that died during the course of the ex periment were autopsied and the results or findings recorded (Table 7), In a number of the control chicks the renal tubules .were greatly distended, and the kidneys f i l l e d with accumul ations of urates. Several control chicks, however, exhibited ataxia, without any v i s i b l e accumulation of urates in the ur inary t r a c t . The chicks s t i l l l i v i n g on the control diets at the end of the experiment (8 weeks) were k i l l e d and post- mortemed. The two chicks l i v i n g on Ration No. I I , and one on Ration No. I l l , showed urates In the kidneys and ureters, and the i r general appearance and condition indicated that they would have died, probably during the ensuing week. Those on Rations No. I and No. TV shoed sl i g h t accumulations of urates in the kidneys, and no doubt, they would have died in one or - two weeks. The fact that several of the c ontrol chicks lived to the end of the eighth week would indicate that the basal rations used i n these experiments were not absolutely free from Vitamin A. Such results are in agreement with the findings of other investigators (169)* Of the four rations used, Ration No. 1 (containing 12^% milk and 5% meat scrap) and Ration No. IV (containing 12% commercial casein) apparently had appreciable amounts of Vitamin A. There were seven and six chicks, respectively, l i v i n g on these control diets at the end of the experiment. On the other hand, i t would appear that Rations No. I l : and No. I l l (10% and 7 if* of milk respectively) contained the least amount of Vitamin A, since only two chicks on Ration 138. No. I I , and one chick on Ration No. I l l survived t i l l the end of the eighth week. The survival of only one or two chicks on these l a t t e r two diets Is most l i k e l y due to the individual ? reserves of Vitamin A in the chick as received from the mother hen. The mortality i n the groups which received the various supplements of Vitamin A during the experiment was less than 22%, demonstrating that the chicks were brooded under s a t i s  factory conditions* Up to four weeks of age, the chicks in a l l groups, except the controls, grew at an almost uniform rate. After t h i s , with one exception, the rate of growth of the different groups varied d i r e c t l y , according to the percentage of Pilchard O i l fed. For s ome unaccountable reason, the group receiving • the•• \% Pilchard O i l i n Ration No. I grew faster than the 1% group. This variation was noticeable at the end of the second week of the experiment. As the same sample" of Pilchard O i l was used to supplement a l l rations, the variation in this ration cannot be attributed to the Vitamin A potency of the o i l used. (See Tables No. 5 and No. 6, and Figures 2 to 5 incl u s i v e ) . The growth curves of the chicks which were fed the various basal rations, but the same amount of Vitamin A supp lement, are shown i n Figures 6, 7, 8, and 9. The growth curves of the chicks which r eceived •%% Pilchard O i l i n Rations No. I , No. I I , No. I l l , and No. IV are shown i n Figure 6. It w i l l be seen that Ration No. IV (12% commercial casein) produced the fastest growth while 159* Ration No, II (10% milk) was next i n order. The rate of growth-cm Rations No. I (lSg-% milk) and No. I l l micbk) was p r a c t i c a l l y the same, although considerably behind that produc e d by the other two rations. In the case of the chicks which received Y/° of P i l c h  ard G i l i n the respective rations (Figure 7) i t w i l l be noted that Rations No. I, No. I I and No. IV gave p r a b t i c a l l y the same growth while Ration No. I l l gave the poorest growth . In the case of the chicks which received 1% of Pilchard O i l (Figure 8) i t w i l l be seen that Ration No. IV produced the greatest growth while the other rations were i n the following order; Ration No. I I ; Ration No. I l l ; and Ration No. I producing the poorest growth. The growth curves of the various control groups are found i n Figure 9. A study of these curves reveals that at the end of the eight-week experimental period, the chicks on Ration No. I (7 chicks livings) were rapidly losing weight while those on Ration No. II (2 chicks l i v i n g ) , showed a marked decrease i n weight, weighing considerably less than the chicks fed Rations No. I; No. I l l ; or No. IV. The individual weekly weights showed that the control chick, that l i v e d t i l l the end of the experiment on basal Ration No. I l l , grew at a normal rate. The rapid r i s e i n the growth curve between t he seventh and the eighth week of the control chicks which received basal Ration No I I I i s due to the survival of this exceptionally large chick. The chicks fed Ration No. IV (6 chicks l i v i n g ) showed a gradual 140. r i s e i n the growth curve. This, r i s e i n the growth curve may he taken as conclusive evidence that the commercial casein used i n this experiment contained appreciable amounts of Vitamin A. The mean body weights of the different l o t s were subjected to s t a t i s t i c a l interpretation i n Tables No. 8 to No. 14, inclusive. For the results to b e considered s i g n i f i c a n t , the difference of the two means must be equal to or greater than three times i t s probable error. In a l l rations except Ho, IV, there was a significant difference between the average Y f e i g h t of the \% and groups and the i% and L% groups. Ration No. IV, however, showed only a s i g n i f i c a n t difference between the average weight of the and 1% groups. The fact that i n Ration No. IV the difference between the average weight of the and the \% groups was not s i g n i f i c a n t , indicated further that Vitamin A was presehtrin the commercial casein used i n t h i s experiment. Thus, the superior growth found i n the i% group pf Ration No. IV may be accounted for by the assumption that the Vitamin A - p r e s e n t i n the casein .supplemented that supplied by the Pilchard O i l . There was no si g n i f i c a n t difference betweenthe average weights of the and 1% groups i n any of the rations. This evidnece demonstrated that -§-% of the Pilchard O i l used in this experiment supplied s u f f i c i e n t Vitamin A to promote normal growth of chicks up to eight weeks of age. This further sub- stantiated the findings of Wood (266), that \% of a good grade of Pilchard O i l meets the Vitamin A requirements of growing chicks to.' eight weeks of age. Furthermore, these results show 141. CO to to r H O J 0 2 O J * a « r H r H r H to +.1. - H 1 1 + 1 "sf< <o CO I O to » « O J to co r H r H r H r H <i fa o CO i eis M col co .1 H ipl o |25, E H N ' O CO H CO o M EH CO H EH <J EH C O O •rH H J . CO • r l t > © fH cd T 3 CO -p CO •H © © fc=J 4-» © © rH. 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Oi <3 CO 5 ^ rH .r-i 146. to ml r H © o G CD O •H <H • H a bO •rH CO '£ rH .. © © r H CH ,Q P H CQ a CD ,© o © © f H © CH •H G o CQ ' H CD O o +» H J H J 4 3 H J G G G G G CD CO cd CO cd cd O o o o o o •H • r l •rH • H •H •H <H <£» <H «H «M <H •H •rH •H •H •rH •rH G CJ G G G G bO •bo W W) 550 t>a • H •H •rH •rt •H •H CO CO CO CO CO CO H-> H J H J H J H J - p o O O O o O S2i 125 S2i & i s r-r o CO Oi o 00 CO W © o © « © . e o w o o rH o co CO to O CM iH. o o 0 e « © z> o CO CO r - rH rH rH H rH rH ~H + 1 • + 1 + 1 +! -t- 1 Oi CO co CO O co CO t o rH t o CO o> © o . 9 o 0 o CO CO IO £- CO CO rH •CV2 CO =*fc =*: VS. ^ ^ Oi Oi Oi \ \ \ rH rH r H rH \ rH H rH rH CO > c3 VS. V l Oi -CV2 \ \ rH rH eg Oi c8 to Oi Oi Oi Oi \ \ \ rH ' rH : rH H 147. I f a © o CO: o •rH <H •H a •H CO H o H © O 'S3 © H © 'tHj CO n l o H PH CQ S3 ro © o © o S3 © M © S3 o CQ •H fH cO o o 4-> S3 CO o •PH < H S3 &D •ri CQ 4 J CQ O s r - l oo o « rH + 1 CO t o a 00 4-> 4-> 4 - J 4-> S3 S3 S3 S3 S3 CO ro CO CO CO O o o o o • r i • r i •H • r i •H «H <H <H «H •H •H •H •H •H S3 S3 S3 S3 S3 bO (50 W) ttO &0 •r l •H •H •H •H to CQ CO CQ CQ +» 4-» 4 J 4-> o O O O £H t o t o •<* O O i CO CO o LO CO e • 4 0 e 02 rH t o rH O rH LO Oi . • LO rH + 1 OJ o co CM LO co rH +. ! . to t o LO OJ rH rH + 1 -H< t o O OJ co rH - ( - I O O © tO r H + 1 to o> OJ OJ CO t o «<* =fts H rH «H rH rH c8 <8 c8 rH • rH r H OJ OJ CO . ««*• =*!= ^ ¥ t .rH rH rH rH rH r-i 148. that no immediate advantage was derived from feeding an excess of Vitamin A. • The groups which received the same percentage of •Vitamin A supplement, hut in different rations, were compared s t a t i s t i c a l l y in Tables No. 12 [•£%) , No. 15 (£%), and No. 14 (1%). A study of Table No. 12 ii%) revealed that the average weight of the chicks fed Ration No, IV (commercial casein) was s i g n i f i c a n t l y higher than the average weight of chicks fed Ration No. I {12%% milk). S i m i l a r l y , the average weight of cbicks fed Ration No.. I I (10% milk) was s i g n i f i c a n t l y higher than the average weight of chicks receiving Ration No. I l l ( 7 i % milk) , and the average weight of chicks fed Ration No. IV was s i g n i f i c a n t l y higher than that of those fed Ration No. I l l These results demonstrated the superiority of Rations No. IV and No. II over the other rations. Since i t has been shown above that Ration No. IV contained appreciable amounts of Vitamin A, this ration cannot be regarded as a suitable Vitamin A-free basal ration. Table No. 13 ( i % j does not show any significant difference i n the weights of the. chicks which were fed the various rations. Any trace of Vitamin A that may have been present i n any of the rations did not influence the rate of growth of these groups a.s i t . did. i n the case of the•%% groups. Such evidence again points out the fact $ hat when chicks up to eight weeks of age are given i% Pilchard O i l , their Vitamin A requirements for normal growth are met. 149. The comparisons of the 1% groups are shown in Table Ho. 14. It w i l l be noted that the only significant difference was that between the average weight of the chicks fed Ration "Bo-. IV and the weight of those fed Ration Ho. I. The difference between Ration No. II and Rati on No. I was nearly significant;. As previously stated, there was some unaccountable, reason for the slow growth of the group fed Ration No. I with 1% o i l as compared with the group fed ^ % o i l . It i s doubtful i f any significance should be attached to this difference. Discussion: It w i l l be seen from Table No. 7 that, at the end of the eighth week, the group fed Ration No. I I had two control chicks l i v i n g , and Ration No. I l l , only, one, and that these three chicks showed external symptoms of avitaminosis A. Since several investigators ( i l l ) have demonstrated chat chicks show marked varia t i o n i n their individual stores of Vitamin A at time of hatching, i t ' may be concluded that the variation i n l l v a h i l i t y of the chicks receiving the various rations was due not e n t i r e l y to varying traces of Vitamin A i n the rations, but to variations- i n individual reserves of Vitamin A i n the chicks themselves. Since i t i s apparent that traces, i f any, of Vitamin A i n Rations No. I I and No. I l l were the same, the rate of growth of the groups fed the Vitamin A supplement becomes the most important fa,ctor for consideration. The weights of the chicks i n the various groups at the end ofthe experiment d e f i n i t e l y show that Ration No. I I i s superior to Ration No. 1 5 0 . I l l i h growth promoting powers. The combination of ten pounds of milk and seven and one-half pounds of meat scrap as used i n Ration No. II can, 'therefore, he d e f i n i t e l y accepted as a. satisfactory protein supplement—practically free from Vitamin A — ± n place of casein for b i o l o g i c a l tests with chicks. The growth obtained with Rations No. I and No. IV i s decidely greater than that with Ration No. I I , but i t has already been shown that these former rations contained an app reciable amount of Vitamin A. Thus i n spite of the increased growth obtained, these rations cannot be recommended for use in Vitamin A determinations with chicks. Besides the cost and time involved i n obtaining Vitamin: A-free casein by either alcoholic extraction or by heating i n an oven, there is the p o s s i b i l i t y that some of the amino acids may be changed or destroyed in either or both of these processes. In the event of any such changes taking place, i t can be readily seen how detrimental this would be to growing chicks. On the other hand, when a small quantity of milk, along v/ith a good grade of meat scrap (Ration No. I I ) , i s substituted for treated casein, the cost per pound protein i s lowered considerably, while at the same time animal protein of high b i o l o g i c a l value i s provided. In order to avoid the influence of the individual reserve of Vitamin A, i t would be desirable to extend.the length of the Vitamin A experiments from eight to ten weeks. This extended period would allow the control chicks more time i n 151. which to exhaust their store of Vitamin A, thus assuring 100% mortality. If the supply of Vitamin A fed to the breeding s • stock i s not controlled, i t would appear desirable to establish for chicks a depletion period similar to that used i n the rat tests. While no definite time regarding the most satisfactory length of this depletion period can be suggested here, the idea would appear to be worthy of investigation. The depletion periods of chicks hatched from flocks receiving different amounts of Vitamin A would ha.ve to be ascertained i n each case. Such a procedure, i f adopted, would lead to the more uniform occurrence of avitaminosis A symptoms,death of control chicks, and more precise evaluation of the potency of Vitamin A ca r r i e r s . Frohring•and Wyeno (86) suggested that in Vitamin A tests, chicks from only one breeding flock should be used, or i f they are obtained from different breeding flocks, that they be distributed equally amongst a l l the groups. The l a t t e r suggestion entails the keeping of extensive records by the person or persons hatching the chicks, end would tend to increase the factor of v a r i a b i l i t y due to the different v i a b i l i t y of the chicks from the different flocks. The idea of using only one breeding flock as a. source of chicks for Vitamin A determinations would appear to be more commendable. At present, the chicks used in b i o l o g i c a l assays do not vary only from laboratory to laboratory, but they also vary within a laboratory, according to the season of the year, strain of 152. birds, and other conditions. In Vitamin A assays the indis criminate use of chicks from various flocks i s undoubtedly responsible for the great variation noted in the rate of ' grwth and f i n a l weight of individual chicks. It probably also accounts for some of the v a r i a b i l i t y i n the weight of chicks within a group at the end of the test period. To avoid such v a r i a b i l i t y , i t would be necessary to breed and maintain a s t r a i n of breeding stock genetically pure for rate of growth. Since 'the rate of growth and the f i n a l average weight of chicks at the end of the experimental period are the main c r i t e r i a used i n evaluating the pbtency of Vitamin A supplements, i t i s essential that the chicks i n the various groups should grow at a uniform l e v e l . To accomplish t h i s , the following conditions would have to he assured: (a) The s t r a i n of birds should be genetically pure for rate of growth. (b) The Vitamin A intake of the breeding stock should be controlled. (c) The Vitamin A reserve of chicks should be depleted before the actual assay period Is begun. (d) A Vitamin A - f r e e ration that ensures rapid develop ment of avitaminosis A symptoms should be used, (e) The Vitamin A-free ration in the presence of suf f i c i e n t Vitamin A sould promote rapid, normal growth. (f) The conditions of brooding and rearing should be uniform. With a l l of the above conditions under complete ex perimental control, i t should be possible within four to six weeks following the depletion period to evaluate with great X 53 e precision the potency of Vitamin A ca r r i e r s . 1 5 4 . Exp e r i me nt No. 5» The object of t h i s experiment was to compare the hi o l o g i c a l and chemical methods, of assaying B r i t i s h Columbia Pilchard O i l for Vitamin A. For this purpose, three samples of Pilchard O i l produced at different stages of the fi s h i n g season were used. Thus, information was also secured on the effect of seasonal variation on the Vitamin A content of P i l c h ard O i l . O i l No. 1 was produced early i n the season and O i l No 2, i n the middle of the season, and O i l No. 3, late i n the season. These o i l s were kept i n large volumes i n commercial storage tanks. A. sample of ea.ch o i l was taken from the res pective tanks and brought t o t he laboratory for bio l o g i c a l assay. A portion of. each o i l was tested by the antimony t r i  chloride test (Carr-Price), and the blue unit value recorded. The results' of th i s test showed that O i l No. 1 contained four blue units, O i l No. 2, two blue units, and O i l No. 3, one blue unit. Experimental Methods; Day-old single comb White leghorn cockerels were divided into seven groups of f i f t e e n chicks each, and placed i n battery brooders as i n the previous experiments. They were fed a basal ration which consisted of the following ingredients Ground Wheat . 38-g- pounds . Ground Oats 10 " Wheat Middlings 20 " X 5 %3 o Wheat Bran 10 pounds Skim Milk Powder 10 » Meat Scrap 7 i " Ground Oyster Shell 2 " Salt 1 pound Ix-radiated Yeast 1 11 Total 100 pounds The amount of Vitamin A supplement added to the ration was either 1/8% or 1/4% of each of the three samples of P i l c h  ard O i l . Each o i l was diluted up to 1/2% by weight with Y/esson O i l , while the controls received 1/2% of Wesson O i l only. A l l details of procedure were similar to those describ ed i n Experiment I I . Results : : • . :• The average weekly weights of the various groups are given i n Table No 15. Growth curves of these groups are shown i n Figure No. 10. A study of the growth curves shows that the average weight of the groups which received O i l No. 1 are heavier at the end of eight weeks, than the groups which received O i l No. 2 or O i l No. 3 or the control. Similarly the chicks which were fed G i l No. 2 show higher average weights than the chicks which were fed O i l No. 3 or the ba.sal ration alone. It should be noted, however, that the average weight of the chicks which received O i l No. 3 was only s l i g h t l y higher than the average weight of the control chicks. 156. 01 © © S3 CO to, © © 03 CD.. ©I tO Wt © © LO 01 © © 03 © © to CO © © O J © © « CO o O J . © CO CO o co o OJ o> O J to © LO CO O J CO LO CO o O O J a •E- O J r H O o 9 LO CO r H • CO LO o LO LO t o r H r H o o o E> CO o t o to t o LO o o £> Q o 9 9 « OJ a> £> t o t o to o to o O l to o * o 9 OJ o r H CO •SP to o CO o to CO o a 9 © CO LO to r H LO to CO OJ CO LO LO o t o co co co O J co e 9 to t o t o OJ CO co to LO to O J O J M OJ O J CO LO LO o to LO CO r-i t o 9 9 9 9 a CO 00 o> o o CO t o O J o O J O J O J O J O J O J co CO CO o z> LO OI O J . r H » e 9 9 9 r H co o CO r - to to to HI r H r H r H rH r H o o t o CO o O J to « 9 « a> O J r H O J Oi O J O i Oi O J r-i r H r H r H r H O J CO OJ O © CO o> o ' O 9 4 9 9 o> o H 00 co (> CO oo LO co O J o r - O J CO t o to t o ® 9 o e CO o> r - t> LO tO LO LO LO o o O J to « to to « to to r H O o to o rH to CO co CO CO 9 CO LO •d o | CO] f H © © r H P P a co r H r H o O J =*« **! r H r H r H •ri •H •H O o o • d • d - d ri b co co 3 43 43 43 o o o i H r H r H •H •H •ri f H ft Pi \ r H . e O J t o 0 t o r-i r H r-i • r i •H •ri O O o - d - d • d H fH U cd CO CO 43 43 .si o o o •rH r H r H •ri •H •ri Pi ft ft < \ H I . \ rH \ r-i o fH +». S3 o 157 n- nd 158. , The control chicks showed the early symptoms of avitaminosis A at three weeks of age. On post-mortem exami ation, considerable accumulations of urates i n the kidneys a ureters were observed. There were only three chicks l i v i n g i n this group at the end of the experiment. These three chicks were k i l l e d and examined i n t e r n a l l y . They showed enlarged kidneys containing large deposits of urates. Keratinization of the e p i t h e l i a l tissue of the respiratory tract was observed om several of the control chicks that died during the course of the experiment. The fact that thre e chicks survived to eight weeks of age on the control diet may be due to late hatching (autumn) and to the large stores of Vitamin A that they could, as a consequence, receive from the parent stock at that time. With the exception of the chicks which were fed O i l No. 3 there were no external symptoms of Vitamin A deficiency i n any other groups. The chicks which received 1/8% O i l No. 3 showed d i s t i n c t symptoms of avitaminosis A at four weeks of age. At the termination of the test at eight weeks, 50% of these chicks had died. On post-mortem examination, accumulat ions of urates were found i n the kidneys and ureters of these chicks. At the end of the experiment, several of the chicks that were s t i l l l i v i n g i n this group were selected for post mortem examination. They too, showed topical avitaminosis A lesions of the kidneys. The symptoms of Vitamin A deficiency in the group fed 1/4% O i l No. 3 were not so pronounced as those found in the group which received 1/8% O i l No. 3. The s t a t i s t i c a l analysis of the average mean weights "159. of the different l o t s of chicks are given in Table No. 16, and the significance of the difference between these weights in Tables No. 17 and No. 18. A. study of these tables reveals, tthat at eight weeks of age the difference between the average weight of the chicks which were fed either 1/8% or 1/4% P i l c h  ard O i l No. 3, and the control chicks, is not s i g n i f i c a n t . This indicates that Pilchard O i l No* 3 does not supply s u f f i c  ient Vitamin A at these levels to promote normal growth of chicks up to eight weeks of age. Furthermore, i t w i l l be seen that the difference between the a.veragew eight of the chicks which received 1/8% Pilchard O i l No. 1, end that of the chicks which received either 1/8% or 1/4% Pilchard O i l No. 3 i s s t a t i s t i c a l l y s i g n i f i c a n t . The difference i s even more sig n i f i c a n t between the former and the control group. Similarly the difference between the average weight of the chicks which received the supplement of 1/4% Pilchard O i l No. 1 and any of these three l a t t e r groups, i s s i g n i f i c a n t . Comparison of the other groups did not regeal any s i g n i f i c a n t difference. Discussion: It may be readily seen from the above results that the sample of Pilchard O i l No. 1 (early season) i s decidedly sup erior i n Vitamin A potency to the other two samples of o i l . S i m i l a r l y , the sample of Pilchard O i l No. 2 (middle season) is superior to the sample of Pilchard O i l No. 3 (late season)/ Because these o i l s had been stored immediately upon production, any deterioration that may have taken place during storage would be most pronounced in sample No. 1, and least i n sample 160. o H J £ 4-> © - rH •H rH O -H •H «H' CO © 3 O t> o S 3 o •H 4J cd •H © <H T J M 3 s H J CO CO CO + 1 . o CO e r-i CO r-i o> LO r H CO •IO O J oi OJ O J o CO + l 4-1 -+I +1 CO CO r H r-i CT> « * « « LO co o r-i r H r H OJ co O o LO CO CO CO « • r> • o o rA CO rH rH r-i + 1 +1' + 1 + 1 co CO OJ CO CO o> « e rH CO e> CO D~ CO OJ e c o .+ 1 00 ^' ft LO OJ LO CO co + 1 j> o CO OJ CO rH CO LO 00 e o tt O J LO rH rH OJ t l + 1 + 1 CO CO CO tO « » e to to CO CO o rH H J •H © cd © 4 J © S © rH ft ft CO t O •<* OJ r H t l £ > 0 o> 00 r H • H o fH cd & o rH •H ft Vt- co LO rH o LO LO to to to * « O ft © to <o CO rH rH rH rH OJ CO + 1 + 1 t 1 + 1 - H "H O IO LO O to o LO rH rH co tO o 0 s ' o « © £> CO O J O to CO o rH CO IO CO « o « . r H • O J O J CO CO =fe =fe =te rH r-i rH rH rH •H •H •H •H •ri o O o O O X) •d * t f Xi *1 M fH fH f H CO co CO CO CO si & o o o o o rH rH rH rH rH •H •H •H •H •H a PH ft ft ft ft r-i VI VI VS. 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Therefore, i t may he concluded that Pilchard O i l No. 1 (early season) must have contained more Vitamin A at the t ime of production than the other two o i l s . This evidence demon strates that Pilchard O i l - produced early i n the season i s more potent i n Vitamin A than o i l s that are produced at later stages of the fishing season. It i s important to note that t he results of the blue unit test were i n agreement with the b i o l o g i c a l assay in that they showed the differenced in the Vitamin A content of the three samples of Pilchard O i l . Consequently the blue unit test may be used as a preliminary test for estimating the Vitamin A potency of Pilchard O i l . This i s of considerable commercial importance in;a B much as i t would enable commercial refineries to s elect their o i l s on the basis of this test. Such a practice would permit: the use of the o i l s with the highest Vitamin A content for medicinal purposes, while the o i l s of low Vitamin A potency could be used for paints and other manufactured products. Bailey (6) at Prince Rupert has reported that the blue value of Pilchard O i l i s only a rough estimation of the Vitamin A content of the o i l . Under such circumstances, this itest may be used only as a rough measure of the r e l a t i g e potency of various samples of Pilchard O i l . At the present stage of our knowledge, the actual Vitamin A a c t i v i t y of Pilchard O i l has s t i l l to be determined by the b i o l o g i c a l assay. The foregoing experiment has d e f i n i t e l y s hown that the Vitamin A content of Pilchard O i l varies with the season of the 164. year, and that o i l produced early i n the season has the highest Vitamin A potency. It would he interesting to ascertain i f the vitamin D potency of Pilchard O i l would he p a r a l l e l to the Vitamin A potency at various stages of the fishing season. 165. • SUMMARY. Experiment No. 1 (1) 140 day-old single comb White Leghorn Cockerels were divided into seven different groups. The Vitamin A-free basal r a t i o n used was similar to that of Slvehj em and Neu (75). I t was supplemented with 1/8%, \%, j>-% and 1% Pilchard O i l #1, \% Pilchard G i l #2, and \% Cod Liver O i l . One lot of chicks was fed the basal ration only. (2) When either \% Pilchard O i l #1 or \% Pilchard O i l #2 was added to the basal r a t i o n , the rate of growth was as good as when •§-% Cod Liver O i l was added to the ration. (3) The rate of growth of the chicks i n this experi ment was compared with the rate of growth of chicks that had been fed graded amounts of Vitamin A as supplied by Reference Cod Liver O i l (U.S. Pharmacopoeia, 3000 units of Vitamin A per gram). (4) It was found that Pilchard O i l #1 contained at least 300 International Units of Vitamin A per gram and Pilchard O i l #2 at least 600 International Units per gram. (5) Pilchard O i l #2 would appear to meet' the minimum requirements specified by the U.S. Pharmacopoeia for Cod Liver O i l (600 units of Vitamin A per gram). Experiment No. 2. (1) 400 day-old single comb White Leghorn Cockerels were divided into 4 groups and each group was subdivided into 4 l o t s . Each group was fed a different basal ration. Ration No. 1 contained 12-|% powdered skim milk and 5% meat scrap; Ration No.II 10% powdered skim milk and 7f% meat scrap; Ration ' Ho. I l l 7-g-% powdered skim milk and 10% meat scrap; and Ration No. IV l2-g-% commercial casein. (2) The rations were s upplemented with 0%, ^%, Y/° and 1% of Pilchard O i l . (3) I t was shovra that Rations No.I and No. IV contained appreciable traces of Vitamin A. (4) Since basal Ration No.II (10% powdered skim milk and 7ir% meat scrap) contained the least traces of Vitamin A of a l l the rations used i n t h i s experiment i t was found to be the most suitable for Vitamin A studies. (5) \% Pilchard O i l was found to supply adequately the Vitamin. A requirements of grov/ing chicks up to 8 weeks of age. (6.) At 8 weeks of age the average weights of the different lots of chicks (in grams) were as follows: Ration Control l A % P.O. 1/2% P.O. 1% P.O. #1 279 + 13 667 + 11 774+13 . 736+12 #2 261 705.± 12 766+ 11 785+11 #3, 336 658+11 738±12 7.64+8 #4 324+ 23 718 + 12 759T11 794 it 13 (7) Up to 8 weeks of age, the mortality i n a l l the groups except the controls was less than 2 i % . (8) Several recommendations are suggested for im-proving the b i o l o g i c a l Assay chick as the test animal. of Vitamin A ca r r i e r s . using the ' Experiment No. 5. (1) 105 day-old single comb White Leghorn chicks were divided into 7 groups. The basal ration used i n this experi ment was similar to that of Ration Bo. I I i n Experiment No. 2. (2) Three samples of pilchard O i l that had been produced at different stages of the f i s h i n g season were used as Vitamin A supplements. These o i l s were added to the basal ration i n amounts of 0%, and (3) It was found that the pilchard O i l produced i n the early stages of the f i s h i n g season was a more potent source of Vitamin A than the o i l s produced later in the season. (4) These three samples of pilchard O il were tested by the antimony t r i - c h l o r i d e test (Carr-Price reaction). (5) The results of the arflmony t r i - c h l o r i d e test paralleled the results obtained with the B i o l o g i c a l Assay. (6) I t i s recommended that o i l s that are produced i n the early stages of the fishing season should be used for medicinal purposes, because of their higher Vitamin A content. 168 c BIBLIOGRAPHY. 1. Abelin, I., - N.A. and R., 5: No, 1330, 1935 2. 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Zoltan, I.A., - N.A. and R., 5: No. 866, 1935 U.S.P.X Interim Revision Announcement No. 2 A 1934 REVISION OF THE TEXT AND ASSAYS FOR COD LIVER OIL OF THE PHARMACOPOEIA OF THE UNITED STATES TENTH DECENNIAL REVISION This announcement by interim revision of new standards and assays for medicinal Cod Liver Oil is in recognition of the notable advancement in scientific knowledge concerning vitamins, and for the purpose of officially establishing in this country the new International Units for Vitamins A and D. The Pharmacopoeia has accepted these International Vitamin A and Vitamin D Units as the basis for its new standards, and the new " U.S.P. X 1934 Vitamin A Unit" and " U.S.P. X1934 Vitamin D Unit" are identical with the corresponding International Units. It is hoped that this action by the Pharmacopoeia will largely over come the present confusion in label and literature statements concerning the Vitamin A and Vitamin D potency of many products. This con fusion in the past has been due to the use of unofficial units of varying vitamin value.* Following the established policy of pharmacopceial revision the Com mittee has had the advice and cooperation of many experts in the prepa ration of this new text. Two conferences, with more than thirty recog nized authorities in vitamin work,[present at each, laid the foundations for the assays. There has since been established what is known as the "U.S.P. Vitamin Advisory Board." This Board has completed the assay texts and supervised the preparation of an official "Reference Cod Liver Oil" of known Vitamin A and Vitamin D potency, expressed in International Units. This is now being distributed by the Phar macopoeia Board of Trustees as a basis of comparison in assays. The new assays require the use of this official "Reference Cod Liver Oil" as a basis of comparison. T h i s "Reference Cod Liver Oil" was independently assayed, in com- * A statement of the approximate relationship of various unofficial yitamin units, now employed in the study and labeling of products containing Vitamin A and Vitamin D, has been issued by the U.S.P. Vitamin Advisory Board. Information concerning this can be obtained by addressing the Chairman of the U.S.P. Com mittee of Revision. . 1 parison with the international standards, by fifteen different laboratories who reported their results to the Vitamin Board. The members of this Board compiled and evaluated the data submitted and deter mined from these figures the Vitamin A and Vitamin D potency of the "Reference Oil." The Board has also arranged for the periodic recheck of the potency of this "Reference Oil" so that it may serve as a satis factory standard when used by manufacturers of products containing Vitamin A and/or Vitamin D. The "Reference Cod Liver Oil" is ob tainable through the office of the Chairman of the Committee of Revision. This Revision for the first time establishes an official vitamin standard for Cod Liver Oil. Heretofore, the Pharmacopceial requirement for vitamin content and assay has been optional and reflected the situation when the TJ.S.P. X became official some years ago. At that time there was little or no quantitative information upon which to base the proper minimum Vitamin A potency for a medicinal Cod Liver Oil and the re quirement that, when assayed Cod Liver Oil should contain at least 50 Vitamin A units per gram, was primarily to insure the •exclusion of manipulated oils in which all vitamins had heen destroyed. At that time "Vitamin D " was not even known by that designation but was referred to as "an antirachitic factor." The new official standards insure a Cod Liver Oil of excellent quality, corresponding in potency to what is now largely being sold in this country as "U.S.P. Oil." In addition to the introduction of the vitamin standards and methods of assay, the following physical or chemical standards have been intro duced or amended: A standard for permissible color intensity for the official oil has been added. The limit of free acid and the saponification number take into con sideration the occasional use of carbon dioxideas a preservative for the Oil. The directions for the determination of unsaponifiable matter have been so modified as to eliminate possible sources of error. A chill-test has been introduced to insure the absence of excessive amounts of stearin. The new official vitamin standards and assays and the chemical and physical standards as prepared by the Sub-Committee on Organic Chemicals of the Committee of Revision, have been officially adopted by the General Committee of Revision and the Board of Trustees. By action of the Board of Trustees the new standards for Cod Liver Oil will become official on January 1, 1935. E. F U L L E R T O N C O O K 43rd St. and "Woodland Ave.. Philadelphia, Pa. Chairman oj the Committee oj Revision May 1, 1934 ojthe U.S. Phartnacopatia. "SALT MIXTURES" For Diets i n B i o l o g i c a l Assays In reviewing the s a l t mixtures during the r e v i s i o n of the Pharmacopoeia i t was found that the formulas published i n the U,S,p„ Interim Revision Announcement No,. 2 were not s t r i c t l y i n ocnformity with the fovirulas as o r i g i n a l l y published.!, The formulas 3?.cw l-.apc: bee.n oa.i.oul.vred t«» repre sent the SGHW content of s a l t s and acids as i n the criglna 1., but i n terms of o f f i c i a l products. This w i l l enable those using these formu las to secure more e a s i l y the ingredients and prepare the mixtures. The formulas as they w i l l appear i n the U.S.P.XI are as follows: S a l t Mixtures For preparing the s a l t mixtures the a v a i l a b l e form of each chemical i s taken to f u r n i s h the st i p u l a t e d equivalent o f each chemical. Salt Mixture No. 1 • Calcium Carbonate (U.S.p.)..., 134.8 Gm. Magnesium Carbonate (U.S.P., 40 per cent MgO)....... 28.9 Gm. Sodium Carbonate, anhydrous (U.-.S.P, Reagent)......... «.... 34,2 Gm. Potassium Carbonate (U tS wP„, dried at 180°C)„ 141.3 Gm. Phosphoric Acid (U..S,P,, 86.5 per cent),,.... 119.3 Gm. Hydrochloric A c i d (U.S.P,, 36 per cent).. 148.3 Gm. S u l f u r i c Aeid (U.S,P., 96 per cent)., 9.6 Gm. C i t r i c A c id (u.S.p..). .. 111.1 Gm, F e r r i c C i t r a t e (U.S.P, Reagent, 17.5 per cent Fe) 7.44 Gm. Potassium Iodide (U.S.p.) „„ 0.020 Gm. Manganese Sulfate (U.S.P. Reagent, MnS04.4H20) ,... 0.117 Gm. Sodium Fluoride (U.S.P. Reagent) 0.062 Gm. Potassium Alum (U.S.P.) , , 0.044 Gm. Dissolve the c i t r i c a c i d i n a s u f f i c i e n t quantity of hot d i s t i l l e d v/ater and add the s o l u t i o n to the mixed carbonates. Then add the potas sium iodide, manganese s u l f a t e , sodium f l u o r i d e and potassium alum, pre viously dissolved i n d i s t i l l e d v/ater.. Then add the f e r r i c c i t r a t e d i s  solved i n the hydrochloric acid. Dilute the s u l f u r i c a c i d with d i s t i l l e d water; add the phosphoric a c i d and add t h i s a c i d mixture to the mixture previously prepared and s t i r u n t i l effervescence ceases. Evaporate the f i n a l mixture to dryness, i n a current of a i r at from 90° to 100°C, and reduce the r e s u l t i n g product to a f i n e powder. Salt Mixture Mo. 2 Sodium Chloride (U.S.p.) ....... 1.73 Gm. Magnesium Sulfate (U.S.P. ) 5.45 Gm. Sodium Biphosphate (U.S.P,) j , . . 3.47 Gm.. Potassium Phosphate ( K 2 H P O 4 ) . . . . .* 9.54 Gm. Calcium Biphosphate [ C a H 4 ( P O 4 ) 2 . H 2 O ] . , . . i . 5.40 Gm. F e r r i c C i t r a t e (U.S.p. Reagent, 17.5 per cent Pe)..,,.., 1.18 Gm» Calcium Lactate (U.S.P.) j 13 Gm. Mix the f i n e l y powdered s a l t s uniformly. Respectfully submitted, E. HJLLERTON COOK, Chairman OLEUM MORRHILE Cod Liver Oil O l . M o r r h . The partially destearinated fixed oil obtained from fresh livers of Gadus Morrhua Linne" and other species of the Family Gadidm. Cod Liver Oil may be flavored by the addition of not more than 1 per cent of any one or any mixture of flavoring substances recognized in this Pharmacopoeia. Cod Liver Oil contains in each Gm. at least 600 U.S.P. Units of Vitamin A and at least 85 U.S.P. Units of Vitamin D . * The Vitamin A potency and Vitamin D potency of Cod Liver Oil when designated shall be expressed in "United States Pharmacopoeia Units" per gram of oil and may be referred to as "U.S .P . Units" per gram of oil. To indicate the adoption of the new standards the statement "U.S .P . X —Revised 1934" may be used. Description and physical properties—A thin oily liquid, having a peculiar, slightly fishy, but not a rancid odor, and a fishy taste. Cod Liver Oil is slightly soluble in alcohol, but is freely soluble in ether, chloroform, carbon disulphide, and in ethyl acetate. Tests for identity and purity—Specific gravity 0.918 to 0.927 at 25° C. A solution of 1 drop of Cod Liver Oil in 1 cc. of chloroform, when shaken with 1 drop of sulphuric acid, acquires a violet-red tint, gradually changing to reddish-brown. When viewed transversely in a tall, cylindrical, standard oil-sample bottle of colorless glass of about 120 cc. capacity, the color of Cod Liver Oil shall not be more intense than that of a mixture of 11 cc. of colorimetric cobalt, T.S.,t 76 cc. of colorimetric ferric T.S.f, and 33 cc. of distilled water, in a similar bottle of the same internal diameter. * One "United States Pharmacopoeia Unit of Vitamin A " is equal, in growth pro moting and antiophthalmic activities for the rat, to one International Unit of Vitamin A as defined and adopted by the Conference of Vitamin Standards of the Permanent Commission on Biological Standardisation of the League of Nations in June of 1931; one "United States Pharmacopoeia Unit of Vitamin D " is equal, in antirachitic potency for the rat, to one International Unit of Vitamin D as defined and adopted by the Conference of Vitamin Standards of the Permanent Commission on Biological Standardisation of the League of Nations in June of 1931. t Reagents for Color Determination: Colorimetric Cobalt T.S.—Containing 59.496 Gm. of cobaltous chloride (CoCl 2. 6H 20) in 1000 cc. of solution. Dissolve about 65 Gm. of cobaltous chloride in enough of a fluid made by mixing 25 cc. of hydrochloric acid with 975 cc. of distilled water to make a volume of 1000 cc. Measure 5 cc. of this solution into a 250 cc. flask, add 15 cc. of a solution of sodium hydroxide (1 in 4) and 5 cc. of solution of hydrogen dioxide. Boil the mixture gently for ten minutes, cool, add 2 Gm. of potassium iodide and 20 cc. of sulphuric acid (1 in 4). When the precipitate has dissolved titrate the liberated iodine with tenth-normal sodium thiosulphate, using starch T.S. as the in dicator. One cc. of tenth-normal sodium thiosulphate corresponds to 0.023799 Gm. of C0CI2.6H2O. Adjust the volume of the cobaltous chloride solution so that 1 cc. contains 0.059496 Gm. of CoCl 2 .6H 2 0. Preserve the solution in a bottle with a well-fitting glass stopper. Colorimetric Ferric T.S.—Containing 45.053 Gm. of ferric chloride (FeCl 3.6H 20) in 1000 cc. of solution. Dissolve about 55 Gm. of ferric chloride in enough of a fluid made by mixing 25 cc. of hydrochloric acid with 975 cc. of distilled water to make a volume of 1000 cc. Assay the solution as directed under Liquor Ferri Chloridi, U.S.P. X , using 10 cc. of the solution, accurately measured. Adjust the volume of the solution so that 1 cc. contains 0.045053 Gm. of FeCl 3 .6H 2 0. Preserve the solution in a bottle of amber glass having a well-fitting glass stopper. Dissolve 2 Gm. of Cod Liver Oil, accurately weighed, in 30 cc. of a mixture of equal volumes of alcohol and ether, the mixture having been previously neutralized with tenth-normal sodium hydroxide, using 5 drops of phenol- phthalein T.S. as- the indicator, and boil the oil solution gently under a reflux condenser for ten minutes. Cool and titrate the mixture with tenth normal sodium hydroxide to the production of a i pink color which persists after shaking for thirty seconds. Not more than 1 cc. of tenth-normal sodium hydroxide is required (free acid). ' Weigh 5 Gm. of Cod Liver Oil into a 250 cc. flask, add a solution of 2 Gm. of potassium hydroxide in 40 cc. of alcohol and heat under a reflux condenser for two hours, keeping the alcohol gently boiling. Evaporate the alcohol on a water bath, dissolve the residue in 50 cc. of hot distilled water, and transfer the solution to a separatory funnel, rinsing the flask with' two 25-cc. portions of distilled water which are added to the solution in the separator. Cool the mixture to from 15° to 20° C , and extract with two successive portions of 50 cc. each of ether, adding a few drops of alcohol. Combine the ether extracts in another separator, and wash the ether solution, first with 20 cc. of tenth normal potassium hydroxide, then with 20 cc. of fifth-normal potassium hydroxide, and finally with successive 15-cc. portions of distilled water until the washings are not reddened by the addition of 2 drops of phenolphthalein T.S. Transfer the ether solution to a tared beaker, rinse the separator with 10 cc. of ether, and add the rinsings to the beaker. Evaporate the ether just to dryness on a water bath and dry the residue for thirty minutes at 100° G. *Cool the beaker in the desiccator for thirty minutes and weigh. The residue shall not exceed 1.30 per cent of the weight of Oil taken for the assay (unsaponifiable matter). Fill a tall, cylindrical, standard oil-sample bottle of about 120 cc. capacity with Cod Liver Oil, at a temperature between 23° and 28° C , stopper, and im merse the bottle in a mixture of ice and distilled water for five hours. The oil remains fluid and does not deposit stearin (undestearinated cod liver oil). Saponification value: not less than 180 and not more than 192. When carbon dioxide has been used as a preservative, the oil must be exposed in a shallow dish in a vacuum desiccator for twenty-four hours before weighing . the sample for determination of the saponification value. Iodine value: not less than 145 and not more than 180. P r e s e r v e in a cool place, in well-closed containers which have been thoroughly dried before filling. Cod Liver Oil may be bottled or packaged in a vacuum or in the presence of an inert gas. Assay—Proceed as directed under Assays for Vitamins A, and D in Cod Liver Oil, ' pages 4 to 11. Preparation—Emulsum Olei Morrhua?. A V E R A G E DOSE—(Administer three times daily)—Infants: Metric, 4 cc.—Apothecaries, 1 fluidrachm. Adults: Metric, 8 cc.— Apothecaries, 2 fluidrachms. Assays for Vitamins A and D in Cod Liver Oil (To replace the Vitamin Assay on page 489 in the U.S.P. X) Definitions—As used herein, unless the | context otherwise indicates, the term assayer means the individual immediately responsible for the interpretation of the assay; the term assay group means a group of rats to'which the assay oil shall be administered during the assay period; the term assay oil means a God Liver Oil under examination for its vitamin potency; the term assay period for the Vitamin A assay means the interval in the life of a rat between the last day of the depletion period and the twenty-ninth day thereafter or between the last day of the depletion period and the death of the rat; the term-assay period for the yitamin D assay means the interval in the life of a rat between the, last day of the depletion period and the eleventh day thereafter; the term assemble means the procedure by which rats are selected and assigned to groups for the purpose of feeding, care, and observation; the 4 term control group means a group of rats receiving no Cod Liver Oil during the assay period; the term daily, for the Vitamin A assay, means six days of each week of the assay period; the term daily, for the Vitamin D assay, means each of the first eight days of the assay period; the term declining weight means a condition of a rat when the, body weight of the rat on any given day is equal to or less than the body weight of the rat on the seventh day prior to the given day; the term depletion period means the interval in the life of a rat between the last day of the preliminary period and the first day of the assay period; the term dose means the quantity of the Refer ence Oil or of the assay oil to be fed daily to a rat during the assay period; the term fed means made readily available to the rat or administered to the rat by mouth; the term ground gluten means the clean, sound product made from wheat flour by the almost complete removal of starch, and contains not more than 10 per cent of mois ture, and, calculated on the water-free basis, not less than 14.2 per cent of nitrogen, not less than 15 per cent of nitrogen-free extract (using the protein factor 5.7), and not more than 5.5 per cent of starch (as determined by the diastase method); the term group for the Vitamin A assays means six or more rats maintained on the same required dietary regimen during the assay period; the term group for (he Vitamin D . assay means seven or more rats maintained on the same required dietary regimen during the assay period; the term ophthalmia means a pathological state of the eye and/or the conjuctiva and/or the tissues anatomically related to the eye, readily discernible macroscopically and usually associated with Vitamin A deficiency; the term preliminary period means the interval in the life of a rat between the seventh day after birth and the first day of the depletion period; the term rachitogenie diet means a uniform mixture of the food materials, and in the proportions named, in either of the following formulas: Rachitogenic Diet No. 1 Whole Yellow Make, ground. 33 per cent Whole Wheats ground. 33 per cent Ground Gluten : ". 15 per cent Gelatin 15 per cent Calcium Carbonate (CaCO s) . . 3 per cent Sodium Chloride (NaCl) 1 per cent Rachitogenic Diet No. 2 Whole Yellow Maize, ground. 76 per cent Ground Gluten 20 per cent Calcium Carbonate (CaC0 3 ) 3 per cent Sodium Chloride (NaCl) 1 per cent The term Vitamin A test diet means a food material consisting of the following proportions of the named ingredients of the quality specified: Vitamin A Test Diet casein.,- ••••••• / 18 per cent Salt Mixture (see page 6) : 4 per cent Yeast, dried , 8 per cent S ta rch . . . . . ..65 per cent Vegetable Oil 5 per cent yitamin D, a sufficient amount Not less than 3. U.S.P. Units of Vitamin D shall be provided in each gram of diet and this vitamin shall be carried by the yeast or the vegetable oil. The ingredients of the Vitamin A test diet shall be free from Vitamin A or shall have been treated so as to reduce the Vitamin A content to such a degree that when the Vitamin A test diet is fed to the control group two-thirds or more of the rats shall manifest, prior 5 to the eleventh day of the assay period, symptoms of Vitamin A deficiency charac terized by both declining weight and opthalmia. The dried yeast shall carry the Vitamin B complex in such concentration that a daily dose of 0.15 Gm. shall permit an average gain in weight of at least 3 Gm. per week in rats during an interval of four weeks between the twenty-fifth and sixtieth days of age, when the rats are provided ad libitum with a ration which is adequate for optimal growth, except that the ration shall be devoid of the Vitamin B complex. Salt Mixture No. 1 Calcium Carbonate (CaC0 3 ) 134.8 Gm. . Magnesium Carbonate (Mg.COs) 24.2 Gm. Sodium Carbonate (Na 2 C0 3 ) 34.2 Gm, Potassium Carbonate ( K 2 C O s ) . . 141.3 Gm. . Phosphoric Acid (100 per cent) 119.3 Gm. Hydrochloric Acid (100 per cent) . . . .166.9 Gm. Sulphuric Acid (100 per cent) 9.8 Gm. Citric;Acid (1H 2 0) . . 111.1 Gm. Ferric Citrate (1V 2 H 2 0) 5.7 Gm. Potassium Iodide ( K I ) . . . . . . 0.020 Gm. Manganese Sulphate (MnS0 4 ) 0.079 Gm. Sodium Fluoride (NaF) 0.248 Gm. Potash Alum [ K J A I ^ S C M ) * ] 0.0245 Gm. The available form of each chernical substance is taken in sufficient quantity to furnish the stipulated equivalent quantity of each chemical. The mixed carbonates and ferric citrate are added to the mixed acids. The specified quantities of K I , . MnS04, N a F , and K2A1 2(S04)4 are added as.solutions of known concentrations and the resulting mixture is evaporated to dryness in a current of air at from 90 ° to 100 ° C. and ground to a fine powder. Salt Mixture No. 2 Sodium Chloride (NaCl) 0.173 Gm. Anhydrous Magnesium Sulpha te . (MgS0 4 ) . . . . . . . . 0.266 Gm. Sodium Phosphate (NaH 2 P0 4 .H 2 0) 0.347 Gm. Potassium Phosphate ( K 2 H P 0 4 ) 0.954 Gm. Calcium Acid Phosphate [CaH 4 (P0 4 ) 2 .H 2 0] 0.540 Gm. Ferric Citrate ( i y 2 H 2 0 ) 0.118 Gm. Calcium Lactate 1.300 Gm. Uniformly mix the finely powdered salts. The assay of Cod Liver Oil for Vitamin A and Vitamin D potency shall be by comparison with the U.S.P. "Reference Cod Liver O i l , " by assay procedures con forming in all respects to the following specifications: Method of Assay for Vitamin A The Vitamin A assay, comprising the recording of observations of groups of,rats throughout specified periods of their lives, while being maintained on specified dietary regimens, and the interpretation of such data, is as follows: Preliminary period—Throughout the preliminary period each rat shall be raised under the immediate supervision of or according to directions specified by the assayer. Throughout the preliminary period the rats shall be maintained on a dietary re'gimen which shall provide for normal development in all respects, except that the supply of Vitamin A , or precursors of Vitamin A , shall be limited to' such a degree that rats weighing between 40 and 50 Gm. and not: exceeding twenty-eight days of age and • 6 subsisting on a suitable V i t a m i n A deficient rat ion and water for an interval not ex ceeding forty-f ive days shall manifest symptoms characteristic of V i t a m i n A de ficiency. Depletion period—A rat shall be suitable for the depletion period when the age of the rat does not exceed twenty-eight days, and if the body weight of the rat shal l exceed 39 Gm. , and does not exceed 50 Gm. , and if the animal manifests no evidence of injury, or disease, or anatomical abnormality which might hinder growth and de velopment. Throughout the depletion period each rat shal l be provided w i th the V i t a m i n A test diet and disti l led water ad l ib i tum, and during this period no other dietary supplement shall be available to the animal. Assembling rats into groups for the assay period-—Rats which are suitable for the .. assay period shall be assembled into groups. F o r each assay o i l there shall be one or more assay groups. In the assay of one assay o i l there shall be provided at least one control group and at least one reference group, bu t one control group and one reference group may be used for the concurrent assay of more than one assay oi l . The interval of assembling rats into groups shall not exceed sixty days. On any one day during the interval of assembling rats into groups, the tota l number of rats that shall have been assigned to make up any one group shall not exceed by more than two the number of rats that shal l have been assigned to make up any other group. W h e n the assembling of a l l groups shal l have been completed the total number of rats in each group shall be the same, and the number of rats of one sex in each group shall be the same. N o t more than three rats from one l i t ter shall be assigned to one group. W h e n the assembling of a l l groups shal l have been completed, the average weight of the rats in any one group on the day beginning the assay period shall not exceed by more than 10 G m . the average weight of the rats in any other group on the day be ginning the assay period. Assay period—A rat shall be suitable for the assay period, provided that the de pletion period shall have exceeded twenty-four days and shal l not have exceeded forty-f ive days, and provided that a ra t shal l manifest evidence of V i t a m i n A de ficiency characterized %y declining weight and /o r ophthalmia. Throughout the assay period each rat of the control, reference, and assay groups shall be kept i n an ind iv idual cage and shal l be provided w i t h the V i t a m i n A test diet and dist i l led water, ad l ib i tum. Throughout the assay period each rat in any assay group shall be fed dai ly a dose of the assay oi l , and throughout the assay period each rat in any one reference group shal l be fed dai ly a dose of the reference o i l . T h e reference o i l a n d / o r the assay o i l may be di luted before feeding w i th an edible vegetable o i l free f rom V i t a m i n A . D i lu ted o i l shall be stored i n the dark at a temperature not ex ceeding 50° F . T h e period of storage shal l not exceed seven days. N o t more than 0.1 cc. of the di luted o i l shall be fed as a dai ly dose. Dur ing the assay period a l l conditions of environment shall be maintained as uni formly as possible w i th respect to the assay, reference, and control groups. Recording of data—-On the day beginning the depletion period and at intervals of not more than seven days for the first twenty-one days of that period there shall be a record made of the body weight of each rat. F r o m the twenty-f i rst day of de pletion period unt i l the end of the assay period a record shall be made of the body weight and eye condition of each rat at intervals not exceeding five days. T h e eye condition shall be. designated as normal , watery, sensitive to l ight, swollen, bloody exudate, purulent, opacity of cornea, or any combination of these terms.. A record shall be made of the failure of a rat to consume the prescribed dai ly dose of reference or assay o i l . Vitamin A potency of the assay oilr—-In determining the V i t a m i n A potency of the assay oi l the performance of the rats of the assay and reference groups shall be ca lcu  lated for each group on the basis of the difference between the average weight of the 7 surv iv ing rats arid the average weight of the same rats on the day beginning the assay ..period. T h e data f rom the reference group shal l be considered v a l i d for establishing the V i t a m i n A potency of the assay o i l only when two-th i rds or more of the tota l number of animals compris ing a reference group shal l have made ind iv idua l l y be tween the beginning day of the assay per iod and the twenty-eighth day thereafter an increase i n body weight wh ich shal l equal or exceed 12 'Gm. and shall not exceed 60 G m . , and the data f rom an assay or reference group shal l be considered v a l i d for establishing the V i t a m i n A potency of the assay o i l only when two-th i rds or'more, but not less than six, of the rats of an assay or reference group have surv ived twenty - eight days of the assay period. T h e data f rom an assay group shal l be considered va l i d for establishing that an assay o i l conforms w i t h the U.S. Pharmacopoeia standard for V i t a m i n A in cod l i ver o i l only when two-th i rds or more but riot less t h a n six rats shal l have made ind iv idua l l y between the beginning day of the assay period and the twenty -e ighth day thereafter an increase i n body weight wh ich shal l equal or exceed 12 grams. T h e data f rom a rat shal l be considered va l i d for establish ing the average performance of a reference or assay group only on the condition- that the rat has consumed the prescribed dose of o i l for at least twenty - two days of the assay per iod. A V i t a m i n A assay shal l not be considered va l id unless two-th i rds or more of the tota l number of animals comprising the control group shal l , pr ior to the eleventh day of the assay period, manifest symptoms of V i t a m i n A deficiency charac terized by both dec l in ing weight a n d ophthalmia. T h e V i t a m i n A potency of the assay o i l is then calculated according to the fol lowing procedure: L e t " R " equal the da i ly dose in -mi l l igrams of the reference oi l necessary to pro  duce i n a reference group an average gain i n weight, " G , " of not less than 12 G m . and not more than 60 G m . L e t " A " equal the dai ly dose i n mil l igrams of the assay o i l that w i l l produce in an assay group an average gain in weight equal to or greater than " G . " If the product of — X [units per gram of V i t a m i n A contained i n the refer- ence oi l ] is equal to or greater than 600, then the assay o i l contains 600 or more units of V i t a m i n A. per gram of o i l and complies w i t h the U.S. Pharmacopoeial requirement for V i t a m i n A potency. ; Method of Assay for Vitamin D T h e V i t a m i n D assay, compris ing the recording of observations of groups of rats, throughout specified periods of their l ives, whi le being maintained on specified dietary regimens, and the interpretat ion of such data, is as fol lows: Preliminary period—Throughout the prel iminary per iod each rat shal l be raised under the immediate supervision of or according to directions specified b y the as- sayer. Throughout the pre l iminary period the rats shal l be mainta ined on a dietary regimen wh ich shal l provide for normal development i n a l l respects, except that the supply of V i t a m i n D shal l be l im i ted to such.a degree tha t rats, weighing between 40 and 60 G m . at an age of twenty-one to twenty-eight days, and subsisting for ari inter  v a l of three weeks on a suitable rachitogenic diet, shal l manifest evidence of severe r ickets. Depletion period—-A rat shal l be suitable for the depletion per iod when the age of the rat does not exceed th i r t y days, and i f the body weight of the ra t sha l l exceed 44 G m . , and does not exceed 60 G m . , and if the an imal manifests no evidence of in jury , or disease, or anatomical abnormal i ty wh ich might hinder growth and de velopment. Throughout the depletion period each rat shall be prov ided w i t h t i e .8' rachitogenic diet and disti l led water ad l ib i tum, and during this period no other dietary supplement shal l be available to the animal. Assembling rats into groups for (he assay period—Rats wh ich are suitable for the assay period shall be assembled into groups. F o r each assay o i l there shal l be one or more assay groups. I n the assay of one assay o i l there shall be provided at least one reference group, but one reference group may be used for the concurrent assay of more thdn one assay o i l . T h e interval of assembling rats into groups shal l not exceed sixty days. On any one day during the interval of assembling rats into groups, the to ta l number of rats that shal l have been assigned to make up any one group shal l no t exceed b y more than two the number of rats tha t shall have been, as signed to make up any other group. W h e n the assembling of a l l groups shal l have been completed the total number of rats i n each group shall be the same, and the number of rats of: one sex in each group shall be the same. N o t more than three rats from one l i t ter shall be assigned to one group. W h e n the assembling of a l l groups shal l have been completed the average weight of the rats in any one group on the day beginning the assay period shal l not exceed b y more than 8 G m . the average weight of the rats i n any other group on the day beginning the assay period. Assay period—A rat shal l be suitable for the assay period, provided that the de pletion period shall have exceeded eighteen days and shal l no t have exceeded twenty - five days, and provided that a rat shall manifest evidence of rickets characterized by a distinctive, wabbly, rachit ic gait and b y enlarged joints. T h e presence of rickets may also be established b y examination of a leg bone of one member of a l i t ter by the " l ine test" described below. E a c h rat shall be kept in an ind iv idua l cage and shall be provided w i t h the rachitogenic diet and dist i l led water, ad l i b i tum. O n any calendar day of the assay period the assay and reference groups shal l receive a rachitogenic diet compounded f rom the same lots of ingredients. Throughout the first eight days of the assay period each rat i n any one assay group shal l be fed dai ly a dose of the assay oi l , and throughout the first eight days of the assay period each rat in any one reference group shall be fed dai ly a dose of the reference o i l , except that the fol lowing deviation f rom the dai ly feeding shal l be permissible: that the dai ly dose may be doubled on the day preceding a one-day hol iday fal l ing wi th in the first eight days of the assay period. D u r i n g the remainder of the assay period (i. e., the n in th day and tenth day) neither the assay o i l nor the reference oi l shal l be fed. On the eleventh day of the assay per iod each rat shal l be k i l l ed and one or more leg bones examined for healing of the rachit ic metaphysis according to the " l ine test" described below. The reference oi l a n d / o r the assay o i l may be d i luted before feeding w i t h an edible vegetable o i l free f rom V i tamins A and D. T h e d i luted o i l shal l be stored i n the dark at a temperature not exceeding 50° F . and the duration of th is storage shal l not exceed th i r ty days. N o t more than 0.1 cc. of the d i luted o i l shal l be fed as a dai ly dose. Du r ing the assay period a l l condi  tions of environment (particularly w i t h reference to physiologically active radiations) shall be maintained as uni formly as possible w i th respect to the assay and reference groups! Line test—The line test shal l be made on the proximal end of a t ib ia or distal end of a radius or ulna. The end of the desired bone is removed from the animal and cleaned of adhering tissue. A longitudinal median section shal l be made through the end of the bone w i t h a clean, sharp blade to expose a plane surface through the junct ion of the epiphysis and diaphysis. I n any one assay the same bone of a l l the animals must be used and sectioned through the same plane. B o t h sections of the bone shall be rinsed in disti l led water and shall then immediately be immersed i n a 2 per cent aqueous solution of si lver nitrate for one minute. The sections shal l then be rinsed in disti l led water and the sectioned surfaces of the bone shall be exposed 9 i n water to dayl ight or other source of act inic l ight un t i l the calcined areas have de veloped a clearly defined stain wi thout marked discoloration of the uncalcif ied areas. • Records' shal l be made immediately of the extent and degree of calcif ication of the rachit ic metaphysis of every section. I t shal l be permissible to use modifications of the described procedure for staining, provided tha t such modif ied procedures clear ly differentiate between calcified and uncalcif ied areas. Recording of data—On the day beginning the assay per iod a n d on the tenth day thereafter a record shal l be made of the body weight of each rat. A record.shall be made of the quant i ty of rachitogenic diet consumed per ra t per diem during the assay period. N u m e r i c a l values shal l be assigned to the extent and degree of calcif ication of the rachi t ic metaphysis of the bones examined b y the l ine test so that i t w i l l be possible to average the performance of each group. Vitamin D potency of the assay oil—In determining the V i tamin L> potency of the assay o i l the average performance of groups w i t h respect to heal ing of the rachit ic metaphysis shal l be considered, prov ided that the average performance of a reference group w i t h respect to calcif ication of the rachi t ic metaphysis shal l be determined b y the data f rom rats wh ich ind iv idua l ly show an extent and degree of calcif ication i n the rachit ic metaphysis as determined b y the l ine test equal to or greater than a condi  t ion described as a posit ive macroscopic evidence of calci f ication, but less :than an extent and degree of calci f ication described as complete healing. T h e data f rom a reference group shal l be considered Val id for establishing the V i t a m i n D potency of the assay o i l on ly when two-th i rds or more, bu t not less than seven rats, show i n  d iv idua l l y an extent and degree of calcif ication of the rachi t ic metaphysis equal to or greater than a condit ion describ8d=as posit ive macroscopic evidence of calcif ication, but less than an extent and degree of calcif ication described as complete healing. T h e data f rom an assay group shal l be considered v a l i d for establishing that an assay o i l conforms w i th the U.S . Pharmacopoeia standard for V i t a m i n D i n cod l iver o i l on ly when two- th i rds or more, bu t not less than seven rats, show ind iv idua l l y an extent a n d degree of calcif ication of the rachi t ic metaphysis equal to or greater than a con  d i t ion described as posit ive macroscopic evidence of calcif ication. T h e data f rom a rat shal l be considered v a l i d for establishing the average performance of a group on ly on the condit ion that the weight of the rat on the eleventh day of the assay period shal l equal or exceed the weight of the rat on the beginning day of the assay period; and tha t the ra t has consumed 2 or more G m . of the rachitogenic diet on each day of the assay per iod a n d 40 G m . or more of the rachitogenic diet dur ing the assay per iod a n d on the condit ion that the ra t has consumed each prescribed dose of cod l i ver o i l w i t h i n twenty- four hours f rom the t ime it. was fed. T h e V i t a m i n D potency of the assay o i l i s then calculated according to the fol lowing procedure. L e t " R " equal the dai ly dose i n mi l l igrams of the reference o i l necessary to pro  duce i n a reference group an average extent and degree of calcif ication " C " not less than a condi t ion described as a narrow continuous l ine of calcif ication but less than an extent a n d degree of calci f ication described as complete healing. L e t "A" equal the da i ly dose i n mi l l ig rams of the assay o i l that w i l l produce i n a n assay group a n average extent and degree of calcif ication equal to or greater than " C . " I f the product of J^^  J * [units per gram of V i t a m i n D , contained i n the refer ence oil] is equal to or greater than 85, then the assay o i l contains in . each gram 85 or more units of V i t a m i n D and complies w i th the U.S. Pharmacopoeial require ment for V i t a m i n D potency. 10 

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