UC-NRLF B 3 1E7 TD7 EXCHANGE 1.9 '974 A Critical Investigation and an Application of the Rat Growth Method for the Study of Vitamin B. BY ADELAIDE SPOHN Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University. f V) (O 36> 38. The method of testing for vitamin B by measuring the acceleration of yeast growth is open to the question whether the culture medium is adequate in all other respects and whether it is safe to assume that increased yeast is attributable entirely to the vitamin 23-35. The nutrition re- quirements and rate of growth of 'the rat have been exten- sively studied by Hopkins *» 2j 36, Osborne and Mendel 3~7, McCollum 8-11 and others. The rat method is therefore the best standardized by previous work. Dr. Harriet Edge- worth 37, working in this laboratory, after a careful analysis of the yeast and rat growth methods concluded : "The rat growth method involves somewhat larger probable errors than the yeast method, but can be interpreted in terms of B vitamin with much greater certainty and is therefore the preferable method." Skimmed milk in the form of dry powder was chosen as the source of vitamin B, since it furnishes the vita- min in a typical natural state, readily available and uniform. It is easily and accurately manipulated in either large or small quantities and is convenient for study in either the fluid or dry state. EXPERIMENTAL PROCEDURE Selection and Care of Animals. The rats used were twenty- eight or twenty-nine days old, this being the time of weaning adopted in this laboratory, and weighed from thirty to sixty- five grams when placed on the experimental diet. The experi- mental animals all came from mothers on a diet of two-thirds whole wheat, one-third whole milk and sodium chloride, two per cent, of the weight of the wheat (diet 13) or this diet with an addition of ten grams of raw lean beef per adult rat per day (diet 13M). In each experiment, however, care was taken, that equal numbers of comparison and control rats came from mothers on the same diet. The lots were made up in the following manner. If, for example, in one experiment there were five variations of the diet and ten rats -for each variation, then fifty rats were required. Six to eight litters of from five to ten animals each would be needed to supply this number of rats. Each litter was distributed over the five vari- ations as far as it would go. For example, a litter of nine rats would furnish two animals each for four variations and one for the fifth. Then a litter of six rats would provide for one rat where there were two before, and two rats where there was one before, making a total of three rats for each variation of the diet. The remaining thirty-five rats from the various lit- ters were portioned out so that there were ten animals in each lot, and in such a way that the total weight of the rats and the number of males and females on each modification was prac- tically the same. The experiments were continued for eight weeks, from the begining of the fifth to the end of the twelfth week of the rat's life, this being the most rapid growth period. A number of experiments were continued for four weeks longer. These last four weeks, however, did not show any significant differ- ences which were not brought out in the growth curves from the fourth through the twelfth week. When the milk, which was the source of vitamin B, was fed separately from the rest of the diet, the animals were kept in round galvanized iron wire cages, eight inches high and nine inches in diameter, one rat in each cage. In addition to the milk these rats received a vitamin B-free basal ration, of which they ate ad libitum. In the experiments in which the rats received the milk mixed with the rest of the diet, which was always before them, six to eight animals were housed in square iron wire cages 11" x 14" x 10". Fresh water was always available. The cages were cleaned as often as seemed necessary, the large cages usually every day and the small round cages three times a week. Each animal was weighed once a week and the food remaining weighed and subtracted from the amount fed, the difference being the amount con- sumed by the rat or rats during that week. Basal Ration and its Preparation. The basal ration used was planned to contain all the essential food factors in optimum amounts, with the exception of vitamin B which was lacking. It had the following composition : Diet 94 per cent. Casein - 18 Butter fat 10 Starch 68 Salt mixture (Osborne and Mendel)40 - 4 Later, merely as a factor of safety, cod liver oil was substi- tuted for one-fifth of 'the butter fat, making diet 107. The in- gredients of the diet were weighed out and intimately mixed first by hand and then transferred to a mechanical mixer and stirred for five minutes. The casein was prepared from the commercial product by purification in the manner suggested by Sherman and Husa (unpublished experiments) as follows : One liter of 60 per cent, alcohol (by weight) was added to 200 grams of casein and the mixture stirred for one-half hour by means of a mechanical stirrer and was then allowed to stand for five and one-half hours. After this period the casein was filtered off through a Buchner funnel and washed once in the funnel with 500 cc. of 60 per cent, alcohol. The casein was again treated with one liter of 60 per cent, alcohol as before but this time was left to stand for eighteen hours. It was filtered off and washed as before with 500 cc. of 60 per cent, alcohol and finally with 500 cc. of 90 per cent, alcohol. The last washing merely facilitates the drying. The casein was removed from the filter and spread out in a thin layer and allowed to become air-dry. Throughout this paper the strength of alcohol used is always given in per cent, by weight. The butter was melted at a temperature not exceeding 45° C. All the curd, salt and water settles to the bottom of the vessel and may be easily removed when the butter solidi- fies. The butter fat is again melted at a low temperature, and filtered through filter paper using a hot water funnel. The salt mixture described by Osborne and Mendel 40 was used. Commercially pure corn starch, which according 8 to these authors contains no appreciable quantities of the water soluble vitamin furnished the carbohydrate of the diet39. Source of Vitamin B, Treatment and Manner in Which it Was Fed. Dried skimmed milk was used as a convenient source of vitamin B. It permits the study of the effect of heat on the vitamin both in the wet and dry state. By using skimmed milk powder from the same lot throughout one experiment, possible variation in vitamin content is eliminated. The dry milk was heated at 100° C. for six, twenty-four and forty-eight hours in a constant temperature oven. It was spread out in thin layers about one-half inch in thickness and the temperature taken by means of a thermometer extending into the dry milk. Two hours were allowed, which was the time found to be necessary for the milk to come to 100° C. The portions heated six, twenty-four and forty-eight hours respectively actually remained in the oven eight, twenty-six and fifty hours. The reconstructed milk was prepared by weighing out 100 grams of the air-dry powder and making it up to 1,000 cc. with* water. For the heat treatment 250 cc. of this was poured into an Erlenmeyer flask and tightly stoppered with cotton. This was heated in a vigorously boiling water bath for six hours. A thermometer extending into the milk actually re- corded a temperature of 99.5° C. to 99.8° C. At the end of six hours the flask was removed from the bath and rapidly cooled under the tap. Since the volume diminished, due to evaporation through the stopper, the milk was again made up to volume before using. The milk was fed in doses of 8 cc. per rat per day for seven days a week, this, as will later, be shown, being the amount which would best reveal any variation in vitamin con- tent. The milk was measured into a small cup by means of a graduated pipette and placed into the cage each day. If the rat did not drink the milk readily, the water cup was removed for the day and replaced in the evening. Usually this treat- ment brought the desired results. After two to three days all the rats took all the- milk that was given them. The milk heated dry was fed in two ways: (1) mixed with the rest of the diet, replacing 25 per cent, of the starch in diet 94. This ration was always available and the rats ate ad libitum. (2) In another series of experiments the milk, which was heated dry, was reconstructed before feeding by making 25 grams up to a volume of 250 cc. with water. This was fed in doses of 8 cc. per rat per day. The advantage here is that all the rats re- ceived exactly the same amount of vitamin per day. Positive controls, animals receiving in the one case the diet containing 25 per cent, of unheated milk and in the other case 8 cc. per day of the reconstructed unheated milk, were run in every case in comparison with the rats receiving the heated food. Nega- tive controls, receiving only the vitamin-free diet were run in almost all but not every experiment. This was done in order to make sure that the basal diet did not vary. THE ADEQUACY OF THE BASAL DIET In order to prove that the basal ration contained the optimum amounts of the various constituents, it was fed to a series of rats in comparison with diets in which each ingredient of the basal ration, the casein, butter fat and salt mixture, were in turn used in larger amounts. It was thought that perhaps another protein such as ex- tracted meat might prove to be a more adequate protein, or the meat might be more palatable and that therefore the rats would eat more of the basal ration. Consequently diet 94 was also compared with a diet in which extracted meat residue replaced the casein. Commercial extracted meat residue was purified according to the method described for casein (page 7). TABLE I. The following diets were used in the comparison : Diet 94 102 103 105 104 % % % % % Casein 18 18 -23 18 18* Butter fat 10 15 10 10 10 Starch 68 63 63 67 68 vSalt mixture (O. &M.)40.... 44454 * Meat residue. 10 The average growth curves on these various diets were practically identical and the average duration of life varied by only four days. Table II briefly summarizes the results of this series of experiments. TABLE II. Summary of average results on diets 94, 102, 103, 104, 105. Number Average Average Average of initial weight age at Diet rats weight at death death 94 12 47 gms. 34 gms. 61 days 102 8 46 " 31 " 65 " 103 8 47 " 34 " 64 " 105 8 47 " 34 " 63 " 104 8 46 " 33 " 62 " It was thought that the results might be different if vita- min B were added to the diets, in other words if this vitamin was not the limiting factor in the diet. At about this time in the course of the work, it was also thought wise, merely as a factor of safety, to replace 2 per cent, of the butter fat in the diet by cod liver oil. Each rat in this comparison received either diet 107, 108, 109, or 110 with an addition of 7 cc. or 8 cc. of reconstructed skimmed milk. These doses were used, since from experiments reported later in the paper it became apparent that one or the other of these amounts would prob- ably give the best results in the study of the destruction of water-soluble B by heat. 8 cc. was later decided upon as the best dose but the results for both are here given, both being valuable in this connection. TABLE III. The following diets were used in the second comparison: Diet 107 108 109 110 % % % % Casein 18 18 23 18 Butter fat 8 13 8 8 Cod liver oil 2 2 2 2 Starch 68 63 63 67 Salt mixture (O. & M.)40 4445 11 Table IV gives a summary of the results of the compari- son of diet 107 with diet 108 containing 5 per cent, more but- ter fat, diet 109 containing 5 per cent, more casein and diet 110 with an excess of 1 per cent, salt mixture over that in diet 107. TABLE IV. i -0 •S.e-8 1313 (rt .Is i^-s 2^,* •£ aj "^ 'S ^ *^ _c Q « § 6 2> 42 and the possible seasonal variation in the vitamin content of the milk 1» 2> 41> 42> 43> 44 are eliminated here, since the experiments (Tables V and VI) were carried on at the same time in the early spring, late winter milk from the same lot being used in both series. The four rats receiving 7 cc. of reconstructed milk which had been heated, died at the ages of 50, 60, 66 and 66 days respectively, while the rats getting the same amount of unheated milk lived till the end of the twelfth week when they were killed. This is additional evidence of the destruc- tion of vitamin B by this heat treatment. Dry heat applied for 6, 24 and 48 hours seems to have little or no deleterious effect, as the weight curves of the control rats receiving unheated milk and of those receiving the heated milk are approximately the same. Experiments with Milk Mixed with the Rest of the Diet. Preliminary experiments reported elsewhere in this paper in- dicate that a change in the vitamin B content of skimmed milk powder may be most easily observed if the milk is mixed with the rest of the diet in the proportion equal to 25 per cent, of the entire mixture. Therefore the unheated milk of Diet 100 was replaced by the milk heated dry for various periods, 6, 24 and 48 hours. The animals from .mothers on Diet 13 M and Diet 13 placed on these diets have been summarized separate- ly in Tables XV and XVI and the combined results are given in Table XVII and Chart II, Figure II. It will be noted here again, as was observed from the results of the experiments in which different amounts of milk replaced the starch in Diet 94 (Tables IX and X) that the rate of growth of the animals from Diet 13 M is greater than that of the animals from Diet 13. The total food consumed by the animals on Diet 100 and the diets in which the unheated milk of this ration was re- placed by the milk heated 6, 24 and 48 hours was practically the same in each case. The rats therefore received essentially the same number of calories and equal amounts of protein, of the mineral elements and of the vitamin containing food on the four different diets. 20 TABLE XV. . Rats from mothers on Diet 13. No. of rats Diet Milk Milk % Treatment 4 Age in weeks 567 8 9 10 11 Average weight in grams Total 12 gain for 8 weeks 6 94 0 43 49 50 47 41 33* 8 100 25 Unheated 39 58 79 90 93 97 99 99 98 59 8 100 25 Heated dry 6 hrs. 39 53 75 87 93 101 103 102 106 67 8 100 25 Heated dry 24 hrs. 39 52 64 81 90 100 105 112 118 79 8 100 25 Heated dry 48 hrs. 39 53 69 83 87 97 100 104 108 69 * 33 gms.=average weight at death, the average age at death being 60 days. TABLE XVI. Rats from mothers on Diet 13 M. No. of rats Diet Milk % Milk Treatment 4 Age in weeks 5 6 78 9 10 11 Average weight in grams 12 J Total gain for 3 weeks 7 94 0 52 57 59 54 49 42 35* 7 100 25 Unheated 52 71 92 109 129 142 149 164 166 114 7 100 25 Heated dry 6 hrs. 53 73 92 111 122 135 147 162 166 113 7 100 25 Heated dry 24 hrs. 53 70 88 104 116 125 135 141 149 95 7 100 25 Heated dry 48 hrs. 54 67 83 96 111 132 133 145 151 97 * 35 gms.=raverage weight at death, the average age being 67 days. TABLE XVII. (Results of Tables XV and XVI combined.) No. of rats Diet Milk % Milk Treatment 4 s Age in weeks 67 8 9 10 11 Average weight in grams Total 12 gain for 8 weeks 13 94 0 48 53 55 51 45 35* 15 100 25 Unheated 45 64 85 99 110 118 123 129 130 84 15 100 25 Heated dry 6 hrs. 45 62 83 98 106 117 124 130 134 88 15 100 25 Heated dry 24 hrs. 46 60 77 92 102 112 119 125 132 87 15 100 25 Heated dry 48 hrs. 46 59 76 89 98 113 115 125 128 82 * 35 gms.=average weight at death, 64 days being the average age. The conclusions to be drawn from these regarding the heat destruction of vitamin B in the dry state, confirm those of the previous experiment in which the heated milk was fed separately. The average gain in weight for eight weeks on a control diet containing unheated milk and on the diets con- taining the milk heated for 6, 24 and 48 hours is the same. There is evidently no destruction of the vitamin by this treat- ment or the amount of destruction is too small to be detected by either of the methods employed in the present work. 21 SUMMARY A quantitative method for the determination of relative amounts of vitamin B is described. The work comprises eleven comparative studies with a total of thirty-eight varia- tions of the basal diet and includes over six hundred quanti- tative studies of the growth of young rats. An adequate basal ration is described which contains optimum amounts of all the food factors necessary for the growth of young rats with the exception of vitamin B. The most advantageous quantities of milk to feed in order to detect possible differences in the vitamin B content were 8 cc. of skimmed milk or 0.8 gram of skimmed milk powder per rat per day when the milk was fed separately from the rest of the diet ; or 25 per cent, of the total food- mixture when the skimmed milk powder was mixed with the basal ration. Feeding the basal ration under the conditions described to experimental animals of suitable age and size and suffi- ciently known dietary history, it is believed to be possible to detect a variation certainly of 25 per cent, and probably of 15 per cent, in the vitamin B content of the food tested. There was no evidence of destruction of vitamin B in milk powder heated dry at 100° C. even when this heating was con- tinued for forty-eight hours. When the milk was heated in the fluid state for six hours at 100° C. there was appreciable destruction of vitamin B. Apparently about one-fourth of the vitamin was destroyed. Vitamin B is therefore very stable to heating at 100° C. in the dry state, but somewhat less stable when heated at the same temperature in water solution. 22 CHART I. -30 FIGURE I. FIGURE II. Figure I. Average gain curves of rats on diet 94 plus various amounts of skimmed milk. Curve 1 Diet 94+15 cc. milk ' 94+12cc. " 1 94+lOcc. " ' 94+ 8cc. " ' 94+ 6cc. " ( 94+ 4cc. " ' 94+ 2cc. " Figure II. Average gain curves of rats on diet 107 plus various amounts of skimmed milk. Curve 1 Diet 107 + 12 cc. milk ' 107 + 10 cc. " ' 107+ 8cc. " ' 107+ 6cc. " ( 107 no milk K. = killed. D. = died. The last point on the curve is the average age and weight at death. Curves 6 and 7 in Figure I could not be completed as some of the rats died before the end of the experimental period of eight weeks. 23 CHART II. -30 FIGURE I. FIGURE II. Figure I. Curve 1 Average gain curve of rats on diet 101 (30% milk) 100 (25% milk) 99 (20% milk 95 (15% milk) 96 (10% milk) 97 ( 5% milk) 98 (2.5% milk) 94 (no milk) 2 " 3 Figure II. Curve 1 Average gain curve of rats on diet 100 (milk unheated) " " " " " 100 (milk heated 6 hrs. at 100° C.) " " " " " 100 (milk heated 24 hrs. at 100° C.) " 4 " " " " " " " 100 (milk heated 48 hrs. at 100° C.) K. = killed. D. = died. The last point on the curve is the average age and weight at death. Curves 4, 5 and 6 in Figure I could not be completed as some of the rats died before the end of the experimental period of eight weeks. 24 BIBLIOGRAPHY 1 Hopkins: The analyst and the medical man. Analyst 1906 XXXI 385. 2 Hopkins : Feeding experiments illustrating the importance - of accessory factors in normal dietaries. Jour. Physiol. 1912 XUV 425. 3Osborne and Mendel : Feeding experiments with isolated food substances. Carnegie Inst. of Washington 1911 Pub. No. 156. 4 Osborne and Mendel : The relation of growth to the chemi- cal constituents of the diet. Jour. Biol. Chem. 1913 XV 311. 5 Osborne and Mendel : The role of vitamines in the diet. Jour. Biol. Chem. 1917 XXXI 149. c Osborne and Mendel : Nutritive factors in plant tissues. II. The distribution of water-soluble vitamine. Jour. Biol. Chem. 1919 XXXIX 29. 7 Osborne and Mendel : Nutritive factors in plant tissues. III. Further observations on the distribution of water- soluble vitamine. Jour. Biol. Chem. 1920 XLI 451. 8 McCollum : The newer knowledge of nutrition 1922. 9 McCollum and Davis : Essential factors in the diet during growth. Jour. Biol. Chem. 1915 XXIII 231. 10 McCollum and Davis : Nutrition with purified food sub- stances. Jour. Biol. Chem. 1915 XX 641. 11 McCollum and Davis : The nature of the dietary deficiencies of rice. Jour. Biol. Chem. 1915 XXIII 181. 12 Eijkman : An illness of fowls similar to beriberi. Vir- chow's Arch. 1897 CXLVIII 823. 13 Eijkman : Nutritional polyneuritis. Arch. Hyg. 1906 LVIII 150. 14 Eijkman : Polyneuritis gallinarum and beriberi. Arch. Schiffs-Tropen Hyg. 1911 XV 698. 15 Funk : The vitamines. Trans, by Dubin 1922. 16 Funk: Chemical nature of the substance which cures poly- neuritis in birds induced by a diet of polished rice. Jour. Physiol. 1911 XLIII 395, 1912 XLV 75. 25 17 Williams : Chemical nature of the vitamines. I. Antineuritic properties of the hydroxypyridines. Jour. Biol. Chem. 1916 XXV 437. 18 Williams : Chemical nature of the vitamines. III. The struc- ture of the curative modifications of the hydroxypyri- dines. Jour. Biol. Chem. 1917 XXIX 495. 10 Williams : Chemistry of the vitamines. Philippine Jour. Sci. 1916 Ser. A XI 49. 23 Williams and Seidell : The chemical nature of the vitamines III. Isomerism in natural antineuritic substances. Jour. Biol. Chem. XXVI 431. 21 Wildier : La Cellule 1901 XVIII 313. 22 Sherman and Smith : The vitamins. 1922. 23 Williams : The vitamine requirements of yeast. A simple biological test for vitamine. Jour. Biol. Chem. 1919 XXXVIII 495. 24 Williams : A quantitative method for the determination of vitamine. Jour. Biol. Chem. 1920 XLII 259. 25 Williams : Vitamine and yeast growth. Jour. Biol. Chem. 1921 XLVI 113. 20 Bachman : Vitamine requirements of certain yeasts. Jour. Biol. Chem. 1919 XXXIX 235. 27 Eddy and Stevenson : The suitability of the Bachman test for water-soluble B. Proc. Soc. Exp. Biol. Med. 1919 XVII 52. 28 Eddy and Stevenson : Studies in the vitamine content. Jour. Biol. Chem. 1920 XLJII 295. 29 Emmett and Stockholm : Water-soluble vitamines. II. The relation of the antineuritic and water-soluble B vita- mines to the yeast growth promoting stimulus. Jour. Biol. Chem. 1920 XLIII 287. 30 Fulmer, Nelson and Sherwood : The nutritive requirement of yeast. Jour. Amer. Chem. Soc. 1921 XLIII 191. 31 Funk and Dubin : A test for antiberiberi vitamine and its practical application. Jour. Biol. Chem. 1920 XLIV 487. 32 McDonald and McCollum : The cultivation of yeast in solu- tions of purified nutrients. Jour. Biol. Chem. 1921 XLV 307. 26 33 McDonald and McCollum : The bios of Wildiers and the cultivation of yeast. Jour. Biol. Chem. 1921 XLVI 525. 34 Nelson, Fulmer and Cessna : The nutritional requirements of yeast. III. The synthesis of water-soluble B by yeast. Jour. Biol. Chem. 1921 XLVI 77. 35Souza and McCollum : A study of the factors which interfere with the use of yeast as a test organism for the anti- neuritic substance. Jour. Biol. Chem. 1920 XLIV 113. SH Hopkins, Chick et al : Report on the present state of knowl- edge concerning accessory food factors (vitamines). Medical Research Committee Special Report No. 38 1919. 37 Edge worth : Experiments with two methods for the study of vitamin B. Dissertation 1922. 38 McCarrison : Studies in deficiency diseases. Oxford Uni- versity Press. 1921. 39 Osborne, Wakeman and Ferry : Preparation of protein free from water soluble vitamine. Jour. Biol. Chem. 1919 XXXIX 35. 40 Osborne and Mendel : Nutritive value of the wheat kernel. Jour. Biol. Chem. 1919 XXXVII 572. 41 Kennedy and Dutcher : Vitamine studies. IX. The influence of the diet of the cow upon the quality of the vitamines A and B in milk. Jour. Biol. Chem. 1922 L 339. 42 Hopkins : Note on the vitamine content of milk. Biochem. Jour. 1920 XIV 721. 43 Osborne and Mendel : Milk as a source of water-soluble vitamine. Jour. Biol. Chem. 1918 XXXIV 537. 44 Osborne and Mendel : Milk as a source of water-soluble vitamine. II. Jour. Biol. Chem. 1920 XLI 515. 27 VITA Adelaide Spohn was born in Chicago, Illinois, May 25, 1886. In 1908 she received the degree of B. S. from the Uni- versity of Chicago and in 1913 the degree of M. S. from the same University. Since 1908 she has held the following posi- tions : Instructor, Woodstock, Illinois, High School 1908- 1910; Instructor, Joliet, Illinois, Township High School 1910- 1911 ; Research Assistant to Dr. Oscar Riddle at the Univer- sity of Chicago and Station of Experimental Evolution of the Carnegie Institution of Washington, 1911-1915; Instructor in Chemistry, Teachers College, 1915-1916; Chemical Assistant in the Department of Physiology, Columbia University, 1916 to February, 1918; Instructor in Chemistry Northwestern University, February 1918-1919; Instructor in Chemistry, Pratt Institute, 1919-1920; Research Assistant in Food Chem- istry, Columbia University, 1920-1922. She was co-author with Dr. Oscar Riddle of the following publications : Studies on the Physiology of Reproduction in Birds. II. On the Chemical Composition of White and Yel- low Egg Yolk of the Fowl and Pigeon, Am. Jour. Physiol, 1916, XLI, 397 ; Studies on the Physiology of Reproduction in Birds. IV. When a Gland Functions for the First Time is its Secretion the Equivalent of Subsequent Secretions ? Am. Jour. Physiol. 1916, XLI, 419. She has been a graduate student under the Faculty of Pure Science, Columbia University, during the years 1915-16 and 1920-22. 28 THIS BOOK IS DUE ON THE LAST DATE STAMPED BELOW AN INITIAL FINE OF 25 CENTS WILL BE ASSESSED FOR FAILURE TO RETURN THIS BOOK ON THE DATE DUE. THE PENALTY WILL INCREASE TO SO CENTS ON THE FOURTH DAY AND TO $1.OO ON THE SEVENTH DAY OVERDUE. m>° n JDflOllOg'y 25 APR 28 1936 }54 4 1954 LD 21-50m-l,'33 Photomount Pamphlet Binder Gaylord Bros _ Makers Syracuse, N. Y pAT. JAN 21, 1908 ' YD 29358 5, TIN UNIVERSITY OF CALIFORNIA LIBRARY