WWI Ill“ A STUDY {3F fiACHETSfiEMfi NETS WWH SfiéfiiAL REFERENCE TO THE VALUE (3'? LYSéNE SUEMEMEN‘RVHGN “fixaséa for the Qatagme 7:4: it”! 3.. MECHiGAN STATE CEZ‘LLEGE Yéaxé‘aéfig Yang v3.34“; This is to certifg that the thesis entitled 'A.Study of Rachitogenic Diets with Special Reference to the Value of Lysine Supplementation! presented by Te-ehing Yang has been accepted towards fulfillment of the requirements for Ldeqree in Chemist 0.62. /W Major prhrils$r s C '— O - f- r 7".” (flat 2* .; >2 a.- «Java , a w“ a ‘ - 4 w, O t‘H I f . ‘F ’34. ’ 3-." *3 U. - - iéw“:{;.{rj "4 1? 1“ ‘ t 'f‘ - ' (2.7- ‘ _ “:3‘ a.‘. A r, ‘ ”.5" lfa’f'b h-’- M -_I t '1‘ ' ‘ - ‘1 - . - I. . _ . . ‘. . <~ . . 3. l w x jin 5 25 r . 4‘?" . zip “it? ' xi .‘_ ’0 \ .é'l' .1 HQ 1:; v X It ' ‘ f.-' .3} ' firifii‘ffl " \ ‘e ,1. ‘1- I~<§' J. *" V o 1‘ v? V A fit; It". 0 ’. ,g' I I 'P~ ‘ .‘I 1 :J‘." . A . ‘ ‘I \ at; l - .’ ‘fi‘A “Tl. :' 1' ‘x, ,'.*‘~ A} ‘.~ ' ." —._°.4 '- 'm~"i;i$ «If? . 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".)..L}_ n , o \ ‘J _ 3'7" fig" .' 1‘; ." .>Ir~";‘ I a 4.: v 93-- fl‘ D t K. 1 . f 12'" 5 a '. .- ’l ”mu: 3 - , . _ . . .e '3‘. ,, . ‘ [n l _ t I . x A STUDY OF RACHITOGENIC DIETS WITH SPECIAL REFERENCE TO THE VALUE OF LYSINE SUPPLEMENTATION By Te-ehing Yang A Theeie Submitted to the School of Graduate Studies of Michigan State College of Agricultural and Applied Science in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Department of Chemistry June, 1949 v- “.1an n l l,l?,.l"l, 7'6 42 57/5 Y " £93; ACKNOWLEDGMENT The writer of this thesis wishes to eXprBBB her deep gratitude to Dr. Carl A. HOppert, for his en- couragement in carrying out the investigation, and.fcr his kindly assistance and criticism through- out these studies and in the pre- paration of the manuscript. 2.18017 TABLE OF CONTENTS Introduction . . . . . . . . . . . . Historical Review. . . . . . . . . . Experimental Procedures. . . . . . . I. Preparation of Rations. . . II. Determination of Calcium and Phoaphorus. e e e e e 0 III. Care of Animals . . . . . . IV. Line Test . . . . . . . . . «a (0 ea .4 Data . . . . . . . . Table I . . . . Experiment I. . Experiment II . Experiment III. Experiment IV . Experiment V. . Experiment VI . Experiment VII. Experiment VIII Experiment IX . Results and Discussion Summary and Conclusion Bibliography . . . . 15 15 16 18 so 23 e4 25 27 29 so :53 37 38 gggaogpcrton Although many attempts have been made to determine vitamin D with both physical and chemical methods, none of these methods can as yet compete with the biological in sensitivity. The rat assay method is still commonly used and recognized as standard for the determination of vitamin D int ended for use by mammals, including man. This method involves the product ion of rickets by the use of a suitable diet. In spite of the years of extensive use of rachitogenic diets the pro- blem of obtaining uniform composition and subsequent uniform production of rickets has not yet been solved. The classical rachitogenic diets, devised by early workers, have long been subjected to study, but rela- tively little progress has been made in improving them. The variability of the ingredients used in the diet gave rise to different results in different laborator- ies. The animals given these diets usually have low resistance to disease, and frequently a few die during the period of preparation. Some of the animals do not develop satisfactory rickets because of insufficient growth. The need therefore exists of improving the diet both from the standpoint of assuring greater uni- formity and, if possible, of reducing the time needed -1. to develop a suitable degree of rickets and to test for vitamin D in various products. The present study was carried.out in the hope that a more satisfactory diet could be developed. Special attention was given to the use of lysine supplementation. This is based on the fact that cereal proteins are gener- ally low in lysine and growth on cereal diets can be appreciably improved.by the addition of this amino acid. A supply of the BIL-lysine monohydrochloride was made available for this study by Dr. Wadell of the Du Pont Company of New Jersey. HISTORICAL REVIEW A few years before vitamin D was known to the world, experimental rickets was intensively investigated by a number of workers. Among them McCollum, Simmonds, Shiplpy, Park, and Pappsnheimer are all well known in this field. Starting from the year 1920, a series of papers under the title "Studies on Experimental Rickets' was published. (1, 8, :5, 4, 5, 6, 7), Various phases concerning rickets and the antirachitic substance were thoroughly studied. McCollum and his coworkers (1) first used rats to produce experimental rickets. They found that certain diets, when fed to young rats, produced various distur- bances in growth and development of the skeleton. These diets had in common the production of irregularities in the calcification of the intercellular substance of the proliferative cartilage or absence of lime salt deposi- tion from the matrix of the tissue. At that time they attributed the condition to a deficiency in fat-soluble vitamin A and calcium. Following the first experiment, leollum and his coworkers (2) further observed that, with the addition of cod liver oil to the diets, a fresh deposition of lime salt between the cells of the proliferative zone of cartilage could be observed. The deposition of cal- -3- cium salts was linear, the width of the line apparently depending on the length of time that the animals had been fed cod liver oil. A year later McCollum and his coworkers (5) sug- gested the name "line test' for this method of determining the antirachitic activity of a product. The essential point of the test was the ability of a given substance to cause reappearance of a provisional zone of calcification in the epiphyseal cartilage of animals with severe rickets. The reliability of the test depended.upcn a suitable diet which could produce bones with calcium-free epiphyseal cartilage and.mstaphyses. Much work and effort has been put into the development of rachitogenic diets. Hess, McCann and Pappenheimer (8) failed to produce rickets on a diet deficient in vitamin A. After many trials, McCollum and his coworkers (3) in 1931 were able to develop a comparatively satisfactory rickets- producing diet known as McCollum's Diet 3143, which was composed of yellow maize 33%, wheat 33%, gelatin 15%, wheat gluten 15%, sodium chloride 1%, and calcium carbonate 3%. They indicated the diet must have : (l) a specific dispro- portion in the calciumephosphorus ratio, the phosphorus being low, the calcium relatively high, and (8) an insuf- ficiency of the antirachitic substance. With this diet McCollum (4, 6) was able in 1933 to demonstrate the exist- -4- ence of vitamin D. In a more detailed study of the fat-soluble vitamin, Steenbock and Black (9) found that McCollum's Diet 3143 induced too much variation in the production of rickets, due to an insufficiency of vitamin A and too much protein. They suggested that a diet composed.of yellow corn 76%, wheat gluten 20%, calcium carbonate 3%, and sodium chlo- ride 1% would give far more consistent results. It also had the advantage of greater ease of preparation, and re— duced cost. Steenbock and.B1ack's Diet 3965 together with McCollum's Diet 3143 were soon adopted by many labor- atories for the determination of vitamin D from various sources. Due to their wide application these diets were studied.from different angles by many workers. The first point of attack was the phosphorus con- tent and.the calcium-phosphorus ratio. The importance of these elements had been pointed out‘by McCollum and his coworkers (3, 5) in the early studies. Karelitz and.Shoh1 (10) first stated that the addition of phosphate to a rickets-producing diet would cause healing of rickets. Shohl, Brown, Rose, Chapman, and Saurwein (ll, 13) in a series of experiments, varied the amount of calcium car- bonate and sodium dihydrogen phosphate which were added to the modified Steenbock and Black's Diet 2965, causing various levels and ratios. With the same ratio of cal- ~5- cium and.phosphorus, a diet was shown to become progres- sively less rickets-producing as the salt level was raised. Within the range used, the greater the ratio at a given level of phosphorus, the more severe would be the degree of rickets. When phosphorus was very low (0.12%) rickets could be obtained at any calcium phosphorus ratio. The ‘production of rickets with a high calcium diet was impos- sible when the phosphorus content was more than 0.5%. With a still higher calciumpphosphorus ratio, as 12:1, 24:1, or 56:1, a less severe degree of rickets resulted, but such animals would die early without gain or loss in weight. The optimal calcium-phosphorus ratio (13) for the production of rickets probably lay between 4 and 5 to 1. As for the effect of the acidrbase content in a rickets-producing diet, Shohl and his coworkers (14) made a number of observations. By analysis, the bone showed greater ash content with the neutral diet, smaller with the alkaline diets, and least with the acid diets. Later studies by Shchl (15, 16) indicated that the acid diet tended toward the production of more severe rickets, since alkalinity caused a diminished activity on the ion- ization of calcium. This factor was directly related to the blood.a1kalinity, but its mechanism is still not clearly understood. The composition of the yellow corn used in the Steen- book Diet was studied extensively. Holmes and Tripp (17), upon analyzing samples of the Steenbock Diet from different laboratories, obtained.variations in ash, calcium, and phosphorus content. Harris and Bunker (18, 19) determined the calcium and.phosphorus in forty samples of corn and found considerable variation. Rachitogenic diets made with these varieties of corn had calcium-phosphorus ratios ranging from 3.98:1 to 6.7:1. Such variations are of suf~ ficisnt magnitude as to produce different degrees of rick- etc. Other factors which might have influenced.the rachi— togenic diet were the variation of protein and vitamin A content and the fineness of grinding. (13, 17) Modification of the Stsenbock Diet 2965 was made by Ma (20) in 1937. Table corn meal was used instead.of or- dinary ground corn. With the addition of 25% oatmeal and 1% dried yeast, a comparatively satisfactory rachitogenic diet was obtained. In 1947 Bills (13) gave a very comprehensive review of the determination of vitamin D by the rat assay method. He mentioned.that the U.S.P. method still referred to M0- Collum's Diet 3143 and Steenbock's Diet 2965 as standard rachitogenic diets. Young rats weighing just over 44 grams were put on either one of the rachitogenic diets for -7- a preparatory period of 21-24 days. Those rats weighing between 60-80 grams were used and.continued immediately for a 7-day test period. The results were judged by the "line test'. A more recent study by Francis (21) called attention to the amino acid requirement of the rats. By comparing the amino acid content in the Steenbock's Diet 2965 with the rat‘s requirement as determined by Rose in 1937, the data indicated that the diet provides ample quantities of all amino acids save lysine. With the addition of 0.5% lysine to the diet, a satisfactory degree of rickets could be produced in sixteen days. As pure lysine was too expensive to be used in experiments, blood fibrin was tried in the place of lysine and found to be unsatisfactory. The successful practical synthesis of DL—lysine mono- hydrochloride by the Du ant Company has renewed interest in the use of this amino acid product not only for improv— ing the growth-promoting properties of rachitogenic diets, but also of practical mixed feeds containing chiefly cereal products. EXPERIMENT L PROCEDURES I. Preparation of the ratigpgz The rachitogenic diets were prepared on the day that a group of young rats was available for experi- ment. Usually 1 kg. of each ration was prepared at the beginning of the experiment. Each ingredient was weighed carefully on the triple beam balance. Yellow table meal, which composes the bulk of the ration, was weighed out first and transferred to a mixing pan. The other ingredients were weighed out carefully, transferred.to the mixing pan, and mixed thoroughly with the yellow table meal. The portions to be used were immediately transferred to Fisher cups and the rest stored in brown bottles. II. .Deisrmineiion_of-sslsiun-and-nhssnharus3 1. Determination of calcium. Method.from A.0.A.C. 1945, Sixth Edition (22). a. Ingredients other than calcium carbonate and common salt. Triplicate 5-10 grams of finely ground samples were weighed carefully into a porcelain crucible. These were ashed in a muffle furnace until carbon-free. Then the ash was moistened with 1 ml. of concentrated -9- nitric acid. This was followed by drying the contents and again carefully igniting in the muffle furnace till white-colored. After the ash was allowed to cool, 5 ml. of hydrochloric acid was added, allowing the acid to rinse the upper portion of the dish. The material was now'evaporated to dryness on a steam bath. The residue was dissolved.by adding an accurate measure of 2.0 m1. hydrochloric acid. This was heated for five minutes on a steam bath with a watch glass on the dish. Afterwards the watch glass was washed with water and the residue filtered.into a 400-ml. beaker and diluted to 150 m1. Into the solution 10 drops of brcmocresol green indicator were added and than sufficient 20% sodium ace- tate solution to change the PH to 4.8 - 5.0 (blue). Then the beaker was covered with a watch glass and the solution heated to boiling. The calcium was precipi- tated slowly by adding 3% oxalic acid solution, a chap every 3-5 seconds, until the PH was changed back to 4.4 - 4.6, indicated by the appearance of a distinct green shade. This solution was boiled for 1-3 minutes and.allowed to settle overnight or until clear. The supernatant liquid was filtered through a fritted glass crucible and the beaker and.precipitates washed with about 50 ml. of ammonium hydroxide (liso) in small por- tions, using a wash bottle which would deliver a very -lo- small stream. The crucible together with the precipitate was then transferred back to the original beaker, and 125 ml. water and 5 m1. sulfuric acid were added at a temper- ature of 80-900 C. with 0.05N potassium permanganate until a slight pink color was obtahned. The titration result was corrected with a blank titration and the percentage of calcium in the samples calculated. b. Calcium carbonate and common salt. Modified.meth- od from William and Fumman's 'Elementary Quantitative Analysis", Third Edition (23). Triplicate 0.5 grams samples of calcium carbonate (5 grams samples of common salt) were weighed into a 400-ml. beaker. 80 m1. of water and 5 m1. of concentrated hydrochloric acid were added. The beaker was covered with a watch glass and the mixture was then heated until dis- solved. The sides of the beaker and.watch glass were then rinsed and the total volume of the solution was diluted to 150 ml. The precipitation and.titration were carried out as prev iously described. 2. Determination of phosphorus. Method according to A.0.A.C. 1945, Sixth Edition (33). Triplicate 5-10 grams of samples were weighed accur- atcly into SOD-ml Ejeldahl flasks. 5 m1.‘of concentrated - 11.. nitric acid.and 10 ml. of concentratsdsulfuric acid.were added to each flask in the hood. 5 grams of potassium nitrate was added at once and the flask was allowed to stand in the hood until the violence of the reaction was over. Then 50—70 mls. concentrated sulfuric acid was added to each flask and the mixture was then digested to the point where the solution had become nearly colorless. After cooling, 150 m1. distilled.water was added.and the solution boiled for a few minutes. The solution, after cooling, was filtered into a BOO-ml. volumetric flask and diluted to volume. A 50-ml. aliquot was pipetted.into a 250-ml. beaker. Into the solution ammonium hydroxide was added in slight excess. A few dr0ps of nitric acid.were added to dis- solve any precipitate formed. The solution was stirred vigorously and fifteen grams of crystalline ammonium ni- trate added, 60 ml. of the molybdate solutioriwas then added and the solution digested at about 65° C. for one hour. The yellow precipitate was filtered and.washed with ammonium hydroxide solution (1+9) and then dissolved from the filter with ammonium hydroxide (1+1) and hot water into a beaker to a volume of not more than 100 ml. The solution was then neutralized.with hydrochloric acid, using bromothymol blue as the indicator and then cooled. 15 ml. of magnesia mixture was added slowly from a burette -13- (about one dIOp per second) and the solution subjected to vigorous stirring. After 15 minutes, 12 ml. of con- centrate ammonium hydroxide was added. The solution was allowed to stand until the supernatant liquid was clear. The precipitate was then filtered and washed with ammon- ium hydroxide solution (1+9) until the washings were practically free from chlorides. The precipitate was then dried and burned.at low heat and ignited to constant weight in an electric furnace at 950°-lOOO° C. The resi- due was cooled in a desiccator and weighed as Mg2P307. From this the percentages of phosphorus of the samples were calculated. III. C r m : Young rats approximately three weeks of age and weighing 45-55 grams were placed on the rachitogenic diet in separate cages. Tap water was given ad.libitum. Weights were recorded at the end of each week. At the end of two or three weeks, when most of the animals gained at least 20 grams, they were put on a supplemental diet for from 5-10 days. At the end of the test period the rats were killed.by ether vapors. The radii were removed for the line test. -13- IV. Line 3111;: The wrist bones, after being preserved in 95% alco- hol for at least 24 hours, were split and then immersed for 2 minutes in 2% silver nitrate solution. The bones were then transferred to a small porcelain dish contain- ing distilled water and exposed either to natural or artificial light until a desired darkness developed in the calcified area of the bone. Results were then Judged by observing the epiphyseal line of calcification. DATA TABLE I The calcium and phosphorus content of various ingredients. NAME or INGREDIENT 7!; CALCIUM % PHOSPHORUS Yellow table meal 0.00561 0.158 Yeast 0.00167 1.125 Oil meal 0.428 0.925 Wheat gluten 0.0922 0.276 Casein 0.647 1.088 Whole wheat 0.0566 0.328 Gem Iodized salt 0.0236 -- Morton's Iodized salt 0.0379 -- Calcium carbonate 39.85 —- -15- EXPERIMENT I TABLE II Composition of rachitogenic diets. Name of Ingredient No. of Diet ll A 1 L 5 6 Yellow table meal 73% 72% 59% 63% 68% 59% Wheat gluten 20 20 25 30 20 20 Casein (crude) -- -- -- -- 5 -- 011 meal -- -- —- -- -- 5 Yeast 3 4 3 3 3 3 09.003 3 3 3 3 3 3 NaCl 1 l l 1 1 1 Ca 72 1.22 1.22 1.22 1.23 1.25 1.24 P 7: 0.204 0.214 0.210 0.216 0.251 0.243 Ca:P 5.95 5.70 5.81 5.68 4.99 5.13 -15- TABLE III Growth response of young rats fed the above rachitogenic diets. Average Av. Wt. Av. Wt. Av. It. No. of No. of Initial. End.of Gained Gained Diet Rats Weight 3 weeks 3 weeks Daily grams grams grams grams 1 8 47 73 26 1.2 2 8 49 77 28 1.4 3 7 50 76 26 1.2 4 7 50 83 33 1.5 5 8 46 80 34 1.6 6 7 47 77 20 1.0 Ling test: 2 d10ps of standard.cod liver 011 containing 5 U.S.P. unite given by mouth on the first day of the test period. The rats were continued.on the same rachitogenic diets for 10 days. Results: All groups gave good responses. -17- Exgpglurnr 1; W Composition of rachitogenic diets. Name of Ingredient No. of Diet §__ 7 1 Yellow table meal 72% 71% 71.5% Wheat gluten 20 20 20 Yeast 4 4 4 Ca003 3 3 3 NaCl 1 1 1 Lysine -- l 0.5 Ca % 1.22 1.22 1.22 P % 0.214 0.211 0.213 Ca:P 5.70 5.73 5.72 TABLE V Growth response of young rats fed the above rachitogenic diets. Average Av. Wt. No. of No. of Initial End of Diet Rats 'Weight 3 weeks Av. Wt. Av. Wt. Gained Gained 3 weeks Daily grams grams 3 13 51 76 7 12 50 85 8 12 51 84 grams grams 25 1.2 35 1.7 33 1.5 Line i: 31,3 1 drop of standard cod liver oil (5 U.S.P. units) was given by mouth on the first and sixth day of the test period, the rats being kept on the same rachito- genic diets for 10 days. Rgglts: All groups gave very good responses. EXPERIMENT III TABLE VI Composition of rachitogenic diets. No. of Diet Name of Ingredient 1 9 10 11 12 13 Yellow table meal 73% 72.5% 68% 67.5% 63% 62.5% Whole ground wheat -- -- 5 5 10 10 Wheat gluten 20 20 20 20 20 20 Yeast 3 3 3 3 3 3 08.003 3 3 3 3 3 3 NaCl 1 1 1 1 1 1 Lysine -- 0.5 -- 0.5 -- 0.5 Ca % 1.22 1.22 1.22 1.22 1.22 1.22 P % 0.204 0.204 0.203 0.203 0.221 0.221 05:? 5.95 5.95 6.00 6.00 5.52 5.52 TABLE VII Growth response of young rats fed the above rachitogenic dieta Average Av. Wt. Av. Wt. Av. Wt. No. of N0. of Initial End of Gained. Gained Diet Rats Weight 3 weeks 3 weeks Daily grams grams grams grams 1 3 48 76 28 1.4 9 3 53 81 26 1.2 10 4 53 74 21 1.0 11 4 49 82 33 1.5 12 3 46 66 20 1.0 13 4 53 88 35 1.7 Line test: 4 units of vitamin D in 1 cc. corn oil, mixed with 50 grams of each rachitogenic ration, were given to the rats as supplementary diet for 10 days. Results: All groups gave good responses. There was a slight difference in the degree of response with increases in the amount of phosphorus present in the rachitogenic diet. Those groups with 0.5% lysine gave slightly better respon- ses than the similar groups without lysine. -31- EXPERIMENT IV TABLE VIII Composition of the rachitogenic diets. Name of Ingredient N0. of Diet -L‘L___ Yellow table meal 62. 5% Wheat gluten 30 CaCO3 3 Yeast 3 NaCl 1 Lysine 0.5 Ca % 1.23 P % 0.215 Ca:P 5.70 TABLE IX Growth response of young rats fed the above rachitogenic diets. Average Av. Wt. Av. Wt. Av. Wt. No. of No. of Initial End of Gained Gained Diet Rats Weight 2 weeks 2 weeks Daily A grams grams grams grams 14 24 47 75 28 2.0 Ling test: At the end of two weeks all rats were given supple- ments of fluid vitamin D in a small cup. The 24 animals were divided.into 4 groups: (1) 5 m1. vitamin D milk containing 2 U.S.P. units, (2) 7-1/2 m1. ' 7 0 3 n n , (3) 10 m1. ' ' 7 4 . n , (4) 10 m1. ordinary milk. The rats were killed at the end of 5 days. Rggglt : (1) gave broken lines of healing, (2) gave fairly good responses, (3) gave very heavy responses, and most of them tended to oalcify downward. The lines were not clear. (4) gave a negative response. -33- EXPERIMENT V TABLE X Growth response of young rats fed rachitogenic diets No. 4 and.No. 14. Average Av. Wt. Av. Wt. Av. Wt. No. of N0. of Initial End of Gained Gained Diet Rats Weight 2 weeks 2 weeks Daily grams grams grams grams 14 12 45 75 30 2.1 4 12 46 64 18 1.3 Line test: Rats on Diet No. 14 at the end of 2 weeks were given supplements of 7.5 m1. (3 U.S.P. units) vitamin D milk, 'whereas rats on Diet No. 4 were given similar supplements at the end of the 5th day. Rgggltg: Rats on Diet N0. 14 gave a mostly negative line test. Rats on Diet N0. 4 gave fairly good responses. EXPERIMENT VI TABLE XI Composition of rachitogenic diets. Name of Ingredients No. of Dist 15. 1§__ Yellow table meal 52.5% 53% Wheat gluten 40 40 Yeast 3 3 Ca003 3 3 N801 l 1 Lysine 0.5 -— Ca % 1.24 1.24 P % 0.227 0.228 Ca:P 5.46 5.43 -35- TABLE XII Growth response of young rats fed the above rachitogenic diets. Average Av. Wt. Av. Wt. Av. Wt. N0. of N0. of Initial End of Gained Gained Diet Rats Weight 2 weeks 2 weeks Daily grams grams grams grams 15 12 45 69 24 1.7 16 10 45 68 23 1.6 Ling test: At the end of 2 weeks all rats were given supple- ments of 7.5 ml vitamin D milk (3 U.S.P. units) in addition to the rachitogenic diets. All rats were killed at the end of the fifth day. Results: All gave negative response. -26.. EXPERIMENT VII TABLE XIII Composition of rachitogenic diets. Name of Ingredient No. of Diet ._. _JUL_ 11__- Yellow table meal 62.5% 61.5% Wheat gluten 30 30 Yeast 3 4 0a003 3 3 NaCl 1 l Lysine 0.5 0.5 Ca % 1.23 1.23 P % 0.215 0.225 Ca:P 5.70 5.46 -37- TABLE XIV Growth response of young rats fed the above rachitogenic diets. Average Av. Wt. AV. Wt. Av. Wt. No. of No. of Initial End of Gained Gained Diet Rats Weight 2 weeks 2 weeks Daily grams grams grams grams 14 8 45 66 21 1.5 17 7 48 70 22 1.6 Line test: At the end of two weeks all rats were given a supplementary diet containing 10 m1. vitamin D milk mixed.with 40 grams of the ration. All rats were killed at the end of a week. Regglts: Rats on Diet N0. 14 gave slight or negative line tests. Rats on Diet No. 17 showed broken lines of calci- fication. A11 metaphyses were wide and clear. -28- EXPERIMENT VIII Rachitogenic Diet N0. 17 was used, with one varia- tion, numbered 17'. Diet No. 17' had the same composition as No. 17, except that ordinary yellow table meal was sub- stituted for finely ground yellow table meal. T BLE XV Growth response of young rats fed the above rachitogenic diets. Average Av. Wt. Av. Wt. Av. Wt. No. of N0. of Initial End of Gained Gained Diet Rats Weight 2 weeks 2 weeks mily grams grams grams grams 17 6 42 69 27 1.9 17 ' 6 42 64 22 1.6 At the end of two weeks 10 m1. vitamin D milk, incorporated with 40 grams of the ration which did not contain lysine, was given as supplementary diet. The rats were killed at the end of a week. Resultg: A11 gave very slight responses with just the sign of a beginning of line formation. -39- EXPERIMENT IX TABLE XVI Composition of rachitogenic diets. Name of Ingredient No. of Diet _17 W Yellow table meal 61.5% 61.5% 61% 61% Wheat gluten 3O 30 30 30 Yeast 4 4 4 4 05003 3 3 3 3 NaCl 1 1 1 1 Lysine 0.5 0.5 1.0 1.0 1155003 -- 0.2 -- 0.4 Ca % 1.23 1.23 1.23 1.23 P % 0.225 0.225 0.224 0.224 Ca:P 5.46 5.46 5.48 5.48 1A§L§_IEII Growth response of young rats fed the above rachitogenic diets. Average Av. Wt. Av. Wt. Av. Wt. No. of No. of Initial End of Gained Gained Diet Rats Weight 2 weeks 2 weeks Daily grams grams grams grams 17 12 47 75 28 2.0 18 10 46 69 25 1.8 19 12 46 72 26 1.9 20 12 45 75 30 2.1 Lips tegt: At the snd.of two weeks all rats were given supple- ments of 25 ml. of vitamin D milk inoorporated with 40 grams of the ration. Rats were killed at the end of one more week. 5532111: All rats gave very good responses. More uniform and.clearer lines were obtained with the rats receiving Diets No. 18 and No. 20. -31- H H .4 H .196? swam 41130.59 .10 sewn Rrsghrs AND DISCUSSION In this series of studies twenty rachitogenic diets were used, Each diet was Judged by the rate of growth of the rats, by the width and the character of the rachitic metaphyses of the radii and the ulnae, and by the extent and.nature of the calcification induced by administering a vitamin D supplement. The results of these experiments are presented in tables I-XVII. Inasmuch as considerable emphasis has been placed on the importance of the calcium-phosphorus ratios of a rachitogenic diet, calcium and phosphorus analyses were made of all the ingredients. The ratios found varied from 4.99 to 6.00. There was no particular correlation between the ratios and the suitability of the rations for determining vitamin D. The Michigan State College standard rachitogenic diet (Diet No. 2) and five modifications were used for an ini- tia1 exploratory experiment. The rats were fed the various rations for a three-week preparatory period and a vitamin D supplement during the ten-day test period. At the end of the first trial, all animals gave good line-test res- ponses. The casein in Diet 5 definitely helped the growth of the young rate. This was undoubtedly due to the enrich- ment of the diet in phosphorus and in the essential amino acids, especially lysine, in which cereals are strikingly -33- deficient. Increasing the amount of yeast as well as the wheat gluten also showed beneficial effects, whereas oil meal had less influence. The results of Experiment II show that the rate of growth was appreciably increased.with a supplement of 0.5% of DL-lysine monohydrochloride which was a synthetic product supplied by the Du Pont Company. It was espec- ially noticed that growth was more rapidly initiated during the first week. A comparison of the did: with 0.5% and 1% lysine supplementation showed that the higher level did not increase the rate of growth. Some rats on the 1% lysine diet developed inflamed eyelids, but this may have been due to other causes. The responses to vitae min D were good as to the quantity of the calcification. However, a diffused type of calcification occurred which was more noticeable at the 1% level. This type of response is undesirable because it is more difficult to evaluate. As had.been pointed.out by Karelitz and Shohl (10), increasing the amount of phosphorus in the diet would decrease the amount of vitamin D required or increase the response to a given amount of vitamin D. In the experiment with the addition of 5 and.10 percent. whole wheat, the result of the line test showed increasing responses with the increased phosphorus supplied by the whole wheat. Lysine supplementation again resulted.in an appreciable -34- increase in the rate of growth. Further studies were made by the addition of 0.5% ly- sine to Diet 4, which was the simplest and cheapest to prepare and.would support a good growth rate. The test period started.at the end of two weeks, since all the rats had.gained.more than 20 grams. Fairly good results were obtained with 7.5 m1. of vitamin D milk fed at the beginning of a test period of 5 days. By comparing the results with diets 4 and 14, the femorable effect of lysine supplementation is obvious. A higher level of wheat gluten diet was tried. Results showed that a 40% level was too high. In a dir- ect comparison between the 30% and 40% wheat gluten diets, the former gave much better growth. On trying a higher level of yeast, better growth was found at the 4% level. This was undoubtedly due to the larger amount of vitamin B complex supplied in the yeast as well as to the phosphorus and protein. A study was made of the influence of the fineness of grinding of the yellow table meal. The rats showed some- what better growth on the diet containing the finely ground.msal, which was used in subsequent experiments. No particular differences in the line-test response were observed, however. The last phase of the investigation was a study of -35- the influence of the hydrochloric acid introduced.in the ‘use of lysine monohydrochloride. A comparison was there- fore made with a diet supplementation with lysine mono- hydrochloride only and another containing in addition sodium bicarbonate, equivalent to the hydrochloric acid present in the lysine monohydrochloride. All rats could be used for the test at the end of two weeks. 7.5 m1. of vitamin D milk (3 U.S.P. units) incorporated with 40 grams of the diet gave very good line-test responses in a one-week test period. 0.5% lysine supplementation seemed to give clearer lines than the 1% lysine. Slightly better results were obtained with the rations containing sodium bicarbonate. In view of the variability of the biological assay for vitamin D, even under careful management it would be desirable to make further comparative studies in the use of lysine to speed growth and the development of rickets. The results obtained indicate that the time needed to prepare the rate for the test could be shortened to two weeks and.the test period to one week. This would effect an overall saving of 10 days in the determination of vitae min D and simplify the management of the test. Further work is also needed to determine the cause of the diffused character of the calcification rats. observed in the lysine-fed -36.. SUMMARY AND QONCLUSIQN 1. Modifications of the Michigan State College standard basal rachitogenic diet were used in the pro- duction of rickets in rats. Better growth was obtained by increasing the amount of wheat gluten or yeast or by the addition of casein or oil meal. 2. With the supplementation of 0.5% and 1% syn- thetic lysine monohydrochloride to the basal diet, much better growth resulted. However, the 1% lysine level did not show any greater effect than the 0.5%. 3. A gain in weight of over twenty grams at the end of two weeks could be produced by adding 0.5% lysine to the modified diet containing 30% wheat gluten. 4. With the addition of an amount of sodium bicar- bonate equivalent to the hydrochloric acid present in the lysine monohydrochloride, better results were ob— tained.in the line test. 5. As a result of lysine supplementation, rats can be prepared for a vitamin D assay in two weeks. The test psriod.oan be shortened to one week. However, the deposition of calcium salt is somewhat diffused instead of linear, making the evaluation of the response more difficult. -37- 5. BIBLIOGRAPHY McCollum, E. V., Simmonds, H., Parsons, H. T., Shipley, P. G., and Park, E. A., Studies on Experimental Rickets: I. The Production of Rachitis and Similar Diseases in the Hat by Deficient Diets. J. Biol. Chem., 45, 333, 1920—21. Shipley, P. G., Park., E. A., MCCollum, E. V., Simmonds, H., and Parsons, H. T., Studies on Experimental Rickets: II. The Effect of Cod Liver Oil Administered to Rate with Experimental Rickets. J. Biol. Chem., 45, 343, 1920-21. McCollum, E. V., Simmonds, H., Shipley, P. G., and Park, E. A., Studies on Experimental Rickets: VIII. The Pro- duction of Rickets by Diet Low in Phosphorus and Fat Soluble A. J. Biol. Chem., 47, 507, 1921. McCollum, E. V., Simmonds, H., Shipley, P. G., and Park, E. A. ., Studies on Experimental Rickets: XII. Is There a Substance Other than Fat Soluble A Associated with certain Fats which Plays an Important Role in Bone Devel- opment? J. Biol. Chem., 50, 5, 1922. McCollum, E. V., Simmonds, H., Shipley, P. G., and Park, E. A., Studies on Experimental Rickets: XVI. A Delicate Biological Test for Calcium-depositing Substances. J. Biol. Chem., 51, 41, 1922. McCollum, E. 'V., Simmonds, H., Becker, J. E., and Ship- ley, P. G., Studies on Experimental Rickets: XXI. An experimental Demonstration of the Existence of a Vitamin which Promotes Calcium Deposition. J. Biol. Chem., 53, 293, 1922. McCollum, E. V., Simmonds, H., Becker, J. E., and Ship- ley, P. G., Studies on Experimental Rickets: XXIII. The Production of Rickets in Rate by Diets Consisting essen- tially of Purified Food Substances. J. Biol. Chem., 54, 249, 1922. Hess, A. E., McCann, G. E., and Pappenheimer, A. H., Experimental Rickets in.Rats: II. The Failure of Rats to Develop Rickets on a Diet Deficient in Vitamin A. J. Biol. Chem., 47, 395, 1921. -33- 10. 11. 12. 13. 14. 15. 16. 17. 18. Stecnbock, H., and Black, A., Fat soluble Vitamins: XXIII. The induction of Growth-promoting and Calci- fying Properties in Fats and their Unsaponifiable Constituents by Exposure to Light. J. Biol. Chem., 64, 263, 1925. Karelitz, S, and Shohl, A. T., Rickets in Rats: II. The Effect of Phosphate added to the Diet of Ricketic Rats. J. Biol. Chem., 73, 665, 1927. Shohl, A. T., Brown, H. B., Rose, 0. S., Saurwein, E., Does the Ratio of Calcium t0 Phosphorus of the Diet determine whether Rickets is produced.in the Rat? J. Biol. Chem., 97, X, 1932. Brown, H. B., Shohl, A. T., Chapman, E. E., Ross C. S., and Saurwein, E. H., Rickets in Rats: XIII. The Effect of various Levels and Ratios of Calcium to Phosphorus in the Diet upon the Production of Rickets. J. Biol. Chem., 98, 207, 1932. Bills, 0. E., Vitamin D Assay: Line Test and Chem- ical Methods. Biol. Symposia, Vol. XII, 409, 1947. Shohl, A. T., Bennett, H. B., and Week, K. L., Rick- ets in Rats: IV. The Effect of varying the Acid-base Content of the Diet. J. Biol. Chem., 78, 181, 1928. Shohl, A. T., Brown, H. B., Rose, C. 8., Smith, D. H., and Cozad, F., Rickets in Rats: XII. The Acidrbase Equilibrium of the Blood in Rickets and Tetany. J. Biol. Chem., 92, 711, 1931. Shohl, A. T., Brown, H. B., Chapman, E. E., Ross, C. S., and Saurwein, E. H., Rickets in Rats: XIV. A Diet -which Demonstrates the Effect of the Acidrbase Content upon the Production of Rickets and.also causes Idio- pathic Tetany. J. Biol. Chem., 98, 215, 1932. Holmes, A. D., and.Tripp, E., The Influence of the Come position of Yellow Corn on the Effectiveness of a Each? itogenic Ration. Cereal Chem., 10, 313, 1933. Harris, R. S.,and,Bunker, J. W. E., Variability in the Corn Component of a Rachitogenic Diet. J. Lab. and Clin. Med., 19, 390, 1934. 19. 20. 21. 22. 23. Harris, R. S., and Bunker, J. W} M., The Phytin Phos- phorus of the Corn Component of a Rachitogenic Diet. J. Nutrition, 9, 301, 1935. Ma, F. L., Studies on the Production of Rickets in Rats and the Mode of Action of Vitamin D. Ph.D. Thesis of Michigan State College, 1937. Francis, P. 8., Improvement of Steenbock Rachitogenic Diet by a Supplement of Lysine. J. Assoc. Official Agr. Chemists, 30, 364, 1947. Official and Tentative Method of Analysis of the Asso- ciation of Official Agricultural Chemists. Sixth Edi- tion, ppe 23-24, 240, 1945. William, H. H., and Furman, H. H., Elementary Quantit- ative Analysis. Third Edition, p. 342, 1940. -40- -. T612 .015 ' 218017 Y22 ' ‘ Yang ”f " " 4'.M'°f'7m1t°8931 .. diets with special reference to the value of lysine supple- mentation. ~51 w. I .- I; .3 3 .Tfi‘ N a ‘ ,. .s 41 3;. I. "I \ -. 3 1293 024467