LIBRARY, A 8: H COLLEGE- CfiMPQS. A240-6M-1231-L1 80 TEXAS AGRICULTURAL EXPERIMENT srmou ULLETIN NO. 441 DECEMBER, 1931 DIVISION OF CHEMISTRY he Relation 0f Lime and Phosphoric A Acid t0 the Growth and Bone Development of White Rats w T. O. WALTON, President ,- AGRICULTURAL AND MECHANICAL COLLEGE OF TEXAESj‘. STATION STAFFT ADMINISTRATION: A. B. CoNNER, M. S., Director R. E. KARPER, M. S., Vice—Director CLARIGE MIxs0N, B. A., Secretary M. P. HoLLEMAN, JR., Chief Clerk J. K. FRANCKLOW, Assistant Chief Clerk CHEsTER HiGGs, Executive Assistant HowARn BERRY, B. S., Technical Assistant CHEMISTRY: G. S. FRAPs, Ph. D., Chief; State Chemist S. E. AsBuRY, M. S., Chemist J. F. FUDGE, Ph. D., Chemist E. C. CARLYLE, M. S., Assistant Chemist T. L. OGIER, B. S., Assistant Chemist A. J. STERGEs, M. S., Assistant Chemist RAY TREIGHLER, M. S., Assistant Chemist W. H. WALKER, Assistant Chemist VELiviA GRAHAM, Assistant Chemist JEANNE F. DEMoTTIER, Asst. Chemist R. L. ScHwARTz, B. S., Assistant Chemist C. M. PoUNDERs, B. S., Assistant Chemist H(S)RTICULTURE: VETERINARY SCIENCE: *M. FRANcIs, D. V. M., Chief H. SCHMIDT, D. V. M., Veterinarian I. B. BOUGHTON, D. V. M.. Veterinarian I **F. P. MATHEws, D.V.M., M.S., Veterinar W. T. HARDY, D. V._ M., Veterinarian ' — ————, Veterinarian A PLANT PATHOLOGY AND PHYSIOLOG A J. J. TAUBENHAUS, Ph. D., Chie . N. EzEKIEL, Ph. D., Plant athologist . W. J. BAGH, M. S., Plant Pathologist C H. RoGERs, Ph. D., Plant Pathologist FARM AND RANCH ECON MICS: L. P GABBARD, M. S., Chief _ W. E. PAULSON, Ph. D., Marketing C. A. BoNNEN, M. S., Farm Management **W. R. NIsBET, B. S., Ranch Management **A. C. MAGEE, M. S., Farm Management RURAL HOME RESEARCH: JEssIE WHiTAcRE, Ph. D., Chief _ MARY ANNA GRIMEs, M. S., Textiles _ ELIZABETH D. TERRILL, M. A., Nutrition S. P. DAvIs, Wool Grader S. E. WOLFF, M. S., Botanist ENTOMOLOGY: SWINE HUSBANDRY: F. L. THQMAs, Ph. D., Chief: State FRED HALE, M. S., Chief Entomologist _ DAIRY HUSBANDRY: - H. J. REINHARD, B. S., Entomologist O. C. COPELAND, M. S., Dairy Husbandman ‘ R. K. FLETGHER, Ph. D., Entomologist POULTRY HUSBANDRY: W. L. _O WEN, JR, M. S., Entomologist R. M. SHERWOOD, M. S., Chief . J. N. RoNEY, M. S., Entomologist _ J. R. CoUcH, B. S.,Asst. Poultry Husbandman J. C. GAINES, JR., M. S., Entomologist AGRICULTURAL ENGINEERING: S. E. JoNEs, M. S., Entomologist H. P. SMITH, M. S., Chief F. F. BIBBY, B. S., Entomologist MAIN STATION FARM: S. W. CLARK, B. S., Entomologist _ G. T. McNEss, Superintendent **E. W. DUNNAM, Ph. D., Entomologist _ APICULTURE (San Antonio): ' **R. W. MoRELAND B. S., Asst. Entomologist H. B. PARKs, B. S., Chief C. E. HEARD, B. ., Chief Inspector A. H. ALEx, B. S., Queen Breeder C. SIDDALL, B. S., Foulbrood Inspector FEED CONTROL SERVICE: S. E. McGREGoR, B.S., Foulbrood Inspector F. D. FULLER, M. S.,_Chief AGRONOMY: JAivIEs SULLIVAN, Assistant Chief E. B. REYNoLDs, Ph. D., Chief S. D. PEARCE, Secretary R. E. KARPER, M. S., Agronomist _ J. RocERs, Feed Inspector P. C. IVIANGELSDORF, Sc. D., Agronomist K. L. KIRKLAND, B. S., Feed Inspector D. T. KILLoUGH, M. S., Agronomist S. D. REYNOLDS, JR., Feed Inspector H. E. REA, B. S., Agronomist _ P A M00RE, Feed Inspector B. C. LANGLEY, M. S., Agronomist E. J. WILsoN, B. S., Feed Inspector PUBLICATIONS: H. G. WicKEs, B. S., Feed Inspector A. D. JAcKsoN, Chief SUBSTATIONS No. 1, Beeville, Bee County: No. 9, Balmorhea, Reeves County: R. A. HALL, B. S., Superintendent J. J. BAYLEs, B. S., Superintendent . H. YARNELL, Sc. D., Chief **L. R. HAWTHORN, M. S., Horticulturist H. M. REED, M. S., Horticulturist SOIL SURVEY: **W. T. CARTER, B. S., Chie E. H. TEMPLIN, B. S.,_Soi Surveyor J. F. W0oD, B. S., Horticulturist A. H. BEAN, B. S., Soil Surveyor L. E. BROOKS, B. S., Horticulturist R. M. MARsHALL, B. S., Soil Surveyor RANGE ANIMAL HUSBANDRY: **M. W. BEcK, B. S., Asst. Soil Surveyor J. M. JoNEs, A. M., Chief BOTANY: B. L.WARwIcK, Ph.D., Breeding Investigations V. L. CoRY, M. S., Act. Chief No. 2. Lindale, Smith County: _ . R. JoHNsoN, M. S., Superintendent **B. H. HENDRIGKsGN, B. S., Sci. in Soil Erosion **R. W. BAIRD, B. S., Assoc. Agr. Engineer No. 3, Angleton, Brazoria County: R. H. STANsEL, M. S., Superintendent H. M. REED, 1W. S., Horticulturist No. 4, Beaumont, Jetferson County: R. H. WYGHE, B. S., Superintendent **H. M. BEAGHELL, B. S., Jr. Agronomist No. 5, Temple, Bell County: HENRY DUNLAvY, M. S., Superintendent C. H. R0GERs, Ph. D., Plant Pathologist W. T.- HARDY, D. V. M., Veterinarian H. E. REA, B. S., Agronomist - O. L. CARPENTER, Shepherd S. E. WOLFF, M. S., Botanist **O. G. BABcocK, B. S., Asst. Entomologist **H. V. GEIB, M. S., Sci. in Soil Erosion No. 15, Weslaco, Hidalgo County: **H. O. HILL, B. S., Jr. Civil Engineer W. H. FRIEND, B. S., Superintendent No. 10, College Station, Brazos County: . SHERwo0D, M. S., In charge L. J. MGCALL, Farm Superintendent No. ll, Nacogdoches, Nacogdoches County: H. F. MoRRIs, M. S., Superintendent **No. 12, Chillicothe, Hardeman County: J. R. QUINBY, B. S., Superintendent **J. C. STEPHENs, M. A., Assistant Agronomist No. 14. Sonora, Sutton-Edwards Counties: . DAMERoN, B. S., Superintendent I. B. BQUGI-ITON, D. V ., Veterinarian No. 6, Denton, Denton County: - S. W. CLARK, B. S., Entomologist _ P. B. DUNKLE, B. S., Superintendent W. J. BAcH, M. S., Plant Pathologist **I. M. ATKINs, B. S., Jr. Agronomist J. F. Wo0D, B. S., Horticulturist No. 7, Spur, Dickens County: No. 16, Iowa Park, Wichita County: R. E. DICKSON, B. S., Superintendent H. McDowELL, B. S., Superintendent B. C. LANGLEY, M. S., Agronomist . E. BROOKS, B. S., Horticulturist No. 8, Lubbock, Lubbock County: No. 19, Winterhaven, Dimmit County: D. L. JoNEs, Superintendent E. MoRTENsEN, B. S., Superintendent FRANK GAINEs, Irrig. and Forest Nurs. **L. R. HAWTHORN, M. S., Horticulturist Teachers in the School of Agriculture Carrying Cooperative Projects on the Station: W. ADRIANCE, Ph. D., Horticulture W. BiLsiNG, Ph. D., Entomology F. R. BRIs0N, B. S., Horticulture Ff. LEE, Ph. D., Marketing and Finance W. R. HoRLAcHER, Ph. D., Genetics DCOATES, A. E., Agricultural Engineering J. H. KNox, M. S., Animal Husbandry i. MAGKEY, M. S., Animal Husbandry A. L. DARNELL, M. A., Dairy Husbandry J. S. IVIOGFORD, M. S., Agronomy Dean School of Veterinary Medicine. _ _ _ TAs of January 1, 1932. ‘In cooperation with U. S. Department of Agriculture. The importance of a sufficient quantity of lime, phosphoric acid, and other minerals in the food of man and animals is generally recognized. The effect of the quantity and of the ratio of lime to phos- phoric acid in the feed was studied with rats because the re- sults might have some relation to the needs of man and do- mestic animals for these minerals. The ratio of lime to phosphoric acid of 1:1 was found to be favorable for rats when the ration contained 1.0, 1.5, and 3.0 per cent of each mineral. Small deviations of either lime or phosphoric acid from the favorable ratios or amounts decreased the growth of the rats. The ash in the femurs was in some cases affected by these changes, but not always. The minimum amounts in the ration which produced nor- mal growth and good bone development were found to be 0.75 per cent of lime and 1.0 per cent of phosphoric acid. Less than the minimum amounts of lime or phosphoric acid caused decreased growth, produced less than the normal per- centage of bone in the rat, and lowered the percentage of ash in the femur. The excessive percentage of 6.0 per cent of phosphoric acid fed with 1.5 per cent of lime in the ration decreased the growth of the rats considerably, but produced femurs contain- ing practically a normal percentage of ash and developed a large quantity of bone out of proportion to the total weight of the body. On the other hand, 6.0 per cent of lime with 1.5 per cent of phosphoric acid produced normal growth and normal bones. The ratio of lime to phosphoric acid in the femurs had no relation to the amounts of lime and phosphoric acid fed in the ration. CONTENTS PAG; Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. I Historical . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. i Methods and procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . Requirements for normal growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Z"- Eifect of deviation from requirements for normal growth . . . . . . . . . . 12 Amounts of minerals which are distinctly unfavorable. .p . . . . . . . . . . 1/1 The relation of lime and phosphoric acid to the percentage of femur and femur ash ‘in rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Ratio of lime to phosphoric acid in femur ash . . . . . . . . . . . . . . . . . .. 15p Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 i Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1'7 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 18o‘ BULLETIN NO. 441 DECEMBER, 1931 RELATION OF THE LIME AND PHOSPHORIC ACID i IN THE FEED TO THE GROWTH AND DEVELOP- MENT OF BONES OF WHITE RATS* By J. K. BLUM It is a recognized fact that minerals are a very important part of p. animal foods. Not only the presence and quantity of minerals play a part, but also the ratio of one to the other seems to be a factor in the » growth and health of the body. Of the mineral elements studied, phosphorus and calcium have re- ceived the greatest amount of attention. Recent investigations show that the insufficient quantity or an unsuitable ratio of these elements results in retarded growth and the development of diseases related to the skeleton. Osteoporosis, or porous bone, has been traced to an in- sufficient quantity of either calcium or phosphorus in the diet. Rick- i, ets also has been found to be caused by an improper ratio of calcium ‘~ _ to phosphorus. While vitamins are important in bone development, they cannot correct deficiencies in mineral. Poultry, cattle, and other domestic animals as well as man have dis- eases which are traceable to insufficient quantities or incorrect ratios of calcium to phosphorus in the diet. It is becoming necessary to grow animals at a rapid rate in order to derive a profit from feeding. To - do this every food requirement must be supplied in proper quantity. The study upon which the following discussion is based was under- taken to determine the effect of varying the lime and phosphoric-acid content of the ration upon the growth and the bone development of white rats. While results from experiments on rats cannot be applied directly to other animals, they can be used as a foundation for more extensive study. A ration was used which was believed to contain all the necessary factors for growth and well-being of white rats, with the exception of lime and phosphoric acid, which were supplied in various amounts. The results of such a study should show the extent to which lime and phosphoric acid affect the health of the animal when all other factors are supplied in proper quantity. HISTORICAL McCollum (5) discovered about 1914 that calcium supplied as the lactate when added to a diet made up of parts of the wheat plant in- *A Thesis submitted to the Faculty of the Agricultural and Mechanical College of Texas in partial fulfillment of the requirements for the degree of Master of Science in Science. 6 BULLETIN NO. 441, TEXAS AGRICULTURAL EXPERIMENT STATION creased the growth of pigs and rats. Thiswas confirmed later by Mc- Collum and co-workers (13) (14) using a different ration and adding: calcium carbonate. Steenbock and associates (239 observed that a ration made up off- cereals and gelatin was improved by the addition of one per cent of? This addition produced better growth in rats than i These results were confirmed by Shohl 1 calcium carbonate. the ration without the mineral. and Karelitz (20). McCollum and Simmonds (13) found that rats fed a ration con- taining a high percentage of calcium (3 per cent, calcium carbonate) i and a low percentage of phosphorus developed signs of rickets and died early. They implied that the ratio between calcium and phosphorus ' had more to do with the production of rickets and other similar dis- eases than the actual quantities of the mineral. Steenbock (23) concluded that a ration which contained 1.08 per cent , of calcium and .254 per cent of phosphorus produced rickets in rats. McCollum and co-workers (14) produced rickets on rats with a ration made up of purified food substances containing 1.202 per cent of cal- i“ This was confirmed by further a cium with .217 per cent of phosphorus. experimentation (13). They found that a disproportion between cal- cium and phosphorus with a limited amount of cod liver oil resulted in A; rickets while an insufficient quantity of calcium and phosphorus pro- duced osteoporosis. Simmonds (19) claimed that the rats require about .64 gram of cal- cium per hundred grams of ration. Nelson, Irwin, and Peet (16,) state that 0.5669 gram of calcium and 0.4556 gram of phosphorus in a calcium-to-phosphorus ratio of 1:.80 per hundred grams of ration were sufficient for rats both as to quantity and ratio of calcium to phosphorus. According to the work of Holmes and Pigott (18), a ratio of cal- cium to phosphorus of 1.8 to 1.74 produces the best growth in chicks. These same ratios also produced tibiae which contained greater percent- ages of ash than other ratios. One per cent of cod liver oil was fed to the chicks. Josef Schneider (17) states that if a normal person receives calcium and phosphorus in the ratio of 1:444, no vitamin D is necessary. According to Grieves (4) 0.41 gram of phosphorus per 100 grams of ration is optional for rats. He found that good growth, well-being, and normal osseous tissues were induced in rats with 0.64 gram of cal- cium and 0.41 gram of phosphorus to 100 grams of rations when vitamin D was added in the form of butter fat or cod liver oil. The work of Steenbock (23) and associates shows that a ratio of Ca: P within a range of 1: .75 to 1: .50 produced the more favorable quantity of ash in the femurs of rats. r Karelitz and Shohl (10) discovered that rickets could be cured in rats in some cases by correcting the ratio of calcium to phosphorus to 1:.95. _ ‘main. GROWTH AND DEVELOPMENT OF BONES OF WHITE RATS 7 Moritz and Krenz (15,) state that if the diet of rabbits contains cal- cium and phosphorus in the proper ratio, no vitamin D was needed to prevent rickets. _ Sherman and Steebeling (18) concluded that either vitamin D, cal- cium, or phosphorus may prove to be the limiting factor in the devel- opment of bone in rats. About the year 1923 Touverud (25) found that rats Which were fed a low calcium diet developed bones which contained smaller percent- tages of ash than normal rats. McCann and Barnett (11) observed that the total ash of rachitic bones is lower than in normal bones, while the ratio of calcium to phos- phorus in the ash is not changed. The work of Steenbock and associates (23) (24), Dutcher and asso- ciates (2), (3), McCann and Barnett (11), McCollum and Adams (12), Shohl and co-workers (20) (21), Hess and others (7), Holmes and Pigott (8), all go to show that either a disproportion of calcium to phosphorus in the diet or a lack of sufficient vitamin D results in pro- ducing a bone containing less ash than normal. However, there is lit- tle variation in the ratio of calcium to phosphorus in the ash of the bone from that of normal, which is approximately 1: .50. METHODS AND PROCEDURE Rats about twenty-three days old and of a weight of thirty-seven to forty grams were placed in separate cages and given a ration made up of the following: 20 parts of casein, 10 parts of dried yeast, 67.5 parts of starch, 1 part of sodium chloride, and 1.5 parts of a salt mixture made up of the following: 68 parts of potassium sulphate, 444 parts of potassium chloride, 96 parts of magnesium carbonate, 62 parts of sodium carbonate, 3'7 parts of potassium carbonate, 252 parts of citric acid, 24.- parts of iron citrate, 0.32 part of manganese sulphate, 1.0 part of sodium fluoride, 0.08 part of potassium iodide, 0.08 part of potas- sium alum. The percentage of lime (CaO) in this ration was found ' by analysis to be 0.250 (Ca: 0.178 per cent) and of phosphoric acid (P205) 0.670 (P I 0.292 per cent). Different levels of lime and phos- ‘phoric acid were obtained in the rations by substituting in place of starch, calcium carbonate or disodium phosphate. Each rat was given plenty of distilled water and three or four drops of cod liver oil (vitamin-tested) per day. Records were kept of the amount of food consumed and the weight gained each week. At the end of eight weeks the rats were killed with chloroform and the femurs dissected out. The procedure used for determination of the ash in the femurs was b-ased upon that of Dutcher and Honeywell (2) (9). The femurs were cleaned free of flesh and connecting tissues and then weighed. After being dried at a temperature of 70-90° C to constant weight they were weighed again. The samples were then extracted for twenty-four hours 8 BULLETIN NO. 441, TEXAS AGRICULTURAL EXPERIMENT STATION with ninety-five per cent alcohol and then twenty-four hours With ether if after Which they were dried and weighed. The ash was prepared fromi this dried fat-free bone by combustion in an electric furnace, and Weighed. The ash Was dissolved in 1O cc. of dilute hydrochloric acid (1:1) and the volume made up in 100 cc. An aliquot was taken and, the phosphoric acid (P205) determined by precipitation with ammonium“ molybdate and titration of the precipitate with potassium hydroxide and nitric acid. The lime was determined in another aliquot of the solution by pre- cipitating it with ammonium oxalate in the presence of acetic acid. T The precipitate after being burned was heated With ammonium sulfate and the calcium Was Wreighed as calcium sulfate, and calculated to cal- cium oxide. The Terms Lime and "Phosphorus In making the calculations and ratios the calcium has been expressed as lime (CaO) and the phosphorus as phosphoric acid (P205). While i this is not the custom in animal physiology, the terms lime and phos- . phoric acid are ordinarily used in agriculture, instead of calcium and phosphorus. The analysis of fertilizer is always, by legal requirement, - expressed in terms of phosphoric acid. It Was thought that the results might be better interpreted in relation to feeding experiments, Where it is customary to use agricultural analyses. REQUIREMENTS FOR NORMAL GROWTH The gain in weight of each individual rat is presented in Table 1, along with the percentages of lime and phosphoric acid in the ration. This table shows some variations in the Weights of the rats on the same ration. There are several factors that aifect the health and growth of rats, such as the inherent vitality and ability of the animal to grow, and this may account for the variation. In Table 2 there is found the weights of femurs and other data on the bones along With the lime and phosphoric acid in the rations fed. The percentages of the femur and of the femur ash in the rat are given. It is thought that these figures might have some relation to the value of the rations. In Table 3 some of detailed results in Tables 1 and 2 are averaged and arranged in four groups, according to the Weights of the rats. 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Iv. ohoo 89.. hoohm SE. ohho m hhgm S; one Z2 Q N3. hoho S? hRm. 25w. $5 m ohJm 9.: oo.o ohv. film ovov. h»? ooow 3S m ohJm ootm oo.o .2 S» $5.2m w55m 503m.“ 53 3am ~53 SQ 53 Eon m53m 3:52 w53m 3:58 E 5253 53 Hon o3 E 3:53 .3558 ovoomb 3:53 o noamnowU E Eon aofimu 3on5sz “mm 55am: E =3 S fi< LEM 3cm E $3 22.5 28.3 E BEA $2.3 Eon uioanwonn 3 v5: oo @033 #50555 at? 530.5 33 o0 Emma 558 15w 558 .3 359w? .h 01mm m. 11 GROWTH AND DEVELOPMENT OF BONES OF WHITE RATS XON. . - . . . . . . . . ~ . . . . - . . . . . . . - - - . . - . . . - - . . - - . . . . . . - - . . . - . . . - . . - - . N NQm. 2w. 8.2 w“ QQNQ ooo omQ ooQwQ N oQQ. mom. woov 3 moQQ $. mN. woNQ Q» wt. 8a. 8.3 QQNQ Qo. Q m». on. omQQ N wi. oNQX 3S om 2w Q omQ oo.N m“. Q m wQN. wow. Qmtvm mNQ om. Q mm. omQ om. Q . . . . . . . . . . . . . . . . . . . . . . . ......._NQ|w.\l .- u v - - - . - . - - . - . .- -|- 0-. nn-u-u- . . . . ~ ~ . . . - . . . . . . . . . . . . N m2. wow. 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IonQlooQ Esau :5 Eva Ea Eco E5 Eve 3Q 3.3 i3 E i3 E .553 wEEm c033 c033 E3 “on 205E E Qo Ems his»: wvoombxo wow‘. Q0 E nQHwU E Eva c055 woumuomU mEmEU 55.2.4 oZ gsEohQ QECQ E ~14 onwmwk/ oSwwQ 012E E v55 Q0 oGwMQ $25 Ema v55 o0 iQwBB o5 E E3 3E we 3:395 Qo 5.3a“ E Eon uiosamosn E5 0E: Qo coifiofl m. ES. 12 BULLETIN NO. 441, TEXAS AGRICULTURAL EXPERIMENT STATION average percentage of ash in the femurs was 58.85. The bones seemedi to be normal in content of ash and i11 percentage of ash in the femur, though the ash in the femur from the rat receiving 1.0 per cent each, of lime and phosphoric acid Was above normal (60.79 per cent). The three rations with a lime-to-phosphoric-acid ration of 1: 1 gave“, good growth and a normal percentage of femur ash. These rations contained 1.0 to 3.0 per cent of each mineral. The ration containing; 0.75 per cent of lime and 1.0 per cent of phosphoric acid also gave nor-f, mal growth and bones, as did the rations containing per cent and 6.0 per cent of lime With 1.5 per cent of phosphoric acid and ratios of l5 1: 0.33 and 1: 0.25. EFFECT OF DEVIATION FROM REQUIREMENTS FOR NORMAL GROWTH The weights of the rats in the second group of Table 3 are below 1 normal, though the deficiency is small in some cases. The ash in the femurs, while a little less than in the first group, could be termed nor- p. mal and the same could be said regarding the ash content of the femurs. This indicates that slightly unfavorable percentages of lime and phos- v phosoric acid may not affect the structure or quantity of the bones, ~ while they may decrease the growth of the animal. The rats on the ration which contained 0.7 5 per cent of lime and 0.75 per cent of phosphoric acid (Group 2) weighed 144 grams, while the rats which received the same amount of lime and 1.0 per cent of phosphoric acid made normal growth (Group 1). This shows that less than 1.0 per cent of phosphoric acid or less than 0.75 per cent of lime is below ‘the minimum for good growth, since this ratio of 1: 1. has proven favorable for good growth and normal quantity of ash in the femurs when larger amounts of the minerals are fed. Either decreasing the lime from 1.5 to 1.0 per cent in the ration when 1.5 per cent of phosphoric acid was present or increasing it to 2.5 per cent, decreased slightly the gain in weight of the rats. The final weights of the rats were 145 a11d 146 grams compared with 161 it grams of the other rations. The graph (Fig. 1) brings out the relation between the percentage of lime and phosphoric acid in the ration and the average Weights at- 5’ tained by the rats. The average normal weights are in circles. Three of the normal weights are on rations with the ratio of lime to phos- f phoric acid of 1: 1, and percentages of 1, 1.5, and 3. One of the nor- mal weights is at the percentage of 0.75: 1, and the two others at 4.5: 1.5 . and 6: 1.5. The graphs showvery clearly that small increases or de- creases in either lime or phosphoric acid from the favorable amounts decrease the weights of the rats. In the third group in Table 3 are shown the rats which) attained a final average weight of 136 to 140 grams. The rats on the ration con- K taining 1.5 per cent of lime and 2.0 to 3.0 per cent of phosphoric acid v made practically the same growth (138 and 110 grams), while the per- GROWTH‘ AND DEVELOPMENT OF BONES OF WHITE RATS 13 itages of ash in the femurs were the same. The ration containing Y. per cent of lime and 1.50 per cent of phosphoric acid produced rats a: a final weight of 138 grams and with bones which contained 57.7 cent of ash, while the ration which contained only 0.25 per cent of i and 0.75 per cent of phosphoric acid produced rats With a final ht of 136 grams and bones which contained only 51.3 per cent of m» >...-...iv.i.e.i¢=.~i-ez..;w.,siwpa=immi i “o ‘ ,l’l'l ' R "IN _ z S‘ 3Q "AN 1 s‘ x .» E n52 l; x ;_e"' 14s X 99 X 7‘ X X X X nzs n54 @ :37 :47 :39 7s X H6 I44 — I23 x I70 x 3a eggs - I Pcncammca or Pnospuonuc ACID p i é 5 4 5 é igure 1.—Re1ation of weight of rats to percentages of lime and of phosphoric acid in the ration. p It is prob-able that the low percentage of lime and phosphoric in the ration was the important factor in producing a bone with a percentage of ash. It is probable that in the ration containing ijper cent of lime and 0.75 of phosphoric acid, both lime and phos- 'ic acid are below the minimum, while in the ration containing 0.50 fcent of lime and 1.50 per cent of phosphoric acid, lime only is be- 14 BULLETIN NO. 441, TEXAS AGRICULTURAL EXPERIMENT STATION l 10W the minimum required for good growth and proper bone develops ment. g AMOUNTSOF MINERALS WHICH ARE DISTINCTLY UN FAVORABLES In the last group, Table 3, are placed all rats which attained a final weight of less than 135 grams. The rats on the ration containing 2.0. per cent of lime and 1.5 per cent of phosphoric acid, attained a final Weight of only 99 grams and had bones which contained only 51.7 per cent of ash. Thus, the addition of 0.5 per cent of lime to a ration con- taining 1.5 per cent each of lime and phosphoric acid resulted in a seri- ous depression in growth accompanied by a deficiency in the ash of the" bone. The graph (Fig. 1) brings this point out very clearly. This sudden decrease in weight caused by a slight increase in lime might seem to be an error, but it occurred with both rats, and is confirmed by the low percentage of ash in the bones: _ The ration containing 0.25 per cent of lime and 0.67 per cent of phosphoric acid gave rats with a final weight of 83 grams. These rats had an ash content of the bones of only 41.0 per cent. These data can be compared with the data on the ration containing 0.25 per cent of lime and 0.75 per cent of phosphoric acid, which produced rats weigh- ing 136 grams with 51.3 per cent femur ash. Both of these rations are deficient in the minerals. The ration containing 1.5 per cent of lime and 6.0 per cent of phos- phoric acid produced rats of a Weight of 78 grams, while the ash per- centage in the femurs was 57.9 per cent, which is what could be called about normal. The percentage of the ash in the bones did not give any indication of the unfavorable ratio between the lime and phosphoric acid in the ration as shown by the growth. The lack of proper growth was probably caused by the large quantity of phosphoric acid supplied to the ration by the disodium phosphate. THE RELATION OF LIME AND PHOSPHORIC ACID TO THE PER- CENTAGE OF FEMUR AND OF FEMUR ASH IN RATS The average percentages of dried femurs and of femur ash in the ‘ rats are given in Table 3 along with the lime and phosphoric acid in the ration. l The ration which had 1.0 per cent each of lime and phosphoric acid f gave 0.407 per cent of extracted femur and 0.229 of femur ash in the _ rat. With 1.5 per cent each of lime and phosphoric acid in the ration, the rats contained 0.441 per cent of femur and 0.238 per cent of femur I ash. These appear to be above the normal percentages. The rats on a ration containing 0.50 per cent of lime and 0.75 per ' cent of phosphoric acid had only 0.348 per cent of femurs and 0.176 . per cent of femur ash. The ration containing 0.25 per cent of lime a and 0.67 per cent of phosphoric acid produced rats which had 0.333 l per cent of femurs and 0.116 per cent of femur ash, although the aver- ‘ l GROWTH AND DEVELOPMENT OF BONES OF WHITE RATS 15 age weight of the rats was only 82.5 grams. These low percentages of lime and phosphoric acid in the ration did not produce animals with a proper ratio of bone to the total weight of the rat. y The ration containing 1.5 per cent of lime and 6.00 per cent of phos- giphoric acid grew rats which had 0.619 per cent of femurs and 0.312 per cent of femur ash. If about 0.420 per cent of dried femur in the ‘rat and about 0.230 per cent of femur ash is taken as normal, then the rats on this ration had too great a quantity of bone in proportion to the rest of the body. All the other percentages of femur and of femur ash are fairly con- stant, not varying widely from 0.420 per cent of dried femur and 0.230 _rper cent of femur ash. Of course, there are many factors that affect athe growth of rats as well as the bone content. i‘ RATIO OF LIME TO PHOSPHORIC ACID IN THE FEMUR ASH l1’- In Table 4 the percentages of lime and of phosphoric acid in a few rmples of ash of the femurs are given along with the ration of lime to hosphoric acid. Although the percentages of lime and of phosphoric Encid in the bone are not constant, the ratio does not range far from 1:12, which compares favorably with the results of Holmes and Pigott (8) and McCollum and Adams (12). There is some variation, out the variation of samples from rates on the same ration is almost as ; eat as samples from rats on different rations. Table 4. Relation of lime and phosphoric acid in the ration to the ratio of lime to phosphoric acid in femurs. Ratio of Ratio of Lime in Phosphoric Lime in Phosphoric lime to_ Ca0zPz0s ration acid in femur ash acid 1n phosphoric in ration per cent ration per cent femur ash acid in per cent" per cent femur ash 1: .5 1.50 .75 53.37 42 3'7 1.26 1:1.0 1.50 1.50 49.78 3 5 1.25 1:1.0 1.50 1.50 49.30 40 28 1.22 1:1.0 .75 .75 47.56 41 44 1.12 ' 1:1.0 75 .75 51.83 41 03 1.23 1:1.50 1 00 1.50 44.03 39 26 1.12 1:1.50 1 00 1.50 41.83 37 98 1.10 1:1.50 50 .75 56.01 45 O0 1.24 1:1.50 5O .75 53.51 44 54 1.20 1:3.0 50 1.50 52.27 41 60 1.25 1:3.0 5O 1.50 53.05 41 74 1.27 123.0 2a .75 49.40 33 1.17 1:3.0 25 .75 53.34 44 71 1.19 There seems to be no relation between the_ratio of lime to phosphoric jacid in the ration fed and the ratio of lime to phosphoric acid in the x DES. ‘ ACKNOWLEDGMENT l» The writer wishes to acknowledge the kind assistance, criticisms, and “~ 'mely advice of Doctor G. S. Fraps, under whose supervision this study was carried out. 16 BULLETIN NO. 441, TEXAS AGRICULTURAL EXPERIMENT STATION Special thanks and appreciation are extended to Doctor C. C. Whose encouragement enabled me to perform this Work, and to 1 Alice Ward, Who aided in preparing the manuscript. SUMMARY ,_ Forty-nine rats in three series Were fed rations containing fou, different ratios of lime to phosphoric acid. 1- The percentages of lime in the rations ranged from 0.25 per ce V. 6.00 per cent, while the percentages of phosphoric acid ranged q 0.67 per cent to 6.00 per cent. Ratios of lime to phosphoric acid vats from 1: .25 to 1:4.0. This is equivalent to a range of calcium: phosphorus ratio from 1: .15 to 1:2.44. Cod liver oil was fed to? ~ rats. Rations containing 1.0, 1.5, and 3.0 per cent of lime gave growth when the ratio to phosphoric acid was 1: 1. Rations cont_ ing 4.5 or 6.0 per cent of lime and 1.5 per cent of phosphoric ac also produced rats with a normal Weight or above, and With femurs? a normal ash content. The minimum percentages which gave no p growth was found to be 0.75 per cent for lime and 1.0 per cent phosphoric acid. Rations containing 0.25 per cent of lime and per cent of phosphoric acid produced rats which had only 51.32 per q of ash in the femur, and rations containing 0.67 per cent of phospho" acid With 0.25 per cent of lime produced rats with only 40.98 perc of ash in the femur. The normal percentage of ash in femurs about 58.00 per cent. A ration containing 1.5 per cent each of l" and phosphoric acid gave normal growth, but When the percentage“ lime Was raised to 2.0, the growth was decidedly decreased and the a centage of ash in the bones Was less than normal. Percentages of lime or of phosphoric acid slightly lower than p most favorable percentages, in some cases decreased the growth, W the ash content of the femurs in some cases remained practically L mal. Small increases in either lime or phosphoric acid from the fav’, able amounts in some cases decreased the growth, especially when ration was low in minerals. '1 When the amounts of lime or phosphoric acid fed were below ' minimum, the growth was decreased and the femur ash was lower normal. A percentage of 6.0 per cent of phosphoric acid in a ration with I‘. per cent of lime caused a decided decrease in growth of the rats, b, the percentages of ash in the femur was practically normal. The pe7 centage of femurs in these rats was above normal. . - No relation was found between the ratio of lime to phosphoric aci in the ration and the ratio of lime to phosphoric acid in the ash of I‘, femur. ' GROWTH AND DEVELOPMENT OF BONES OF WHITE RATS 17 BHHJOGRAPHY 1. Bennett, Helen, and Shohl, Alfred T., 1930. Bickets in rats. The alteration of calcium and phosphorus. Metabolism of normal and rachitic rats produced by irradiated ergosterol. Jour. Biol. Chem. 86:246. 2. Dutcher, B. A., Creighton, Mattie and Bothrock, Henry A., 1925. Vitamin studies. Inorganic blood phosphorus and bone ash in rats fed on normal rachitic and irradiated rachitic diets. Jour. Biol. Chem. 66:401. 3. Dutcher, B. A., Ely, J. 0., and Honeywell, H. E., 1927. The calcifying potency of cod liver oil. Penn. Sta. Bul. 213, 4. 4. Grieves, C. l, 1922. Jour. Amer. Med. Assoc. 79:1568. 5. Hart, E. B., and McCollum, E. V, 1914. Influence on growth of rations restricted to the corn or Wheat grain. Jour. Biol. Chem. 19:373. 6. Hess, A. E., McCann, G. F., and Pappenheimer, A. M., 1921. Experimental rickets in rats. The failure of rats to develop rickets on a diet deficient in vitamin A. J our. Biol. Chem. 47 2395. 7. Hess, Alfred F, “leinstock, Mildred, Riviken, H., and Gross, l, 1931. The development and cure of rickets and the inorganic phos— phorus concentration of the blood. J our. Biol. Chem. 87 :38. 8. Holmes, Arthur D., and Pigott, Madeliene G., 1931. Effect of cod liver oil on calcium metabolism of young chicks. Ind. and Eng. Chem. 23:190. 9. Honeywell, Hannah E., Dutcher, B. A., and Dahle, Chester D., 1931. Vitamin studies. Ossifying potency of raw and evaporated milk. Jour. of Nutrition 2:252. 10. Karelitz, S., and Shohl, A. T., 1927. Bickets in rats. Metabo- lism studies on high calcium low phosphorus diets. J our. Biol. Chem. 73:665. 11. McCann, Gertrude F“ and Barnett, Marion, 1922. Experi- mental rickets in rats. The distribution of phosphorus and calcium be- tween the skeleton and soft parts of rats and rachitic and non-rachitic diets. Jour. Biol. Chem. 54:203. 12. McCollum, E. V, and Adams, G., 1928. Biological assay of cod liver oil. Jour. Biol. Chem. 78:512. ' 13. McCollum, E. T, Simmons, Nina, and Becker, J. Ernestine, and Shipley, P. G., 1922. Experimental rickets. The production of rickets in the rat by diets consisting essentially of purified diets. Jour. Biol. Chem. 54:249. - 14. McCollum, E. Y, Simmons, Nina, and Shipley, P. G., and Park, E. A., 1921. Studies in experimental rickets. The production of rickets by diets low in phosphorus and fat soluble A. Jour. Biol. Chenr 48:507 15. Moritz, Allan R, and Krenz, Carl, 1930. Experimental rickets in rabbits. Jour. Xutrition 2:257. 18 BULLETIN NO. 441, TEXAS AGRICULTURAL EXPERIMENT STATION 16. Nelson, Mabel P., Irwin, Margaret, and Peet, Louise J ., 193 Meat in nutrition. Jour. Nutrition 2:52. » ' 17. Schneider, Josef, 1930. Chem. Listy 24:181. p 18. Sherman, H. C., and Stiebeling, H. K., 1930. The relation 0 vitamin D to deposition of calcium in bone. Soc. Exp. Biol. and Me: Proc. 27:663. ' 19. Simmonds, Nina, 1924. Amer. Jour. Hyg. Supplement 1:23. 20. Shohl, A. T., and Karelitz, S., 1927. Rickets in rats. Meta‘ olism studies on high calcium low phosphorus diets. J our. Biol. Chem,’ 73:665. ' . 21. Shohl, A. T., Bennett, Helen, and Weed, Katharine L., 1928. Rickets in rats. Metabolism of calcium and phosphorus of rats fed‘ upon non-ricketogenic diets. J our. Biol. Chem. 79:257. ‘ 22. Shohl, A. T., Bennett, Helen B., and Weed, Katharine L., 1927;. Effect of phosphate added to the diet of non-rachitic rats. Proceed. of? Soc. Exp. Med. and Biol. 25:669. 23. Steenbock, H., Bethke, B. 1L, and Nelson, Mariana T., 1923. Fat soluble vitamines. XV calcium and phosphorus relation to growth and composition of blood and bone with varying vitamin intake. J our. . Biol. Chem. 58:71.. ‘ 24. Steenbock, H., Hart, E. B., Sell, M. T., and Jones, J. H., 1923. The availability of calcium salts. J our. Biol. Chem. 56:375. _ 25. Toverud, Guttarm, 1923. The influence of diet on teeth and bones. Jour. Biol. Chem. 58:583. ?