TH . STUDIES WITH RATS ON THE AVAILABILITY OF VARIOUS CALCIUM SALTS Thesis for the Degree of M 5. Keith D. Crane 1936 .. '0 ‘ \ .I\;.'v* ._ .: ~. ' 1 ‘ . ‘1‘ ,I"..|." .‘ . .51)! y... . _ , ‘ v . - u‘ _ ' U {91" o _ ', " l t . I “f: "V ,v -.‘ I' .‘ ‘ _' .- -_' ~'-7‘| "’ I.‘.' .I_I I .- : "‘.1.“:'.'.:-" "I ‘1 ‘1'. - 1"“ .‘. . ‘1 it ‘2" '. .. ‘ ... . ‘. Irhfl.‘ . ' ' _ ' 1' -~"_ '.'I'7... . “I’wf ..’.' v'_' “ . , N . , ‘ "' ,.' . - _\ ‘v ‘ , . ”7.40m;- J. ,1 . .‘ '.J I. * w ..v",.' “1“ ‘13:: ‘5‘ ' ° .3‘: '1.tfi:,‘f“ ‘4 Nuk‘. l . 6"" at ' b. ' AB? ' . '.‘ '. _.~.~“Jf ‘c‘ ‘fi‘lt, r. 1" “- STUDIES W 1TH RATS 01‘] 1‘13 AVAILABILITY OF VARIOUS CALCIT'IL SALTS SQUDIES VITH RATS ON Thfi AVAILABILITY OF 'ARIOUS CJLCIUE 3: TS Submitted to the Faculty of the Llichigan State College in partial fulfillment of the require— ments for the degree of Easter of Science. BY ‘ ,\ 1‘ 1 x\ ‘j .‘ : i \.' EL TH D. CRASE 1936 dCKHOULEDGMEflT -—— The author of this thesis wishes to acknow- ledge the assistance of Dr. Car1.A. Hoppert, Associate Professor of Chemistry, for his aid in directing the experiment, and his kindly criticism of the manuscript. NTRODUCTION The study of calcium metabolism has received a great deal of attention both in human and animal experimentation. Probably this has been due to the fact that many of the natural foods and food mixtures are deficient in this element, so that particularly in animal feeding, use has been made of various inorganic salts to enrich the diet in this element. In 1918, (1) Osborn and.ten— del showed that diets low in calcium have a marked inhibitory action on growth and bone development in the rat. They remedied this by feeding calcium.in the form of the inorganic salt, calcium carbonate. In 1921 (2) Forbes and coaworkers at Ohio showed that the addition of calcium salts was advantageous in feeding swine. They fed rations supplemented.with (l) pulverized limestone, (2) precipitated bone flour, (5) raw rock phosphate floats, (4) special steamed bone flour, 1d (5) calcium carbonate. They state that in feeding a basal cereal ration alone the prominent characteristics were lack of calcium re- tention, sub-normal retention of magnesium and phosphorus, and high acidity and ammonium salts in the urine. In the animals receiving a supplement the efficiency to cause increased retention of calcium, as indicated by the percentage of calcium retained, was manifested by four of the supplements namely, pulverized limestone, precipitated bone flour, steamed bone flour, and calcium carbonate which differed but little, while the raw rock phosphate was dedidedly the least ef— ficient. According to the authors, the most efficient of the mineral supplements, probably because the most readily soluble, were the cal— cium carbonate and precipitated bone flour. In 1922 (3) Hart, Steen— AK -2... book, HOppert, and Bethke supplemented various stock rations with bone meal and found that it greatly reduced the negative calcium and phosphorus balances in dairy cattle although it did not estab- lish positive balances. These studies and many others of a similar nature indicate that calcium salts of different kinds may generally serve to supply the needs for this element. Relatively little work has been done to show whether or not there may be a difference in availability of the various calcium salts. This question has recently been revived by the claims of the Bay Chem- ical Company that calcium in the form of di calcium phosphate is super- ior to other calcium salts. The above claims are contrary to the results reported by (4) Steen— book and couworkers who showed that on a synthetic diet practically de- void of calcium this element could be supplied by the feeding of calcium carbonate, calcium lactate, calcium sulfate, tri calcium phosphate or calcium silicate. The results of these experiments show conclusively that when calcium is fed in liberal amounts to the rat, the latter can cover its requirements with any of the well known calcium salts. So far as indicated there was no difference in their relative availability. These investigators stated however that the question of comparative availability is an open one because it is apparent that even the slight- est solubility of the most insoluble calcium salt might still allow the solution of enough calcium if the irregation of the intestinal contents - 3 - with digestive secretions was sufficiently large. In the above experiments growth alone was taken as a criterion of the availability of calcium in its various fomns. Since no spec— ific data was presented as to the ash content of the bones and since there is a paucity of information on this subject, it seemed desirable to add to the studies on availability with the idea of substantiating in a more decisive manner the conclusions of the above investigators. Another factor that must be taken into consideration in a study of the utilization of calcium is the effect that other constituents may exert. hany authorities feel that there exists a definite relation— ship between calcium and certain other elements of the diet. (5) Hart and Steenbock say that the interrelations existing between mineral el- ements are important factors for consideration in studying the specific role of a mineral element in animal nutrition. (6) Hoag and Palmer state that they believe a more or less balanced condition exists between the calcium, phosphorus, and magnesium salts of the ration. Their mineral balance data points definitely to the fact that magnesium may be a dis— turbing factor, interferring with normal bone development. The selection therefore of the constituents of a diet for compar— ing the availability of various calcium campounds is an important matter. The conclusion arrived hy (7) Hawk in comparing di calcium phosphate with tri calcium phosphate and calcium carbonate are believed to be misleading because of the fact that the basal diet used in his experiments was pat— terned after the Steenbock rachitogenic ration. This ration while ad- mirably suited for the production of rickets is believed to be unsuited L‘ »J h a . ' a C . , . . \ 4 \—r -‘- \z -4... for a comparison of the availability of calcium salts at the levels selected by Hawk because the ration is characterized by a fairly low phosphorus content. For this reason the two percent level which was used in Hawk's experiments caused calcium carbonate to show up to a disadvantage when compared.with di calcium phosphate. This was the basis for Hawk's claim that the calcium in the form of di calcium phosphate is utilized much more efficiently than is the calcium from equivalent quantities of calcium supplied in the form of calcium car— bonate, tri calcium phosphate and calcium citrate. It seemed desir— able to make comparisons using various levels of the salts with the ration used by Hawk, as well as with other practical rations. The purpose of this thesis, then, was to make a comparison of the availability of various calcium salts in several types of rations, using not only growth of animal but also the ash content of the femurs and hum— eri as criteria of the availability. _ 5 - - PROCEEDUEE — Albino and piebald rats were used in all of the experiments. They were about twenty—eight days of age, from fifty to sixty—five grams in weight. The animals were placed in round metal one piece cages with raised.wire mesh bottoms to prevent access to excreta. The animals were weighed once a week and the weights recorded. The experiments were of six weeks duration, this being the period of most rapid growth and bone development. The rats were then killed and the humeri and femurs were removed. The flesh was removed from the bones as completely as possible. The bones were extracted in a Soxhlet extraction tube for forty-eight hours with ninety—five percent ethyl alcohol. After extraction the bones were dried for one hour at 100° C in an electric furnace. They were then weighed and ashed in muffle furnace for two hours and reaweighed. The percentage of ash was determined by difference. -6... E} 33 KLflT WORK SJRIBS I The rations used in this series were composed of the following: Wheat Gluten 20% Sodium Chloride 1/"; Calcium Salts Equivalent to .5,3 CaCOz Cornmeal to make 1003 The actual amounts of calcium salts fed per hundred grams of ration were as follows: Calcium Carbonate .Efi Gm Dicapho # 1.54 Gm hono Calcium Phosphate 1.36 " Tri Calcium Phosphate .52 " Calcium Sulfate .68 " Calcium Lactate 1.54 " Calcium Fluoride .39 " #Dicapho is the trade name for di calcium phosphate, and is manufactured by the Bay Chemical Company. The basal ration in series I was a modification of Steenbock's rachitogenic ration with the calcium carbonate omitted and supplemented with the varous calcium salts at levels equivalent to one half per cent calcium carbonate. The salts fed were the Baker's and.herck's C.P. salts with the exception of the di calcium phosphate which was a commercial product known as "dicapho" and manufactured by the Bay Chemical Company. TABLE I Heights Ash Analysis Group No. Salt Sup-Initial Final Gain Humeri Femurs 39,. flat; plement l 6 Basal 67 94 27 35.61 41.05 Calcium 2 6 Carbonate 63 125 62 47.12 49.61 3 6 Dicapho 62 105 43 46.54 49.61 Kono Calcium 4 6 Phosphate 62 95 33 45052 47077 Tri Calcium 5 4 Phosphate 62 95 33 45.91 49019 Calcium 6 6 Sulfate 64 111 47 47.65 49.55 Calcium 7 4 Lactate 63 103 40 47.61 49.35 Calcium 8 4 Fluoride 68 73 4 37.76 41.45 An examination of table I indicates that tie control group receiving the basal ration containing no calcium supplement made poor growth and showed deficient skeletal development. any im- provement over the basal ration would have to be due to the cal- cium salt added and would be a measure of the availability of that salt. The results in series I show that at one half percent levels there was no appreciable difference in the ash content of the bones of the rats receiving the various calcium salts except in the case of calcium fluoride. The animals receiving the calcium fluoride supplement showed very little growth and extremely poor bone de— velopment, the ash content of the bones being quite comparable to that of the basal group. The failure of the rats to utilize the calcium from the calcium fluoride is probably explained on the basis of the relative insolubility of this compound. This conclusion is all the more plausible because of the absence of the effects on the teeth which an equivalent amount of fluorine in a more soluble form would have caused. —9- 3‘ BRIZBST' WORK SERIES 2 The rations used in this series were composed of the following: Wheat Gluten 20,3 Sodium Chloride 1;: Calcium Salt Equivalent to 1% CaC03 Corn real to llake loo/Z The actual amounts of calcium salts fed per hundred grams of ration were as follows: Calcium Carbonate l Gm Dicapho 3.08 Gm Mono Calcium PhOSphate 2.52 " Tri Calcium Phosphate 1.04 " Calcium Lactate 3.08 ” Calcium Fluoride .78 " The basal ration in this series was the sane as that used in the preceding one. Here again the calcium.carbonate was omit— ted and supplemented with the various calcium salts at levels equivalent to one percent calcium carbonate. The salts fed were the same as in series one. TABLE 2 Weights Ash Analysis Group No. Salt §up~ Initial Final Gain Humeri Femurg go, Bats plement 9 6 Basal 59 83 24 35.87 41.66 Calcium 10 6 Carbonate 64 132 68 46.45 50.57 11 6 Dicapho 62 119 57 50.99 52.56 Mono Calcium 12 4 Phosphate 55 9O 35 48.46 50.77 Tri Calcium 13 4 Phosphate 61 120 59 49075 53002 Calcium 14 6 Lactate 60 121 61 49.44 2.30 Calcium 15 3 Fluoride 60 95 35 37.33 39.08 The animals in the control group were comparable to those in the preceding series, the growth, appearance, and ash content of the bones being very similar. The results in series two again showed no striking differences in the ash content of the bones although the values in the calcium carbonate group were slightly lower than those obtained.with the other salts, excluding calcium fluoride. When the results are considered with those of a later series in Which calcium carbonate was fed at a two percent level it is apparent that when the carbonate is fed at levels higher than one percent in this particular ration there is a gradual tendflncy for poorer bone development. This is not to be interpreted as indicating an inferiority in the availability but rather that a disturbance is caused by excessive intakes of calcium -11- carbonate in a diet low in phosphorus. The calcium fluoride group again showed poor growth and poor bone deve10pment. EKPSRIEJKTAL WORK SERIES 3 The rations used in this series were composed of the following: Wheat Gluten 20% Sodium Chloride 1% Calcium Salts Equivalent to .5, 1, & 2% CaCOs Corn 0:1 5% Corn.Eeal to Rake 100% The actual amounts of calcium salts fed per one hundred grams of ration were as follows: Calcium Carbonate .5 Gm Calcium Carbonate 1 " Calcium Carbonate 2 " Dicapho Equiv. to .5% 03003 1.54 " Dicapho Equiv. to 1% CaCOS 3.08 " Dicapho Equiv. to 2% Ca003 6.16 n The above series was the first of three carried out for a direct comparison between calcium carbonate and d1 calcium phos- phate (dicapho) in various rations. The basal ration was the same as the one used in the preceding series except that five percent corn oil replaced the same percentage of corn meal. This was done with the idea of improving the palatibility and also to pre- vent a sifting out of the salts. The calcium carbonate was fed at levels of one half, one, and two percent and compared with equivalent amounts of dicapho. -13— TABLE 8 ‘."T'IGHTS ass ANALYSIS Group No, Salt Sup: Initial Final Gain Humeri .Egmggg No, Eats Element 50 5 Basal 57 77 :30 59.49 39.58 :3ng Calcium 31 4 Carbonate 61 112 51 50.37 49.37 1,2: Calcium :52 4 Carbonate 55 105 50 45.01 48.62 2,3 Calcium 33 4 Carbonate 55 89 34 35.92 40.47 54 4 1.543; dicapho 58 111 55 53.98 52.54 55 4 5.085% dicapho 55 10:5 » 48 52.44 55.88 55 4 5.1875 dicapho 55 105 50 57.02 57.75 The animals receiving the dicapho showed slightly better bone development than the calcium carbonate animals which can no doubt be accounted for by the influence of the phosphorus introduced by the dicapho. This is especially apparent at the two percent levels. The rats at the two percent level of calcium carbonate showed skeletal development comparable to that of the control group receiving no sup— plement. It is obvious from these results that the inclusion of two percent calcium carbonate in this diet constitutes an excessive use of the salt, whereas in the case of dicapho the two percent level is perhaps Optimal with this particular ration. .— 14 .- EX?SRILEKTAL HORK 353135 IV BASAL RATIOS: 11 1.11131) 3413‘! 31.2101! Yellow Com 25;; Alfalfa 20% Rolled Oats 25$ Bran 15% Cottonseed meal 4% Linseed meal 53 Fat (crisco) 5% Sodium Chloride 1% Salt supplement 733', 1 and 2;; 5315523 ASH AKaLYSIS Group No, Salt sun— Initial Final Gain numeri Femurs Ho: Rats element 57 4 55351 50 148 98 52.52 54.05 “3373 Calcium 38 4 Carbonate 54 144 90 53.04 57.11 1% Calcium 59 4 Carbonate 55 155 100 54.07 55.94 23 Calcium 40 4 Carbonate 51 141 90 50.74 52.23 41 4 1.54% dicapho 55 159 85 54.11 57.92 42 4 5.08% dicapho 50 140 90 55.55 55.48 45 5 5.15% dicapho 52 157 115 52.59 55.91 ...15.. In this series a mixed dairy ration was used, the composition of which is given above. This ration was much more varied than the one used in series III and contained some feeds that are good sources of calcium and some that are good sources of phOSphorus. It might be expected therefore that the addition of calcium salts would prob— ably not have as much effect as in series III. The results are in accord with this idea, for the basal ration showed growth and bone development that is comparable with the supplemented rations. In the case of calcium carbonate the two percent level again constituted an excessive amount of the salt for poorer growth and bone development was obtained than on the basal ration. Although the ash content values on the one—half and one percent dicapho were slightly higher than on the other groups the differences were too slight to be considered significant. So far as the rat is concerned it is questionable whether a ration of this type is improved at all by a supplerent of either calcium or phosphorus. These results are in accord with the work of (8) Reed and Huffman that a balanced ration probably needs no inorganic supplement. Corn teal Middlings Powdered _ 15 _ BKFERIXJETAL'HQRK 42.11.45 v BASAL RAE? ION (wheat) Buttermilk Heat Scraps Fat (cris co) Sodium Chloride Salt Supp lement %, 1 and 2% 58% 20% 10% 5;”. 5% 175 In this series calcium carbonate was compared with dicapho using the Ohio Poultry ration. The bone meal was omitted and calcium carbonate and dicapho were supplemented at the same levels as in the preceding two series. 5518523 155 courses .Qassa. Ear. .éali_asa: 1511151 .2185; .QEEE. 2555551 gaeeeaa 52.. 45551 :8555512 44 4 Basal 55 158 85 55.99 54.09 %fl Calcium 45 4 Carbonate 54 151 97 55.25 55.55 1% Calcium 46 4 Carbonate 58 173 115 54.89 54.59 2; Calcium 47 4 Carbonate 55 179 114 55.55 55.02 48 4 1.54% Dicapho 57 144 87 55.24 55.54 49 4 5.08% Dicapho 59 157 108 57.74 58.20 50 4 5.15% Dicapho 54 175 121 57.51 58.04 _ 17 _ The results obtained, as given in the table, sow that the addition of various amounts of calcium carbonate and dicapho slightly incleased the growth of the rats and the ash content of the bones. The groups receiving the dicapho at the higher levels seemed to be slightly superior to those receiving the cal- cium carbonate. However, the difference between the two is so slight that it does not warrant the conclusion that the dicapho is far superior to the calcium carbonate in this ration. -18.. Discussion The results obtained in the study of the availability of var- ious calcium salts in series one and two are in agreement with those of (4) Steenbock and Jones and indicate that of a number of calcium salts, covering a wide range of solubility all are available to the rat for bone building,'with.the exception of calcium fluoride. The lack of bone development in the latter is undoubtedly due, at least largely, to the extreme insolubility of fihe calcium fluoride, which makes it impossible for it to be absorbed by the rat. The insolu- bility of tie calcium fluoride is further indicated by the fact that no such changes were noticed in the teeth as would have been observed had the fluorine been given in a more soluble form. In the third series in which calcium carbonate and dicapho were compared at various levels using the Steenbock rachitogonic ration with— out the calcium.carbonate, the results agreed in part with those ob- tained by (7) Hawk. The rats receiving the dicapho equivalent to two percent calcium carbonate showed decidedly superior skeletal development. at the one percent level the difference was much less pronounced and at the one-half percent level the difference was still smaller. In view of these results the far reaching conclusions of (7) Hawk are probably unwarranted. Although there may be little difference in the availability, the choice of a particular mineral supplement depends largely upon the other constituents of the ration. This is certainly the case with supplements containing calcium, phosphorus, or both of these elements. The rations used in feeding domestic animals and fowl may be divided into four general types. .A ration containing no appreciable amount of roughage or poor roughage and relatively little concentrate would be low in both calcium and phosphorus. Such a situation would call for a supplement of calcium and phosphorus, which - 19 - might be supplied in the form of di calcium phosphate, tri calcium phos- phate, or possibly a mixture of calcium carbonate and di or tri calcium phosphate. A ration consisting of a good roughage with little concentrates would represent a case in which the supply of calcium would be adequate, whereas the supply of phosphorus would not. Such a ration would be best supplemented by a salt high in phosphorus, as mono calcium phosphate or d1 calcium phosphate. If, on the other hand, a ration is fed which consists chiefly of grains and concentrate with little or no roughage, the ration is relatively high in phosphorus but low in calcium so that a suitable mineral supplement would be one containing chiefly calcium. In this case calcium carbonate would be the most economical and appropriate mineral supplement. The fourth type of ration characterized by containing both an abundance of goal reujhzge and a liberal supply of concentrate would ordinarily not require any mineral supplement at all. (8) Lindsey and Archibald point out that in A ’5. V Q4 dairy feeding, if the cows are average producers and are on good quality ’— roughage no mineral supplement is necessary. The results of series four and five add further proof to this point of view. The ration in series four repres— entating a good mixed dairy ration was not significantly improved by the add— ition of either calcium carbonate or dicapho. In the case of the Ohio poultry ration some improvement was obtained with the addition of either calcium car— bonate or dicapho. It is evident, then, that one must be careful in generalizing about the use of mineral supplements in the feeding of farm animals and poultry. It is obviously improper to make a comparison of two minerals in one particular ration and then to apply the results to all other types of rations. To do justice to a comparison betneen two minerals or salts it is necessary to use them in a variety of typical, practical rations. Only such a comparison will enable one to determine the actual feeding values of these compounds. _ ho _ S"IZARY In experimenting with young rats, using the ash content of humeri and femurs as a criterion, no difference was found in the availability of calcium carbonate, calcium lactate, calcium sulfate, mono, di, and tri calcium phosphate when these were fed in amounts equivalent of oneéhalf percent of calcium carbonate. Calcium fluoride when fed in similar amounts appeared not to be available to the rat for growth and bone deveIOpment. This was probably due to the extreme insolubility of the salt. When feeding calcium carbonate and dicapho at various levels using the Steenbock rachitogenic ration the results showed that dic- apho was definately superior to calcium carbonate when fed at a two perceit level. At the one-half percent level the differences were much smaller, the one percent level being intermediate. Extra calcium and phosphorus added to a practical dairy ration in the form of calcium carbonate or dicapho and fed to rats failed to increase materially the growth or bone development in the animals. The addition of calcium carbonate or dicapho to a typical poultry ration resulted in some improvement in the bone deveIOpmcnt and growth of the animals, the latter being slightly superior. BIBLIOGRAPHY Osborn, T.B., and L.B Kendal: Inorganic Elements in Nutrition. Jour. Biol. Chem. 1918,34,131—39 Forbes, 3.3., and J.O. Halverson, L.E. Morgan, J.a. Schulz: hetabolism of Calcium Compounds by Growing Swine. Ohio Agric. BXper. Sta. Bull. 347, 3-36, 1921. Hart, 3.3., and H. Steenbock, C.&. HOppert, Ram. Bethke: Dietary Factors Influencing Calcium Assimilation. Jour. Biol. Chem., 1922, 54, 75. Steenbock, H., and 3.8. Hart, H.T. Sell, J.H. Jones: availability of Calcium Salts. Jour. Biol. Chem., 1923, 56, 375. Hart, 3.3., and H. Steenbock: The Effect of a High Magnesium Intake on Calcium Retention by Swine. Jour. Biol. Chem. 1913, 14, 75. Hoag, J.R. and Leroy S. Palmer: Effect of Variations in the PrOportions of Calcium.hagmesium and Phos- phorus Contained in the Diet. Jour. Biol. Chem. 1928, 75, 567. Hawk, P.B.,: Researches on Utilization of Dicapho. Bull. by Food ReSearch Laboratories. Lindsey, J.B., and J.G. Archibald: Studies on Mineral Nutrition. hass. Exper. Sta. Bull. 255, 155-166, 1929. ’I I to 10 1 Reed, 0.3., and C.H. Huffman: Results of Five Year hineral Feeding Investigation with Dairy Cattle. Xichigan State College Exper. Sta. Bull. 105, 1935. | IV . I. .11.! 1 y I . I ... ,1 A . a \ . . . ,\ , i v ' . . 7 . 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