THE EFFECT OF THE CALCIUM-PHOSPHORUS RELA- TIONSHIP ON GROWTH, CALCIFICATION, AND

BLOOD COMPOSITION OF THE RAT”

BY R. M. BETHKE, C. H. KICK, AND WILLARD WILDER

(From t&e Department oj Animal Industry, Ohio Agricultural Experiment Station, Wooster)

(Received for publication, June 13, 1932)

Numerous investigators have shown that the three dietary com- ponents, calcium, phosphorus, and vitamin D, are especially con- cerned in normal bone formation. McCollum and associates (1) and Sherman and Pappenheimer (2) showed independently that the quantitative relation between the calcium and phosphorus in the food supply is, within certain limits of concentration, of con- siderable significance in determining whether the rat will develop bones which are normal or pathological. Likewise, Goldblatt (3) reported that the change in the Ca: P ratio from 1: 0.88 to 1: 0.20, in a purified synthetic diet low in phosphorus (0.22 per cent) and the fat-soluble organic factor, through the addition of calcium car- bonate had the effect of changing it’ from a non-rickets-producing to a rickets-producing diet.

Karelitz and Shohl (4) reported that rats made rachitic with a high calcium-low phosphorus ration, devoid of vitamin D, were cured by the addition of monobasic sodium phosphate to the ration.

Bethke, Steenbock, and Nelson (5) pointed out that with rats on low calcium rations the quantity of cod liver oil necessary to furnish the antirachitic factor in sufficient amounts varied inversely with the calcium content of the ration. The same conclusion was reached in the case of the rat on moderately high phosphorus rations. Haag and Palmer (6) also showed the importance of a more or less balanced condition of calcium, phosphorus, and mag- nesium salts in the ration for good growth and mineral retention in

* Published with the permission of the Director of the Ohio Agricultural Experiment Station.

359

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the rat. The same investigators call attention to the importance of the vitamin-mineral relationship by stating, “The response of various groups of animals [rats] to certain vitamin levels points to even more important vitamin-mineral interrelationships than have heretofore been recognized.”

Medes (7), in studies of the effect of varying mineral ratios on the mineral composition of the rat, reported that the normal com- position of the rat with respect to calcium and phosphorus (mg. per gm. of weight) cannot be altered by increasing the amount of phosphorus in the diet above that needed for normal growth. She also reported that altering the P:Ca ratio from 0.73 to 1.87 did not have any effect as long as both elements were present in adequate amounts and the calcium was not present in massive doses. The same investigator reported that rats which received diets containing 0.12 per cent phosphorus and 0.37 per cent calcium developed severe rickets. This is in accordance with the report of Pappenheimer, McCann, and Zucker (8) who state that when the phosphorus of the diet is reduced to 0.135 per cent, rickets begins to appear and is active when phosphorus is present only to the amount of 0.11 per cent.

The practical significance of adequate amounts of calcium and phosphorus in the food supply is imperative. It is also recognized that a marked disproportion in the calcium and phosphorus of the diet, in absence of vitamin D, seriously interferes with calcification and constitutes one of the dietary essentials for the production of experimental rickets in the rat. Information, however, on the interrelation of these elements at different levels or concentrations, in the absence or presence of vitamin D, is meager. The present report deals principally with some of these fundamental questions with respect to bone formation in the rat. The data are, in part, from a series of investigations started several years ago to study the importance of the calcium-phosphorus relationship in the nutrition of the rat, pig, and chick.

EXPERIMENTAL

At the start of the series of investigations it was thought advis- able, for experimental reasons, to use comparable rations as much as possible. To this end, we had in mind a ration which could be relied upon to produce incipient rickets in the rat in 3 to 5 weeks

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Bethke, Kick, and Wilder 391

and still, in the case of the pig, constitute a comparatively practical ration. After a great deal of preliminary investigation, a ration of 79.5 parts of yellow corn, 20 of soy bean oil meal, and 0.5 of sodium chloride, was chosen. This ration, when supplemented with 3 parts of calcium carbonate, produced severe rickets in rats of our breeding in 3 to 4 weeks.

In all of the experiments rats reared on our stock ration were used. They were started on experiment when 23 to 25 days of age and weighing from 45 to 60 gm. All groups were confined in wire cages provided with false screen bottoms and cared for in a room devoid of sunlight. With few exceptions, the sex distribution was equal in all groups in a particular experiment.

For calcification studies, the femurs, ulnae, and radii were removed from each rat. The femurs were freed of adhering tissue, dried, and then extracted for 18 to 24 hours with hot alcohol and subsequently with ether for a similar period. The dried extracted bones were then ashed in an electric muffle furnace. The per cent ash is calculated on a moisture and fat-free basis. The ulme and radii were fixed in 4 per cent formalin and examined for degree of calcification by the silver nitrate procedure.

The first experiment consisted of five groups of rats, six in each group. They received the yellow corn-soy bean meal ration, supplemented with varying amounts of calcium and phosphorus, as shown in Table I. At 5 weeks, two animals from each group, except Group 2, were killed for bone analysis. The remaining animals were continued on experiment for another 12 weeks, when they were killed for calcification studies. The results are pre- sented in Table I.

The group that received the unsupplemented basal ration, exhibited signs of a marked mineral deficiency, in the form of a rough hair coat and stiffened, staggering gait, at an early date. The addition of 3 parts of calcium carbonate to the basal mixture produced severe rickets, as attested by the low ash content (21.2 per cent) and gross examination of the ulnae and radii. These animals were in an extremely rachitic condition and probably would not have survived longer. When the basal mixture was supplemented with 3 parts of secondary sodium phosphate and 3 parts of calcium carbonate, the ash content of the bones and their histological picture were normal. Similar results were ob-

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392 Effect of Ca:P Ratio in Diet

tained when the ration was fortified with calcium and phosphorus in the form of ground limestone and bone meal. The beneficial effects of ultra-violet irradiation on growth and bone formation are revealed in Group 3 (Table I). It is interesting to note that the growth of the irradiated group did not equal that of the groups that received additional phosphorus in the form of bone meal or sodium phosphate, even though the latter groups received little if any vitamin D.

TABLE I

Effect of Varying Calcium and Phosphorus. Intake on Growth and Ash Content of Femurs

“goyp Additions to basal ration

1

2 3*vt

4*

5t

pad3

None

3 calcium carbonate 3 ‘I ‘I

(irradiated) 3 calcium carbonate,

3 disodium phos- phate

1 ground limestone, 1 bone meal

T

Ca in ration

P in ration

oer em t 7er em

0.06 0.33

1.16 0.32 1.16 0.32

1.14 0.62

0.62 0.44

-

t

Ca:P ratio

0.18

3.62 3.62

1.84

1.41

-7 - Wks. on atior

5 17

5 5

17 5

17

5 17

?a 0 ani- mals

verag ain in weight

.-

am. 9er cent

38 37.1 66 37.0 36 21.2 56 45.5

134 62.9 75 49.4

173 65.1

2 88 45.1 4 156 62.6

* Group irradiated 10 minutes daily, six times per week, with quartz mercury vapor lamp at a distance of 30 inches.

f One female in group had young but they were not nursed.

The data, in general, are in agreement with previously reported investigations-that vitamin D and the quantitative relation between the calcium and phosphorus in the ration are important factors in calcification. The next logical step appeared to be to study the influence of small progressive increments in calcium and phosphorus on growth and bone formation. For this purpose the aforementioned basal ration of yellow corn, soy bean oil meal, and salt was supplemented with increasing amounts of calcium carbonate alone, and calcium carbonate and secondary sodium phosphate and fed to rats in groups of four each. All groups, unless otherwise indicated, were on experiment for 12 weeks, when they were sacrificed for bone analysis.

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The data recorded in Table II show that without the further addition of phosphorus to the basal mixture, the ash content of the femurs was progressively reduced when more than 1 per cent of calcium carbonate was added to the ration. When the per cent phosphorus in the ration was increased, with corresponding in-

TABLE II

Effect of Varying Calcium and Phosphorus Intake on Growth of Rats and Ash Content o.f Their Femurs (No Vitamin D Added)

Mineral addition to ration

.s I

8 ii d z

parts parts

0.5 1.0 2.0 3.0

1.0 0.5 1.0 1.0 1.0 2.0 1.0 3.0 1.0

2.0 0.5 2.0 1.0 2.0 2.0 2.0 3.0 2.0

wr cm ilm. per cent 0.06 0.18 44 32.4 0.24 0.73 108 51.1 0.42 1.27 103 51.4 0.79 2.39 48 28.5* 1.16 3.62 39 23.5* 0.06 0.14 48 33.3 0.24 0.56 86 45.3 0.42 1.00 104 60.6 0.78 1.86 116 52.2 1.15 2.74 71 29.0 0.06 0.11 45 31.4 0.24 0.45 88 41.5 0.41 0.79 112 55.6 0.78 1.50 90 59.2 1.14 2.23 89 57.1

* Killed at 5 weeks. 7 One animal died in 2nd week. $ One animal died in 12th week. Q Two females had 3 litters of young, not nursed.

,er een

0.33 0.33 0.33 0.33 0.32 0.43 0.43 0.43 0.42 0.42 0.53 0.53 0.52 0.52 0.51

Condition of animd

Stiff, poor Good

‘I

Rachitic Very rachitic Stiff, poor Some stiffness, fair Good

“ t

Rachitic Stiff, very poor1

“ but fair Goods Good

“

creased amounts of calcium, the animals tolerated a greater per cent of calcium, as reflected in increased growth and bane ash- again showing that the proportion of calcium to phosphorus in the ration is of great significance in calcification. The addition of 0.2 per cent of phosphorus, in the form of secondary sodium phos- phate, to the basal ration, supplement’ed with 0.5, 1.0, 2.0, and

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394 Effect of Ca:P Ratio in Diet

3.0 per cent of calcium carbonate, further reflected the importance of the inorganic relationship in bone formation. A ration which normally produced incipient rickets in 3 to 4 weeks was made non- rachitogenic by adding 2 parts of secondary sodium phosphate. It is also evident that the concentration or per cent of phosphorus in the ration had some effect on bone ash. A Ca : P ratio of 2.39 at a phosphorus level of 0.33 per cent caused rickets, whereas a 2.23 ratio with the phosphorus increased to 0.51 per cent made for a nearly normal ash content.

Other experiments with the same basal ration confirmed the results of the previous experiments-that the proportion of cal- cium to phosphous in the ration of the rat is of great significance in bone formation. Evidence was also obtained which indicated that the amount of vitamin D required for normal calcification was dependent upon the Ca:P ratio of the ration. The addition of either tertiary calcium phosphate or calcium carbonate and secondary sodium phosphate to the basal mixture to establish a Ca:P ratio between 1.00 and 2.00 made for a practically normal ash value which was but slightly increased by the addition of vitamin D.

Although the results showed rather conclusively the importance of the Ca : P ratio in the nutrition of the rat, it still remained to be determined whether the concentration or level of these two ele- ments at a particular proportion also exerted an effect on calcifica- tion. It being assumed that there exists an optimum calcium- phosphorus relationship at a given level or concentration, are the vitamin D requirements of the particular animal at a minimum at this relationship? Or, to state it in another way, is it possible to reduce the antirachitic requirements of an animal to a minimum by properly adjusting the calcium and phosphorus content of its ration?

With these questions in mind, a fourth experiment, which involved the feeding of the yellow corn-soy bean oil meal ration supplemented with such amounts of calcium carbonate, secondary sodium phosphate, and tertiary calcium phosphate, to obtain eight different Ca:P ratios at three different levels or concentra- tions, was started. The general plan of the experiment, with the per cents calcium and phosphorus in the different rations, are shown in Table III. Each ration was fed to two groups of four

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rats each. Irradiated ergosterol dissolved in linseed oil was added to the ration of one group in each pair, while the other group served as a negative control. The oil solution was added on a 0.5 per cent basis. 1 cc. of the solution was equivalent to approxi- mately 200 Steenbock units of vitamin D. All groups in one particular series were started at the same time. The foodstuffs and salts used in the low and the medium concentration series were from the same stock. In the high series a new batch of yellow corn, soy bean oil meal, calcium carbonate, and secondary sodium phosphate were used.

TABLE III

Plan of Experiment, Showing Per Cent o.f Calcium and Phosphorus in Rations

Low concentration*

Ca:P ratio Calcium

5.00 1.79 4.00 1.44 3.00 1.09 2.00 0.74 1.00 0.37 0.50 0.37 0.33 0.36 0.25 0.35

Phosphorus Calcium Phosphorus

0.36 2.64 0.53 0.36 2.14 0.53 0.36 1.63 0.54 0.37 1.10 0.55 0.37 0.56 0.56 0.73 0.55 1.09 1.08 0.53 1.60 1.42 0.52 2.08

I Medium concentrationt High concentrationf

Calcium Phosphorus

3.24 0.65 2.64 0.66 2.01 0.67 1.36 0.68 0.69 0.69 0.67 1.34 0.65 1.95 0.63 2.53

-

* Basal ration plus varying amounts of calcium carbonate and secondary sodium phosphate.

t Basal ration with 1 per cent calcium phosphate plus varying amounts of calcium carbonate and secondary sodium phosphate.

$ Basal ration with 2 per cent calcium phosphate plus varying amounts of calcium carbonate and secondary sodium phosphate.

The groups were continued on their respective rations for 5 weeks, when they were sacrificed for blood and bone analyses. To obtain blood samples, the rats were anesthetized with ether and bled from the carotid artery. The blood from each group was pooled and allowed to stand overnight in the refrigerator. The following morning the serum was pipetted off, centrifuged to remove contaminating corpuscles, and immediately analyzed for calcium (9) and inorganic phosphorus (10). The femurs, ulnaa, and radii were treated as previously described.

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396 Effect of Ca :P Ratio in Diet

The inorganic phosphorus content of the blood serum, pre- sented in Table IV, was greatly reduced in the groups which did not receive vitamin D (irradiated ergosterol), when the Ca:P ratio was greater than 2.00. The addition of irradiated ergosterol to the ration of the corresponding groups increased the inorganic phosphorus content to near normal, as reported by Kramer and Howland (11). Added phosphorus, in the form of secondary sodium phosphate, or a change in Ca:P ratio from 1.00 to 0.25, increased the inorganic phosphorus values of the blood serum in

TABLE IV

E$ect of Varying Calcium and Phosphorus Intake, with and without Irradiated Ergosterol, on Inorganic Phosphorus Content of Blood Serum

Results are expressed in mg. per 100 cc.

Ca-P concentrations

Ca:P ratio Controls T Irradiated ergo&ml

Low Medium High LOW Medium High

5.00 2.9 2.4 3.3 5.3 8.1 8.2 4.00 2.5 2.4 3.6 6.8 8.1 7.0 3.00 3.5 4.1 3.8 7.3 7.7 6.9 2.00 6.2 6.3 6.0 8.1 10.5 10.1 1.00 9.2 10.4 8.8 9.3 10.2 9.2 0.50 12.2 10.1 9.6 9.0 10.9 8.1 0.33 11.4 12.2 11.0 8.7 13.0 9.5 0.25 13.5 13.4 11.8 11.5 15.8 11.1

Considerable difficulty was experienced with hemolysis in the samples from some of the high phosphorus groups.

the control groups. The addition of vitamin D (irradiated ergosterol) to the same groups did not appear to affect the phos- phorus content of the serum. It is also significant to note that as the proportion of phosphorus to calcium in the ration was increased a more or less progressive increase in inorganic phosphorus content of the serum was noted, irrespective of the presence or absence of the antirachitic factor. These observations are in general in agreement with those recently reported by Kramer and Howland (11). It should be noted that the inorganic blood phosphorus of the majority of the irradiated ergosterol-fed groups in the medium

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concentration series was somewhat higher than in the low or high series. We are of the opinion that the increased phosphorus was due primarily to the hemolyzed condition of the serum rather than the effect of phosphorus concentrat.ion in the ration.

The calcium content of the blood serum (Table V) was not as much affected as the inorganic phosphorus. All groups, irrespec- tive of the Ca:P ratio or the concentration of these elements in the ration, with the exception of the control groups which received a greater proportion of phosphorus than calcium in their rations, showed a calcium content within the normal range (11). It is of

TABLE V

Eflect of Varying Calcium and Phosphorus Intake, with and without Irradiated Ergosterol, on Calcium Content of Blood Serum

Results are expressed in mg. per 100 cc.

I Ca-P concentrations

Ca:P ratio Controls T

Low

- Medium High Low

5.00 10.7 13.3 11.1 13.0 4.00 9.9 12.4 11.8 12.2 3.09 9.3 11.9 12.4 12.5 2.00 10.5 10.7 9.2 12.3 1.00 9.9 9.6 9.7 11.7 0.50 7.8 7.7 7.3 12.7 0.33 6.4 9.9 6.6 12.8 0.25 5.3 6.1 7.4 11.4

Irradiated ergo&sol -

_ Medium High

11.2 13.4 11.6 12.8 12.5 11.4 11.5 9.5 11.9 10.6 9.0 10.4

12.4 9.4 9.9 10.7

especial interest to note, however, that the calcium percentage of the blood serum was reduced as the Ca:P ratio of the ration was changed from 1.00 to 0.25, in the absence of irradiated ergosterol (vitamin D). When vitamin D was added no such reduction in calcium content was noted. These observations are in accord with those previously reported by Bethke, Steenbock, and Nelson (5), and recently by Kramer and Howland (11). The groups which showed a high phosphorus-low calcium content of the blood serum were nervous and excitable. Whether these animals were border- ing on tetany we are unable to state.

The results of the ash determinations on the femurs are recorded

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398 Effect of Ca:P Ratio in Diet

in Table VI. These data show conclusively that calcification, especially in the absence of vitamin D, is greatly affected by the Ca:P ratio of the ration. Keeping the per cent phosphorus in the ration practically constant and increasing the per cent calcium, through the addition of calcium carbonate, caused a progressive decrease in the ash content of the femurs. The addition of irradiated ergosterol (vitamin D) to the same rations prevented the occurrence of rickets and improved calcification in all instances. However, the same general decline in per cent bone ash, although less pronounced, was observed by increasing the Ca : P ratio from

TABLE VI

Effect of Varying Calcium and Phosphorus Intake, with and without Irradiated Ergosterol, on Ash Content of Femurs

Results are expressed in per cent on a fat-free basis.

Ca:P ratio 1

-

Controls Irradiated erg&em1

LOW Medium High LOW Medium

-- High

5.00 22.6 26.9 30.1 50.8 53.8 51.6 4.00 23.6 27.2 32.5 51.3 54.7 52.0

3.00 32.2 36.5 40.2 54.4 57.7 54.2 2.10 42.2 45.1 48.8 56.3 59.9 58.1 1.10 46.4 52.3 51.3 57.4 59.2 59.8 0.50 46.3 50.9 51.6 58.3 58.8 58.3 0.33 47.2 48.5 51.2 56.9 57.3 55.8 0.25 46.6 48.9 48.6 57.3 55.2 55.2

I- I-

G-P concentrations

1.00 to 5.00. This observation gives further support to the earlier findings of Bethke, Steenbock, and Nelson (5) that the wider the Ca:P ratio the greater are the requirements for vitamin D. When the Ca:P ratio was changed from 1.00 to 0.25 through the addition of phosphorus, in the form of secondary sodium phos- phate, a slight reduction in per cent ash was observed in the medium and high concentration series. The per cent decrease in bone ash was as great in the groups fed irradiated ergosterol (vitamin D) as in the controls. We had anticipated that a ration much higher in phosphorus than in calcium would greatly affect the per cent of bone ash, especially in the absence of the anti-

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rachitic vitamin, since in earlier investigations rations of this type were used for the production of experimental rickets. Appar- ently, in these experiments the per cent calcium in the rations was either too high or the Ca: P ratio was not sufficiently wide to produce changes ordinarily observed on a high phosphorus-low calcium dietary.

Increased levels or concentrations of calcium and phosphorus in the rations exerted a favorable effect on bone ash in the absence of vitamin D, especially when the amount of calcium was equal to or greater than that of phosphorus. The inclusion of the anti-

TABLE VII

E$ect of Varying Calcium and Phosphorus Intake, with and without Irradiated Ergosterol, on Growth of Rats

Results are expressed in gm.

I 6-P concentrations

Ca:P ratio Controls Irradiated ergosterol

LOW Medium

- I High LOW Medium High

5.00 29 40 42 30 49 4.00 30 43 46 53 62 3.00 49 72 57 67 104 2.00 58 72 81 90 101 1.00 54 65 89 86 98 0.50 35 37 59 85 84 0.33 25 31 28 76 50 0.25 15 15 13 74 32

44

52 83 85 96 90 44 33

rachitic factor (irradiated ergosterol) increased the per cent of ash in all instances. The greatNest favorable effects were obtained when the calcium content of the ration exceeded that of phos- phorus.

The degree of calcification in the ulnae and radii in all cases closely checked with the ash determinations.

The average growth of the different groups is presented in Table VII. The data show that growth is greatly affected by the Ca:P ratio of the ration. With few exceptions, better growth occurred when the amount of calcium in the ration exceeded that of phosphorus than when the reverse was true. The rate of gain,

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however, progressively declined as the width of the Ca:P ratio increased, irrespective of the presence of vitamin D, or whichever element was in excess. The inclusion of the antirachitic factor (irradiated ergosterol) increased growth regardless of concentra- tion or ratio in all cases. It is evident from the data that the greatest growth occurred at Ca:P ratios of 2.00 and 1.00, regard- less of the concentration or per cent of these elements in the ration.

DISCUSSION

The data, in general, show conclusively that in the absence of vitamin D the proportion of calcium to phosphorus in the ration, within certain limits, exerts a marked effect upon growth, bone formation, and the percentages of these elements in the blood. Although the concentration or the levels at which calcium and phosphorus were present in the ration had some beneficial effect on growth and bone formation, this effect was not as great as that exerted by the actual proportion between the elements. The inclusion of vitamin D not only tended to stabilize the calcium and inorganic phosphorus concentrations in the blood serum, but made a greater percentage of these elements available for such biological phenomena as calcification and growth. Evidence is also presented showing that the wider the Ca:P ratio the greater are the requirements for vitamin D. With the same amount of irradiated ergosterol, a ration possessing a Ca:P ratio of 5.00 pro- duced bones from 6 to 7 per cent lower in ash than did a similar ration with a Ca : P ratio falling between 2.00 and 0.50.

Since all work bearing on the calcium-phosphorus relationship in calcification brings out t.he importance of the ratio of these two elements in the ration, it is imperative that these facts be taken into consideration when attempts to evaluate a particular ration or foodstuff for its calcifying properties are made. It would also appear to be essential and desirable, from an experimental stand- point at least, to define our more or less standard rickets-producing rations, or those rations employed for calcification studies, in terms of calcium and phosphorus, as well as kind and proportion of ingredients. Such a procedure would certainly tend towards greater uniformity of results between different laboratories or investigators who draw upon different stocks or sources of supply for the ingredients used in the compounding of their experimental

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rations. We know from experience that grains, such as corn, as well as other foodstuffs, vary greatly in their inorganic composition, depending upon such factors as soil fertility, rainfall, climate, variety, etc. The kind of corn-meal, i.e. whether it is a meal pre- pared from the entire corn grain or from the common market “corn-meal” which, in many instances, is devoid of the corn germ, may be a major factor. Data showing great variation in the inorganic composition of the corn grain and commercial corn meals were recently presented by Holmes and Tripp (12).

It also appears that some distinction should be made between calcification studies designed to study the “over-all” effect of a particular foodstuff on one hand and its inherent vitamin D potency on the other. Experiments planned to determine the vitamin D content of foods, like milk, which supply considerable quantities of calcium and phosphorus, by feeding varying amounts of the same, without adjusting the calcium and phosphorus content of the basal ration accordingly, and determining their effect on calcification, in reality do not strictly measure the vitamin D potency of the food under examination. Rather they measure the “over-all” or combined effect of the inorganic elements and vitamin D. Any addition to a ration which makes for a Ca:P ratio approaching 2.00 to 1.00 would of itself make conditions more favorable for calcification and materially reduce the requirements for vitamin D. The actual vitamin D content of a food can only be determined when other inherent factors, such as calcium and phosphorus, are corrected for. Recently Bloom (13) reported that secondary calcium phosphate prevents and cures rickets in rats without vitamin D. This investigator substituted an equiva- lent quantity of calcium in the form of secondary calcium phos- phate for the calcium carbonate in his basal rachitic ration and noted a decided increase in the per cent bone ash. Such a sub- stitution naturally changes the Ca : P ratio from one that induces rickets to one that falls within the optimum value for good bone formation. In our experiments a similar favorable Ca:P ratio gave approximately the same per cent of bone ash (50 per cent). This, however, was not a maximum value since the addition of irradiated ergosterol (vitamin D) made for a 57 to 59 per cent ash content, which, in light of our interpretation, was normal.

It is probable that the form in which calcium and phosphorus

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402 Effect of Ca:P Ratio in Diet

are included in the ration may exert an influence. Experimental work bearing on these points has been reported, but the evidence, in general, is conflicting, and, in the light of present knowledge of the effect of the Ca: P ratio, a great deal of the work needs to be repeated. To what extent the reaction of the ration exerted an effect on calcification we are unable to state. Evidence in this re- spect is at variance and requires a great deal more work before we can come to an intelligent understanding of the problem.

SUMMARY

1. The ratio of calcium to phosphorus in a ration exerts a marked effect on growth, ash content of the bones, and the calcium and inorganic phosphorus values of the blood serum of rats.

2. Increasing the Ca:P ratio from 1.00 to 5.00 causes a progres- sive decrease in growth, bone ash, and the percentage of inorganic phosphorus in the blood serum. The addition of irradiated ergosterol (vitamin D) to the ration of corresponding groups made for increased growth, ash values, and normal inorganic phosphorus of the blood.

3. When the Ca:P ratio was changed from 1.00 to 0.25, growth was depressed and the ash content of the femurs was slightly decreased at the two highest Ca-P concentrations. The same change in ratios caused a decrease in the per cent calcium and an increase in the per cent phosphorus in the blood serum. Added vitamin D (irradiated ergosterol) increased growth and the ash content, but did not influence the decline in ash values or the increase in inorganic phosphorus of the blood serum. Vitamin D also made for normal calcium values of the blood serum.

4. Increased levels or concentrations of calcium and phosphorus in the ration exerted a beneficial effect on growth and bone ash.

5. The ratio of Ca : P had a greater effect on growth and calcifi- cation than the concentration or levels of these elements in the ration.

6. The most favorable Ca : P ratio for growth and bone forma- tion was between 2.00 and 1.00. The vitamin D requirements were at a minimum at these proportions.

7. Further evidence is presented which shows that the wider the ratio of Ca:P the greater are the requirements for vitamin D.

8. The desirability of adjusting the Ca:P relationship in calcifi-

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Bethke, Kick, and Wilder 403

cation studies, and the importance of defining rachitic rations in terms of calcium and phosphorus as well as ingredients, are pointed out.

BIBLIOGRAPHY

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2. Sherman, H. C., and Pappenheimer, A. M., Proc. Sot. Exp. .Biol. and Med., 18, 193 (1920-21).

3. Goldblatt, H., Biochem. J., 18,414 (1924). 4. Karelitz, S., and Shohl, A. T., J. Biol. Chem., 78,665 (1927). 5. Bethke, R. M., Steenbock, H., and Nelson, M. T., J. Biol. Chem., 68,

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R. M. Bethke, C. H. Kick and Willard WilderCOMPOSITION OF THE RAT

CALCIFICATION, AND BLOOD RELATIONSHIP ON GROWTH,

CALCIUM-PHOSPHORUS THE EFFECT OF THE

1932, 98:389-403.J. Biol. Chem. 

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