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STUDiES 0N BONE DEVELDPMENT
IN RATS

THESIS FOR THE DEGREE OF M. S,

Sarah May Shaw
1933

 

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STUDIES ON BONE DEVELOPMENT IN RATS

A THESIS

Submitted to the Faculty of the Graduate School
of
MICHIGAN STATE COLLEGE
in Partial Fulfillment of the Requirements for the Degree
of

MASTER OF SCIENCE

by
Sarah May Shaw

June, 1955

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ACKNOWLEDGMENT

The writer wishes to express full appreciation to
Dr. C. A. Hoppert for his guidance, direction, and
assistance in the conduct of the experiment herein

reported.

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CONTENTS

Historical Review
Plan of Experiment
Experiment

Results

Table I

" II
III
IV
V

333

Discussion
Conclusions

Bibliography

12
13
16
17-18
20-21
22
25
26
28
50

51

HISTORICAL REVIEW

"A perfect experiment in any field of science may be said to be
one that has been planned and conducted in such a way that the results
obtained are susceptible of only one interpretation." (1) Such is
the aim which H. R. Mitchell has set for experimental work expecially
in the biological field. This general idea has been used in judging
the applicability of the conclusions reached by previous workers and
in setting up the experiment which is to be reported in this paper.

Rickets has long been known to be a disturbance in the calcium
and phosphorus deposition in the bone of animals suffering from this
malady. As generally Spoken of now, it refers specifically to the low
phosphorus type i.e. the blood is usually low in inorganic P. The
disease is produced in rats by feeding the Steenbock and Black ration
which is now being widely used in the experimental study of rickets.
It was found necessary to have a high Ca:P ratio in the dietary to pro-
duce the typical change in bone structure. In the Steenbock diet this
ratio is approximately 4 to l.

The blood picture was one of the first factors to be studied.
Since the blood carries all nutritive materials to every portion of
the body, a drop in calcium or phosphorus content would, of course,
influence bone development. Butcher, Creighton, and Rothrock reported
in 1925 that in normal young rats the phOSphorus in the blood was about

10 mg./lOOcc. of serum, and that this level continued, falling slowly

over an eight week period, to 8 mg./100 cc. of serum. The ash of the
femur correspondingly rose from 40% to 62%. On the other hand, with
rats on the Steenbock ration, the phosphorus dropped to 1.6 mg./lOO cc.
of serum within three weeks, rising slightly thereafter presumably due
to a lessened food intake. The ash of the femur became 26.5% in three
weeks and values even as low as 24%have been obtained. (2) Koch and
Cohan feund.results comparable with these. They, however, did some work
on the method of obtaining the blood and on the influence which any
variation would have on the phosphorus content. They concluded that
deep anesthesia increased the inorganic blood phosphate very rapidly,
light anesthesia increasing it only 2-6%. They also found that the
struggle and injury from stunning the animal increased the blood phos-
phorus even more than anesthesia. (3) Shohl and his co—workers have
found this same condition in rickets - low phosphorus content of the
blood serum with normal calcium. (4) Later, they tried adding NaHZPO4
to the basal Steenbock ration changing the Ca:P ratio from 4:1 to 1:1.
The blood calcium remained the same but the blood phosphate became
suddenly very high and then came down to a normal value. This was
accompanied by rapid healing of the bone with a tendency toward tentany.
(5) Working further along this line, these experimenters came to the
conclusion that the ratio of Ca:P and their salt level were interdependr

ant. At any given level of calcium or ph08phorus, increasing the Ca:P

ratio intensified the degree of rickets and at any given ratio of Ca:P,

increasing the level of the salts diminished the degree of rickets. (6)
After producing the disease, the next important thing is to effect
a cure. This can be accomplished by irradiation of the animal or feed-
ing phosphate or vitamin D. Schultzer found the following change in the
phosphorus of the blood: In rachitic rats, the phOSphorus was 5.6 mg./lOO
cc. of serum rising to 8.6 mg./100 cc.on treatment with ultraviolet and
as high as 10.6 mg. by feeding cod liver oil. The calcium content of
the blood rose slightly from 11.9 mg./100 cc. of serum in the basal diet
to 15.2 mg. with irradiation and 15.8 mg. with cod liver oil. Normal
rats did not show this increase of calcium with ultraviolet treatment or
cod liver oil administration. Schultzer also concluded that the healing
of rickets by these agencies differed from the healing by added phOSphate
in that the serum calcium and phOSphorus values reached but did not
exceed the normal values. (7) This does not exactly agree with Shohl's
results as given above. Later, Shohl and coworkers reported that marked
cure of rickets in rats was secured in two weeks using cod liver oil or
.01 mg. daily doses of irradiated ergosterol. (8) In a parallel exper-
iment, they found increased blood phosphorus, increased growth, and
increased calcium and phosphorus retention with diets varying from the
basal rachitic ration to the basal plus Na32P04, basal plus butter fat
(replaced lard), basal plus cod liver oil, to the basal of which the
cornstarch was irradiated. (9) In another article, they report finding
that .01 mg. of irradiated ergosterol improved the normal rat in weight
and well being while in the rachitic rat, it effected a cure but not a

general improvement. 0.5—2.0 mg. was toxic to the normal rat while the

rachitic rat was much more resistant to toxic amounts which is to be
expected. The vitamin D did not, however, alter the ratio of Ca:P
retained and, therefore, these workers concluded that the vitamin pro-
duced calcification by dissolution and deposition of the bone salts and
that the amount of calcium and phOSphorus in the diet controlled the
retention while the vitamin controlled the intermediary metabolism. (10)

In spite of all this evidence, Hess, Weinstock, et a1, recently
published an article attempting to prove the lack of relationship between
the develOpment and cure of rickets and the inorganic phosphorus of the
blood. The question might be asked as to whether they had only one
variable in their experiment. Their data indicated very poor growth in
their animals. (11)

Other workers have studied the problem of bone development from
slightly different angles. LeCocq and Villins report that hypophos—
phites, furnishing 0.7% phosphorus in the feed, were less effective than
0.15% phOSphorus in the form of NazflP04. (12) Haury made some deter-
minations on the calcium content of striated muscle of rachitic rats.

The muscle of the rachitic animals contained 41.6 mg. of Ca/lOO g. of dry
muscle in comparison with 74.0 mg. of Ca/lOO g. of dry muscle in the normal
rat. These results may not be comparable to the rachitic conditions as
usually produced, for the diet was quite different from the one ordinarily
used. (15) Horste reported an interesting experiment on calcification
"in vitro'. The epiphyseal cartilege of rachitic rats took up calcium

and phosphorus from the surrounding medium in the mol ratio of 1:1. The

calcified epiphyseal cartilege from normal rats took up less and the

investigator found that if the phOSphorus content of the calcifying
solution was lovered, phosphorus actually escaped from the tissue into
the solution. (14)

Another phase of the rickets problem which was of particular interest
in setting up the present experiment was the influence of the acidity or
basicity of the ration. Zucker, Johnson, and Barnett reported work along
this line in 1922. They thought that they had conclusively proved that
rickets did.not develop on a rickets - producing ration if calcium chloride
or ammonium chloride were used to make the ration acid. They also stated
that they could produce rickets by making a suitable normal ration alkaline
with sodium carbonate. Here again, the question arose as to whether the
acidity was the only variable factor. (15) In 1924, Martha Jones reported
the use of hydrocloric acid in the therapy of rickets. In a few cases
which came to the hOSpital in San Francisco, the addition of hydrochloric
acid to the diet of the rachitic infants improved their condition. She
states that she used this treatment as the result of an idea taken from
the fact that puppies on a seemingly good ration but slightly alkaline
had developed rickets. With rats, this same ration had not, however,
proved rickets producing. Miss Jones also indicated that Schabad had
previously shown that in active rickets, the distribution of calcium and
phosphorus in the feces and urine was different from the normal, indicat-
ing difficulty in the absorption. (16) Samuel and Kugelmass recently
found that acid - forming diets lacking in vitamin D produced rickets
and generally inhibited growth, development, metabolism, and activity.

Again the question arose, and this time quite prominently, as to the

justification of comparing results from two diets so greatly varying
as those used. (17) Shohl and coworkers, taking up this work, tried
varying the rickets producing diet by adding the same amount of phos—
phate so that the diet became alkaline, neutral, or acid. The blood
picture showed the characteristics of tetany with the alkaline diets
and of rickets with the acid diets. The clinical picture corresponded.
Ash analyses of the bones showed greatest salt deposition with neutral
diet, less with the alkaline diets, and least with acid diets. (18)

In a second experiment, they altered the basal diet stepwise by adding
phosphate until it became normal. Then parallel diets were made acid,
neutral, or basic. Under these conditions, in the intermediate groups,
diets were found which produced the mild type of rickets when acid and
no rickets when neutral or alkaline. (19)

From the question of the effect of the acidity of the ration, the
problem advanced to a study of the gastro-intestional system and.the
mechanism and paths of absorption and excretion of calcium and phosphorus.
In 1924, Zucker and Matzner advanced the theory that the disturbance in
rickets was a gastro-intestional one. They came to this conclusion after
observing that the pH of the feces of rachitic rats averaged 7.6 and
that on the addition of cod liver oil or its active principle to the diet,
the pH of the feces changed to 6.2 or even 5.7. The cod liver oil or the
active principle injected subcutaneously did not produce healing or change
in the pH of the feces. (20) Schultzer made a study of the calcium and
phosphorus retention in rickets and gave his results as "retention quo-

tient'I which was the number of mg. of either element retained per g. of

body increase. His figures indicated an increased retention on treat-
ment with ultraviolet or feeding of cod liver oil, but his "quotient"
has not been used. (21) Shohl et al. have done work along this line by
means of metabolism studies. They have shown in their early experiments
the paths of excretion of calcium to be 25% in the urine, 77% in the
feces; for phOSphorus, 5% in the urine and 95% in the feces. The positive
balance of calciumrduring rickets was 50% of the normal and of phOSphorus
20% of the normal. The ratio of Ca:P retention in the normal was 1.58
and in the rachitic animal 5.9.(4) Addition of phOSphate to the basal
diet of rachitic animals increased the phOSphorus retention from 20% to
70% and changed the Ca:P retention ratio to 1.7 and 0.8. (5) The cure
of rickets by cod liver oil or irradiated ergosterol was accomplished
without great increase in the retention of calcium or phosphorus. (8‘& 10)
Bergeim studied the absorption of calcium by adding ferric oxide to
the diet. Knowing the ratio of Ga:Fe in the ration and determining this
ratio in the feces, the amount of absorption could then be calculated.
By this method, he found that lactose seemed to increase calcium and
phosphorus absorption while starch, glucose, fructose, and maltose had
no effect. By killing the animals at the height of digestion, he was
able to find that calcium absorption took place in the upper small intes-
tine and an excretion took place in the lower intestine bringing about a
negative balance. Phosphorus was actually excreted in the upper intes-
tine. He suggested that this secretion might be important in promoting
calcium absorption since it was most rapid where the P:Ca ratio was

highest. He also showed that animals receiving cod liver oil showed a

positive calcium balance throughout the intestine while the phosphorus
was secreted into the upper tract and was absorbed in the lower intes-
tine, resulting in a positive balance, also. In comparison, the rachi-
tic animal absorbs calcium in the upper intestine and excretes it into
the lower while it excretes phOSphorus into the upper intestine and
fails to reabsorb it. (22 & 25) Peola and Guassardo record similar
results studying the calcium and phosphorus absorption by means of an
intestional fistula. (24) Hesse reports a storage in the body of 10%
of the calcium given in the ration. He also has investigated the
effect of emulsifying the calcium salt in gum arabic which seemed to
increase the absorption markedly, while in a phosphatide emulsion the
absorption remained about the same, 50%. (25)

Still another angle of this problem is a study of the hydrogen
ion concentration of the gastro-intestional tract. Abrahamson and
Miller reported a marked increase in the pH of the intestional contents
in the rachitic animal and the return to normal acidity on administra-
tion of cod liver oil. (26) Yoder studied the relation between pH
and calcium and phosphorus utilization. He concluded that irradiation
or feeding of cod liver oil decreased the large calcium and phosphorus
elimination characteristic of the rachitic animal and lowered the pH
throughout the intestional tract. The rickets which he studied could
not have been typical since he started his animals at 150 gm. More-
over, his pH values did not seem to vary sufficiently to justify such
sweeping conclusions. (27) Redmon, Willimott, and Wokes further cor-

roborate the general observations of these previous workers. (28)

Menville, Ane, and Blackberg have added an interesting note by discover—
ing a general hypomotility of the intestional tract of rats on a D
deficient diet. (29)

This pH work has some practical use. It has been suggested that
the pH of the feces be used as a test for vitamin D. Jephcott and
Bacharach first proposed this method. By using the Zucker ration, they
obtained quite significant differences. Their method consisted in feed-
ing this diet until the pH of the feces became constant at 7.2. The
supplement was then given and after the third day, it was again tested
until the pH became constant at some value below 6.8. They found the
rise in fecal pH invariable on the basal ration and the addition of
supplementary doses of non-anti—rachitic substances to cause no alter—
ation in the alkaline pH. Restoration of acid pH occured by the addi-
tion of the antirachitic vitamin and by irradiation. The latter could
prevent the rise of pH in the first place. (50) Shohl and Bing critisized
this method severely and showed evidence that the pH of the feces varied
too greatly to be used in the quantitative estimation of vitamin D. (51)
Bacharach and.Jephcott replied defending their method. They did not
get sufficient fecal change with the Steenbock ration in the same length
of time as with the Zucker ration, but the difference was suggested to
be ascribed to the different Ca:P ratio in the two diets. (52 & 55)

Deer was inclined to agree with Shohl and Bing. He found that the pH
of the feces of the rachitic rat showed insufficient uniformity to
distinguish it from the normal, that there were large daily fluctuations
on the same ration, and that the time response of different rats varied.

(54) Recently, Heller and Caskey have taken up a defense of the method.

10

They recommend it not as a test for vitamin D but as a complete record
of the progress of an animal on an experiment, thus lessening the
possibility of error from basing conclusions on a single examination.
These workers admit the daily variation in values but point out the

fact that the relative slope of the curve is comparable to the skiagrams
of bone changes. Examination of the pH curves indicated that the drop
in pH precedes recalcification by three or four days. Thus it forms a

a good indication of the time to take x—ray pictures of the conditions of
bone development in the animal. (55)

To complete the picture in the study of rickets, the production of
tetany in rachitic rats throws some light on the blood picture and
absorption question. McCollum and Gavin had both shown that fasting
of rachitic animals caused healing of the bone and was accompanied by
a rapid rise in blood phosphorus. Shohl ran into this question in study-
ing the addition of phosphate to rachitic rations. Limiting the food
intake to one-third of that normally consumed caused only slight changes
in the rachitic animal - a slight increase in blood phosphorus, no heal-
ing in the bone, and reduced balances of calcium and phosphorus. (56)
Later, he and his coworkers came to the conclusion that rachitic rats
develop tetany'with high phosphorus and low calcium content of the blood
when moderate amounts of phosphate were added to the diet. Rats fed with
the same diet plus the antirachitic vitamin did not show tetany or alter-
ation in blood calcium and phosphorus and their bones yielded a normal
amount of ash. (57) Again Shohl and Bing found that the administration

of cod liver oil or irradiation lessened the irritability of the muscle.

The addition of phosphate, on the contrary, increased the reaction to a
condition which could clearly be called tetany. (58) In comparing the
conditions of tetany produced by these two methods, Shohl and Brown con—
cluded that the tetany produced from fasting was probably due to phos-
phate. The tetany resulting from feeding phosphate was demonstrated in
animals which gined weight on the rachitic ration. The symptoms deve10ped
soon after the ingestion of the phOSphate supplemented diet. The most
marked condition was produced when the Ca:P ratio was changed to 1:1.
A change to 2:1 showed slight tetany. (59) Still later Shohl and his
coworkers showed that the acid base equilibrium of the blood in rickets
bordered on alkalosis and the condition of tetany brought about by
either of these methods changed the equilibrium to one of acidosis. (40)
Shipley and Holt have tried to explain this change caused by fast-
ing of rachitic animals by means of experiments "in vitro". They
happened to be studying the effect of salts on calficiation. With the
control rachitic animals, a certain addition of NaCl to the calcifying
solution inhibited calcification of the bones of these animals even to
the point of no calcification. Using the bones of rachitic animals
which had been starved twenty four hours, rapid calcification took place
even in the same solutions which had inhibited the calficiation of the
control rachitic bones. These investigators suggested that the rapid
rise in blood phosphorus was not the only cause of rapid healing in the
case of starvation. (41) Wilder has attempted to determine the source
of the increased phosphorus in the blood during fasting. By blood
analyses, he showed thatthe phOSphorus did not come from the constit-

uents of the blood such as the red cells. A study of the N in the urine

12

indicated a destruction of body protoplasm sufficient to account for the
increase in phosphorus in the blood. A comparison of the N:P ratio in
the urine with that of muscle tissue indicated an extensive retention of
phosphate liberated by the tissue destruction. As a check on the con-
dition produced, the blood sugar value Was determined and it was found
that the convulsions produced in the animal could not be due to hypo-
glycemia. Furthermore, normal rats were checked and did not develop
tetany on fasting, did not show an elevation in blood phosphorus, but
did have a much longer survival period than the rachitic animals. (42)
Recently, Hess, Weinstock, et al. have produced tetany in rachitic
rats by suddenly shifting from the rachitic ration to a normal ration
such as dried milk and wheat. These workers felt that the reaction was
brought about by the sudden shift in Ca:P ratio and not by any absolute
or relative increase in phosphorus in the dietary. Changing the ratio
from 4:1 to 1.5:1 or 1:1 caused tetany. If the ratio was decreased to
only 2:1 the condition did not follow. In their discussion, the authors
make this interesting comment - that "attention should be directed to
the effect of marked alterations in the makeup of dietaries since such
shifts might help to explain nutritional disturbances which are inexplic-

able from the standpoint of adequacy." (45)

PLAN OF EXPERIMENT
In setting up the present series of experiments, the original plan
was to study the effect of varying the acidebase value of the rachitic
diet. The study of such a variation did not seem to be thoroughly brought

out inthe literature. In fact, there was considerable controversy on the

15

subject as has already been indicated and the comparison of experiments
by different individuals was difficult. The plan was to use the same
basal rachitic ration and to vary the alkalinity by the addition of equi-
valent amounts of mono-, di-, and tri-sodium phosphates. After running
the preliminary experiments, the s00pe of the work was increased in an
attempt to correlate the amount of blood phosphorus, the alkalinity of
the blood, the percentage of bone ash, and the pH of the intestional

tract.

EXPERIMENT
The basal ration used throughout this series of experiments was
the M.S.C. variation of the Steenbock and Black rachitic ration. Its
composition is as follows:

Corn meal 58%
Oats, ground 58%

Gluten 20%
NaCl 1

By titrating the total alkalinity of this ration as represented by the
ash, a value of 400 cc. of N/lO alkali per 100 g. of ration was obtained.
Shohl gives 510 cc of N/lO alkali for the Steenbock - Black ration.

The animals used for these experiments were from the laboratory
here. Both albinos and piebald rats were used. The animals were started
when they were 28 days old and weighed approximately 50 — 60 g.

The rations fed to the various groups are listed below.

Group 5 l Basal ration

Group S 2 " " plus 1% NaHgPO ° H20

Group S 5 " ” plus 1.02% Na2HPO4

14

Group S 4 Basal ration plus 2.75 % Na5P04 ' 12H20

t!

S 5 ”
S 6 "
S 7 "
S 8 ”
S 9 '

plus 1% sodium acid pyrophosphate

plus 1% sodium metaphoSphate

plus 1% yeast

with 1% of oats replaced by 1% non—
irradiated yeast. Referred to as Y1
with 1% oats replaced by 1% irradiated

yeast. Referred to as 12

510 11 plus 1% NaHgP04 ' 320

811 Y2 plus
812 Y1 plus
$15 12 plus
S14 11 plus

815 12 plus

1.02 % NagHP04

2.75 % Na5P04 . 12320

$16 11 plus N/20 HCl for drinking water

These rations were given ad libitum to the rats from the time they were

first started on the experiment. They were kept on the experiment for

four weeks and then were killed for the various analyses.

A final series was run in comparison with these to determine the

effect of adding the phosphate after the animal had been on a rachitic

diet for three weeks and to compare any rise in blood phosphate to that

caused by fasting after a similar period on the basal diet. The plan

of this experiment was as follows showing the change of ration after

three weeks on the basal:

Sgl Basal ration for 4 days

Sgl? Fasted for 24 hours

15

Sgl8 Fasted for 48 hours

Sgl9 Basal plus 1% NaH2P0 ° H20 for 24 hours

Sg20 " " " " " " " 48 hours
Sg21 w w w w w w ' w 4 days
Sg22 " " " " ” " plus 1% irradiated yeast for 48 hours

Bone analyses were run on all of the animals. The percentage ash of
the femur was determined on the fat-free bone. In the first groups, the
amount of calcification was compared by use of the silver nitrate test
on longitudinal sections of the wrist bones.

For blood analysis, it was found most practical to anesthetize
the animals slightly and obtain blood by cutting directly into the heart.
Anesthesia seemed to cause very little change in the blood values and
to cause less struggle and excitement than stunning. The blood of all
animals of any one group was pooled to obtain sufficient for analysis.
The blood was citrated and immediately centrifuged to obtain the plasma.
The carbon dioxide combining power and the phosphorous content were
determined on thiSLflasma. The carbon dioxide combining power was deter-
mined immediately after obtaining the blood by using the VanSlyke
apparatus. The phOSphorus content of the plasma was determined color-
imetrically. The protein in this sample was precipitated with tri-
chloracetic acid immediately after obtaining the plasma.

In making pH determinations on the intestional tract, the animals
were killed between eight and ten o'clock in the morning. Another worker
in the laboratory had found this the time for most uniform contents in

the stomach and intestional tract. Both Openings to the stomach were

16

tied off immediately and also both openings of the caecum. The tract
was then out just above the stomach and just below the caecum and the
stomach and intestine placed in physiological salt solution to pre—
vent drying of the tissue. Two sections of the stomach, four equal
portions of the small intestine and the caecum were the divisions
made. The contents were emptied into beakers and enough distilled
water was added to give uniform suSpensions. The determination of
the pH was made electrometrically by use of the quinhydrone electrode.

On two series, the pH of the urine was determined. The separa-
tion of urine and feces was made by means of Special metabolism cages
in use in the laboratory. The pH was determined electrometrically
as above.

RESULTS

The results of the first two preliminary experiments are shown
in Table I. The percentage ash of the fat-free bone from the animals
on the basal rachitic ration varies over a large range but is usually
below 50%. The addition of phosphate in the quantities used produced
uniform bone ash within groups. The mono—, di-, and tri-sodium phos—
phates were utilized equally well. The pyrophOSphate in the amount
used gave as good calcification as the orthophosphate indicating the
utilization of phosphorus in this compound. The metaphosphate seemed
to be utilized slightly less readily than the other compounds. The
addition of yeast to the basal ration had no positive effect on
calcification. From the silver nitrate test and also from the bone

ash values, it seemed evident that yeast causes a more severe degree

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ma.mm mma me .e me .
em.nm oaa mm .s ma . Hem
Hm.0m om an .s N e
mm.m~ om om .s H osoz Hem

 

 

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has a Hanan HmHsHsH .

18

.somsom enhances

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e e z a : chmm NNH mb ca mm a s
a = s a : ®N.HN mOH km .8 ha a a 59m
= z s a : mucom
8 3 3 8 —- gomm «m mm .8 ¢H up .-
demon some moves hapsmflam weaknesses mm.mm am pm .h ma ammo» RH saw
a = s : Owovfl mflH Mb .8 mm = a :
mom moose on oasssmasoo ao.ms hoe mm .s an e e . mom
a = e a H®.¢¢ OHM Hm om NH a a :
sm.mm hm mm .8 HH
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meson means no unanimommm enema mamas: pnwwoa Hmsfism Hmmsm op defivapw4 mdomo
new m HosAh HeapHsH .

 

. . seasons

19

of rickets. This may perhaps be explained by the slightly increased rate
of growth that is effected by supplementing the rachitic ration with yeast.
Table II shows the growth and ash analyses of the bones of the
animals fed the orthophOSphate salts with and without the addition of
vitamin D, the latter supplied by irradiation of the yeast. The ash
of the bones of animals receiving the mono-, di-, and tri-sodium phos-
phate without the vitamin show the same composition as in the previous
series. The addition to the basal ration of the vitamin by means of
irradiated yeast increased the percentage of bone ash but not as much
as produced by the combined use of phosphate and vitamin D. The unusually
close agreement of ash values on the mono-, di~, and tri—sodium phOSphate
diets plus the irradiated yeast, indicate normal calcification. The
combination of the phOSphate and vitamin D increased the percentage ash
above that obtained by the addition of either irradiated yeast or phos-
phate alone.

In parallel, the 002 combining power of the blood and the phosphorus
content of the blood plasma of these same animals was determined. The
results are shown in Table III. For some reason, the inorganic phos-
phorus content of the plasma did now show any uniformity. This maytave
been due to errors in technique. With one series, difficulty was en—
countered in the actual colorimetric comparison.‘ The reason for this was
not discovered, but seemed to involve a fading of color in the standard
solution. Checks on the latter did not clear up the difficulty. However,
the inorganic phOSphorus content of the plasma of the rats on the basal

ration showed a surprising uniformity and the addition of irradiated

TABLE II — Growth and Percentage Bone Ash of Animals Given
Phosphate in Addition to the Basal Rachitic Ration
With and Without Vitamin D

 

 

' . Initial Final % ash
'ngup ‘Addition to basal Animal Weight Weight Femur
Scl None 29 m. 54 99 18.61
" 50 f. 64 101 27.45
" 52 m. 60 95 19.67
" 55 f. 65 89 27.55
Sd8 1% non-irr. yeast 49 m. 66 117 28.42
" " ” 50 f. 75 108 52.72

" " " 51 f. 61 150 -
" " " 52 m. 77 157 24.55

" ” " 55 m. 65 127 -

Se8 1% non-irr. yeast 68 f. 61 92 26.55
” " " 69 m. 68 126 24.47
" " ” 70 f. 69 115 27.55
Sel6 1% non-irr. yeast 92 f. 52 88 26.75
plus HCl for water 95 f. 62 102 29.10
” 94 f. 65 61 54.25
Sc2 1% NaH 4 ‘ H 0 54 m. 62 109 48.72
" 3 a 55 r. 58 104 44.09
" " " 56 f. 56 91 46.04
" " " 57 m. 64 125 44.87
" ” " 58 m. 68 110 47.85
Sle (1% non—irr. yeast 54 m. 65 150 47.89
(plus 1% NaH2P04 ° 55 f. 59 124 55.09
(H20 56 f. 64 129 44.45
” 57 m. 69 150 50.64
SelO (1% non-irr. yeast 74 f. 52 100 40.89
(plus 1% NaHZP04 . 75 f. 54 109 50.11
(H20 76 m. 65 141 51.82
Sc5 1.02% Na2HP04 59 m. 65 118 59.05
a n 40 £., 66 90 46.29
a n 41 m- 67 127 42.54
a w 42 r. 57 101 40.60
" " 45 m- 65 109 41.96

TABLE II — continued

 

 

' Initial 57§inal % ash
'Group __Addition to basal Animal Weight Weight Femur
Sdl2 (1% non—irr yeast plus 59 f. 75 140 59.67
(1.02% NazHP04 60 m. 65 148 47.95

( " " 61 m. 65 148 47.62

( " " 62 r. 75 154 50.21

( " " 65 r. 66 104 50.82

Se12 (1% non-irr. yeast plus 80 f. 57 106 44.71
(1.02% NaZHP04 81 m. 59 157 51.01

( n n 82 m. 75 175 45.65

Sc4 2.75% Na§P04 ° 12H20 44 f. 57 95 49.70
' " 45 m. 65 99 48.06

" " " 46 f. 55 97 50.16

" " " 47 m. 66 107 47.75

" " " 48 f. 69 104 50.75

Sd14 (1% non-irr yeast plus 64 f. 66 151 51.67
(2.75% Na3P04 - 12 H20 65 m. 85 210 52.24

( n n n n 66 m. 74 184 51.94

( u n n n 67 f. 70 105 50375

Se14 (1% non-irr. yeast plus 86 f. 51 110 46.48
(2.75% N85P04 . 12 H20 87 r. 47 89 50.49

( " " " n 88 m. 71 198 51.80

Se9 1% irr. yeast 71 f. 54 108 48.17
" " " 72 m. 75 145 47.78

" " " 75 f. 68 104 50.55

Sell (1% irr. yeast plus 77 f. 61 158 56.90
(" " " 79 r. 71 140 56.79

8815 (1% irr. yeast plus 1 85 m. 57 150 54.25
(1.02 % NagHP04 84 m. 56 157 57.62

( " " 85 r. 65 175 56.55

Se15 (1% irr. yeast plus 89 m. 59 164 56.09
(2°7f% N85§°4 ; 12§2° 90 f- 64 150 52.81

( 91 f. 64 151 56.79

21

TABLE III — Carbon Dioxide Combining Power and Inorganic

22

Phosphorus Content of the Blood of Animals Given
Phosphate in Addition to the Basal Rachitic Ration

 

'Group Addition to Basal
I

——1

Mg. inorganic P
per 100 cc. plasma absorbed

cc. of Co '
5y '

100 cc. plasma'

Scl None 5.55 48.1'**
Sd8 1% non-irr. yeast (2.66) 1.04 42.6 **
Se8 ' " " " 2.90 54.2
Sf8 “ " ” ” 2.69 54.8
Sel6 " " W " plus 1/20
801 to drink 2.91 55.8

Se9 1% irr. yeast 5.52 55.6
Sf9 " " ” 5.84 56.7
802 1% 118321304 ° 320 6 o 55 47 .1“
Sle 1% non-irr. yeast plus NafigPO -

H20 (6.16 7.28 40.8**
8810 " " " " 4.00 58.1
SflO " " " " 4.64* 61.6
Sell 1% Irr. yeast plus NaHaP04 ° H20 8.08 66.7
Sfll " ” " u " 4.96* 58.9
Sc5 1.02% NazHP04 5.70 55.2**
Sdl2 1% non. irr. yeast plus 1.02%

N32HP04 (6.48) 7.04 -
8612 " " " n " " 5.68 58.6
Sf12 " " " " " " 4.48 55.7
8912 1% irr. yeast plus 1.02% N82HPO4 7.44 65.4
Sf15 " " " " " " 6.56 65.1
804 2.75% Na5P04 ~ 12 H20 4.50 48.o**
Sdl4 1% non—irr. yeast plus 2.75%

Na§P04 - 12 H20 (7.76, 7.44 45.6**
8614 ' " 7.12 56.2
Sf14 " " " ” 5.6* 60.7
8815 1% Irr. yeast plus 2.75% NaSP04 -

12 920 (5.56) 7.28 61.7
Sf15 " " _1," " ' " 6.4* 65.4

 

- * Value not accurate due to difficulty in making color comparison.

** Exhaled air supplied by different person.

25

yeast to the basal ration also gave consistent results. In general,

it can be said that the addition of the vitamin increased the inorganic
phosphorus content of the blood even for those animals already receiving
phosphate.

The 002 combining power of the blood was the same regardless of
the phosphate used. Here, again, the irradiation of the yeast
increased the value slightly. An interesting point was encountered
that is somewhat of a side issue but is important in drawing con—
clusions from the data. In supplying the 002 for the saturation of
the blood sample, two different people ran the experiment. The 002
supplied by one of these gave results uniformly 5 to 10 cc. lower
than the values obtained by the other person. This result is again
shown in Table 7. It is important because in order to compare results
from several different types of rations, the same person should run
the determinations.

Table IV shows the results of the determinations of the pH of
the intestional tract of individual animals from the same groups as
given above. In general, these results indicate that the addition
of the most alkaline salt caused the greatest increase in acidity.

This is rather unexpected inasmuch as the addition of a basic salt

to an already decidedly basic ration should probably increase the
alkalinity. The mono-sodium phOSphate gave values corresponding to

the basal. Even in the upper part of the small intestine, the contents
of the tract were slightly alkaline. Normally this would be acid as

a result of the presence of the gastric juices. The lower part of

the small intestine was quite alkaline, approaching neutrality again

24

in the caecum. The di-sodium phosphate seemed to lower the pH through-
out the tract. The tri-sodium salt lowered the pH decidedly with about
three quarters of the small intestine showing a pH less than 7.0. The
ration did not seem to iTect the pH of the stomach to any degree. Again
the irradiated yeast in any of these diets increased the amount of lower—
ing of the pH.

With two series of animals, the pH of the urine was determined.

The values obtained were as follows:

urin
Basal ration
Group Scl 8.50
Group Sd8 8.89
Group se8 8.58
Basal plus 1% irr. yst.
Group Se9 7.57
1% NaH2PO4'1820 addition
Group Sle 8.72
Group Sc2 8.55
1% NaH2P04 ' H20 plus 1%
irr. yeast.
Group Sell 8.58
1.02% NaZHP04 addition
Group Sc5 8.89
Group Sd12 9.16
1.02% N82HP04 plus 1%
irr. yeast
Group Se15 8.29
2.75% N85P04 addition
Group S04 8.65
Group Sd14 9.06
2.75% N85P04 plus 1% irr.
yeast
Group Se15 9.06

Since the values were all so extremely alkaline and appeared to give

no definite information, no further work was done along this line.

25

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66.6 66.6 66.2 66.2 62.2 62.6 66.6 66 6662 66662 .222 26 V6666
22.6 66.6 62.2 66.6 66.6 66.6 66.6 666 666 666 6226
66.6 62.2 66.6 66.6 66.6 66.6 66.6 66 . 662662 662.6 66666 6666
26.2 66.6 66.6 66.2 66.2 66.6 66 .6662 .222:662 666 6266
66.6 66.6 26.2 66.6 22.6 2 .6 66.6 666 6626662 666.22 6626
62.6 62.2 26.2 62.2 66.2 66.6 26.6 66 6626 66662 .222 666 6266
62.2 66.2 66.2 66.2 66.2 66.6 66.6 666 62666 66 V 6626
26.2 66.6 26.2 26.2 66.2 66.6 66 6626662 266.6 66666 6666
66.6 66.6 66.6 66.6 66.6 62.6 66 66662 .2261266 266 6666
66.6 66.6 66.2 66.6 66.6 66.6 66.6 666 666 . 6666666 Raw 6626
62.6 66.6 66.6 66.6 66.6 66.6 26.6 62 6666 66662 .226 66 6666
66.2 66.6 66.2 66.2 66.2 62.6 66.6 666 2 2 6 V 6626
62.6 62.6 26.6 66.6 66.2 66.6 62 6 6 6 my 6666
66.2 66.6 66.6 66.6 62.2 .66.6 26 . 62 662 66 66266 g
66.2 66.6 66.6 66.2 66.2 66.6 66 26662 .222.666 266 6666
66.6 66.2 66.2 66.2 - 66.6 26.6 66 2 2 . 666
66.6 62.2 66.2 66.6 66.6 66.6 62 26662 .226 66 666
66.6 66.6 66.2 66.2 66.2 66.6 66.6 66 g . 2 . 626
66.2 66.6 66.6 66.6 26.2 66.6 66 2 . 2 a 666
66.2 62.6 66.6 66.6 66.2 66.6 66 2 2 . 2 2
66.2 66.6 66.6 62.6 66.2 62.6 66 26662 .222-666 66 666
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. 662666666 66666 6666666 _ .

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26

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6 6 6 66 66 66 .6 666
6 6 6 66 66 66 .a 666
6.66 66.6 6266 6 66666666 oz 66 66 66 .6 666
= = a = a = a = @OH mm mm .8 OmH
6.66 66.6 .626 66.666.226 66 6 6 6 6 66 66 66 .6 666
6 6 6 6 6 6 6 666 666 66 .6 666
2.66 66.6 6666 6 6 6 6 6 6 62 22 66 .6 266
6 6 6 6 6 6 666 666 66 .6 666
6 6.66 66.6 .626 66 6 6 6 6 6 66 66 66 .6 666
6 6 = 6 6 6 26 66 66 .6 666
6.66 66.6 .626 66 66666 666 . 6666666 66 666 666 66 .6 666
6 6 62 26 66 .6 666
n 66.6 .626 66 6 66 666 62 .6 666
6 6 66 62 66 .6 666
6.66 66.6 .626 66 666666 666 666 62 .6 666
. 666666..66666 666666.66666 6666666 666666 .66: 6 666666 .

. 266LW66.66 .66666\ 6.6: 66 6666 666666 66666 66 666666 66666 666666 6666666 666666.

27

The last series was suggested by the studies of tetany in
rachitic animals already referred to in the literature. It was
planned to study the increase in inorganic phosphorus content of the
blood caused by fasting and by the addition of the same amount of
phosphate that had been used in the other rations. Since no apprec-
iable difference had been found in the three orthOphosphates, the
mono-sodium phosphate was used in this experiment. The results are
given in Table V. Tetany in the fasting animals was not observed
while the animals were alive. The first group, however, which were
killed after 24 hours, showed intense muscular contraction after
anesthesia and bleeding but of a more violent type from that ordinarily
shown by the drying of tissues. The second.group showed this same
phenomenon but to a much greater degree. One animal completely doubled
up as soon as it was anesthesitized. The muscular contraction in
this latter group consisted of general twitching, movement of toes,
ears, and jaw. The blood picture of these animals showed rapid rise
of the inorganic phosphorus to an unduly high degree. The value
doubled within the first 24 hours. The 002 combining power of the
blood of the first group showed a very low value. Unfortunately,
there was not sufficient blood obtained from the later group to make
this determination.

Addition of phOSphate to the ration of the rachitic rat resulted
in a slow but decided increase in inorganic phosphorus. Likewise,
the 002 combining power of the blood increased immediately. No ill
effects seemed evident from this change. The new ratio of Ca:P in

the ratio was approximately 1.5:1. A change of the ratio to 1:1 is

28

supposed to produce tetany and Shohl has obtained slight tetany

with a change to 2:1.

DISCUSSION

The limiting factor in bone develOpment on a rickets producing
diet seems to be the amount of available phOSphorus. The Steenbock -
Black ration is known to be low in phosphorus content. The avail-
ability of the element is further hampered by the presence of a large
excess of calcium. As a result, the animals placed on such a ration
are unable to absorb enough phosphorus to supply the needs of calci-
fication during growth, and the condition known as rickets is pro-
duced. Addition of vitamin D to the basal ration increases the
amount of inorganic phOSphorus in the blood and thereby increases
the amount of the calcium phosphate salt deposited in the bones.
Since the amount of phosphorus in the ration has not been altered
in this case, the vitamin has, therefore, increased the availability
and the absorption of the element. In the present experiment it has
been shown that the alkalinity of the intestional tract decreased
from the values obtained on the basal diet when the vitamin was fed.
The indication is that the vitamin aided in the absorption of the
phosphorus by decreasing the alkalinity of the digestive tract and
thereby increasing the solubility of the phosphorus. Supplying
vitamin D does not give the best possible calcification of the bone,
however. Evidently, the small amount of phosphorus in the basal
ration is insufficient for the needs of the growing animal even under

the best conditions. So, the addition of any form of phOSphate to

29

the basal ration already supplemented with vitamin D increased the
mineral deposition in the bone.

On the other hand, the absorption of phOSphorus is dependent on
the acidity of the intestional tract only when that element is present
in the ration in limited quantities. If the phosghorus is supplied in
a soluble form, it is readily absorbed and utilized. The theory might
be advanced that phOSphate in an alkaline medium, such as is found in
the intestional tract of the animal on the basal rachitic ration, in
the presence of a large amount of calcium would form the insoluble
calcium phosphate which would then be in an unavailable form to the
animal. However, even though the reaction of the intestional tract
showed no increase in acidity, if sufficient soluble phosphate is
added, enough is absorbed to increase the calcification above that
found in the bone of the rachitic animal. Nevertheless the amount
of phosphorus and, therefore, the amount of bone ash absorbed is
limited after a certain point by the alkalinity. Addition of vitamin
D to the phOSphate supplemented diet increased the inorganic phOSphorus
in the blood and also the bone ash values above that obtained on the
phosphate supplemented diets. Here again, the change was accompanied
by decrease in the alkalinity of the intestional tract. Beyond a
certain point, the absorption of the phOSphorus seems again to be

dependent on the acidity of the intestional tract.

 

50

CONCLUSIONS

The results of these series of experiments may be summarized as
follows:

1. Deve10pment of rickets on the Steenbock - Black ration seems
to be due to a deficiency in available phosphorus which in turn is
influenced by the reaction of the intestional tract.

2. Addition of vitamin D to the diet increased the acidity and
probably, therefore, increased the amount of available phosphorus.

5. Bone develOpment was improved by simply supplementing the
rachitic ration with soluble phOSphates. In this case, the acidity
of the intestional tract seemed to be of relatively little importance.

4. The mono-, di-, and tri-sodium orthophosphates as well as
sodium pyrophosphate were apparently equally effective in improving
bone development. Sodium metaphosphate was slightly less effective.

5. In all cases, the addition of a phosphate increased the
inorganic phOSphorus content of the blood.

6. Similarly an increase in the alkalinity as determined by the
002 combining power was observed with each phosphorus supplement.

7. Healing of rickets during fasting is probably due to an

increase in inorganic blood phosPhorus.

l.

2.

5.

5.

6.

7.

8.

9.

H.

P.

A.

51

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52

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55

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