STUDIES ON THE PRODUCTION OF RICKETS IN RATS AND
THE MODE OF ACTION OF VITAMIN D

A Thesis

Submitted to the Faculty of Michigan State
College in Partial Fulfillment of the
Requirements for the Degree of
Doctor of Philosophy

By
Frederick Ling-yun Ma

Department of Chemistry
June, 1937

ProQuest Number: 10008468

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ACKNOWLEDGMENT

The writer of this thesis wishes to
express his sincerest appreciation
and gratitude to Dr, C. A. Hoppert,
Professor of Biochemistry at Michigan
State College, for his constant
assistance in the conduct of these
experiments herein reported and for
his kindly criticism in the preparation
of the manuscript.

TABLE OF CONTENTS

STATEMENT OF THE PROBLEM

PART I

Page

STUDIES ON THE PRODUCTION OF RICKETS IN RATS

A.

B.

Some Modifications in the Steenbock
Ration 2965
Vitamin D Content of Irradiated Corn,
A preliminary Report

1

11

PART II
STUDIES ON THE MODE OF ACTION OF VITAMIN D
' A*
B.

C.

Introduction

17

The Effect of Vitamin D on Fecal
Alkalinity etc.
in Both YoungandAdult Rats

19

The Effect of Rickets and Vitamin D on
Plasma phosphatase Activity inRats

33

SUMMARY AND CONCLUSIONS

39

BIBLIOGRAPHY

41

STATEMENT OF THE PROBLEM

Although the various phases of the work on vitamin D and
rickets have attracted much attention of investigators all over the
world in recent years yet few attempts have been made to study the
mode of action of this vitamin, and to modify the few classical
rachitogenic diets used for experimental purposes.

As a result,

a great deal has been discovered about the chemical nature, the
distribution and the clinical and commercial applications of vitamin
D, but the exact nature in which vitamin D operates within the
body in the prevention and cure of rickets is still unknown*
Likewise, the rachitogenic rations recommended by early workers
may prove to be satisfactory in some cases, but wide variations
and irregularities have also been reported by different laboratories
where such rations are employed for producing experimental rickets.
With these in mind, the following studies have been undertaken in
the hope that some light might be thrown upon the problems just
mentioned.
For convenience in presentation, the results of the work
have been divided Into two major parts, each of which has been
again redivided into two subdivisions according to the nature of
the work.

Part I is concerned with the production of rickets in

rats while Part II represents some studies made on the mode of action
of vitamin D.

PART I

STUDIES ON THE PRODUCTION OF RICKETS

K RATS

A.

SOME MODIFICATIONS IN THE STEENBOCK RACHITOGENIC RATION 2965

HISTORICAL AND INTRODUCTION

Since the publication by Harris and Bunker (1931, 1934,
1935) on the "variability in the corn component of a rachitogenic
diet" many similar studies have appeared in the scientific
literature during recent years.

Holmes and Tripp (1933) analyzing

samples of rachitogenic rations (Steenbock ration 2965) from different
laboratories, found variations in ash, Ca and P contents and in the
Ca:P ratios.

Samples of ground maize from representative milling

concerns showed considerable variation in fineness, ash, Ca and P
contents, and in Ca;P ratios.

Studying the effects of cereals and

common salt on the development of rickets GyBrgy (1933) concluded,
that the kind of cereal and the amount of NaCl in the diet are
important factors in determining the development of rickets in
rats.

Zucker, Hall, Mason and Young (1933) believed that in order

to obtain good results in vitamin D assay, rats used for the tests
must have good growth during the testing period so that compli­
cations and the loss of animals may be avoided.

These authors

devised a rachitogenic ration which will give growth comparable
with that of a good stock diet, during the three weeks of experi­
mentation.

In a series of publications Steenbock and co-workers

(Templin and Steenbock, 1933, 1 and 2; Lowe and Steenbock, 1936,
1 and 2) reported that germinated autolyzed maize, immature maize
or maize treated with HC1 were less rachitogenic than mature maize

2

owing to an increased content of inorganic P*

In another paper

Thomas and Steenbock (1936) stated that no significant difference
was observed in the growth produced or in the severity of rickets
produced by any of the cereals (maize, v/hole wheat, patent flour
and polished rice)*

ITijveld (1936) found that the addition of

dried liver or of liver extract to the Steenbock ration 2965 caused
improved appetite and growth in young rats*

These beneficial

effects did not interfere with the development of rickets, and
prevented loss of test animals by intercurrent diseases during
experiments •
From these studies two generalizations may be drawn
concerning the qualities of a satisfactory rachitogenic ration*
In order to produce experimental rickets In rats successfully,
the rachitogenic diet used must posses the following two qualities:
1*

The corn (or other cereal) component must have a
constant chemical composition which is favorably
rachitogenic•

2*

Enough growth-promoting factor must be present in
the rachitogenic diet so that the rats may have a
proper gain in weight during the experimental
peridd*

In view of the fact that there is a possibility that
ground whole corn may contain variable amounts of vitamin D which
would affect the degree of rickets produced on a diet containing
such a corn it was considered desirable to attempt to develop a

z

rachitogenic diet using granulated yellow corn or table corn meal.
Since in the preparation of the latter most of the bran is removed,
any vitamin D which might have been formed in the outer layers of
the corn kernel due to the exposure of corn to sunlight during
harvesting would be eliminated.

Using the Steenbock ration 2965

formula as a basis granulated corn was substituted for whole corn
meal and certain other modifications were made which were intended
to yield a satisfactory rachitogenic diet.

EXPERIMENTAL
The original Steenbock ration 2965 as well as six modified
rations were used in this experiment.

The rations and their modifi­

cations are as follows:
Ration Ho.
I.
II.
III.
IV.
V.
VI.

Mo di fi c ati on s
Steenbock ration 2965, no modification
Same as I using granulated corn
Same as II plus 1% dried yeast
Same as II plus 5% linseed meal
Same as II including 25%> ground oats
Same as V plus 1% dried yeast

VII.

Same as V plus 5% linseed meal

Hote:

Ytlherever yeast, linseed meal, or ground oats were
introduced into the ration, corresponding amounts
of corn were reduced

4

Ten young rats approximately three weeks of age weighing
50 to 60 grains were placed on each ration.

The usual care in

obtaining uniform distribution of rats with regard to litter and
sex was observed.

At the end of three weeks, animals 1-5 of each

group were given 2,7 U, S. P, units of vitamin D standard reference
oil while animals 4-7 received a supplement of M, S, C. vitamin D
milk to supply the same amount of vitamin D,
basal rations only.

Animal s 8-10 received

All animals were killed at the end of ten

days after the beginning of the supplements.

The radii and ulnae

were removed for line test and femura, for total ash analysis.

After having been preserved in 95% alcohol for at least
24 hours the w i s t bones were split and stained for 2 minutes in
a 2% AgNOg solution.

The stained bones were then washed and

immersed in distilled water and were exposed to either natural or
artificial light until a desired darkness in the calcified area of
the bone had been developed.

As soon as this was achieved the bones

were fixed in a 10% sodium thiosulphate solution to prevent further
change in color.

A representative bone from each rat was selected

for making the photographic plate.
The femura were extracted for 40 hours with 95c
% alcohol and
the clean, dried bones were ashed in a muffle fumance, and their
total ash content calculated.

5

Growth during the experimental period, first three weeks,
ash content of femura, the general appearance of the line and the
width of the metaphysis were employed as criteria for the quality
of a satisfactory rachitogenic ration,

RESULTS AND DISCUSSION
Results of the growth response of the animals during the
three-week experimental period are presented in Table I and Graph I
while those of the line tests and bone ash determinations are presented
in Plate I and Table II respectively.

Vi/hen granulated corn was substituted for ordinary corn
in the Steenbock ration 2965 the growth rate of the animals was
greatly reduced.

Rats fed Ration II made an average gain of only

nine grams

in three weeks.

A growth-promoting factor was evidently

lacking in

the granulated corn.

This was believed to be due to the

removal, during the process of milling of the granulated corn, of
both the germ and the outer hull of the corn grain.

A deficiency

in vitamins B and G and of essential amino acids in the granulated
corn seemed to be the main cause for poor growth.

VJhen 1% yeast

(Ration III) or 5% linseed meal (Ration IY) or 25% oatmeal (Ration V)
was added,

growth of the test animals was improved slightly in each

case.

animals made average gains of 16 grams, 18 grams and 19

The

grams respectively, during the three week period.

Since best results

in vitamin D assay are cb tained from animals which will gain 25-55
grams at the end of three weeks after they have been placed on a

6

rachitogenic ration the above improvements were not considered
sufficient*

The most satisfactory rachitogenic ration in this

experiment, from the standpoint of promoting growth was found to
be Ration VI in which both dried yeast and oatmeal were added.
Animals fed Ration VI gained an average of 35 grams in three weeks*
Ration VII was the second best as far as the promotion of growth
is concerned*

Animals fed this ration gained 28 grams*

Plate I shows the results -of the line tests*

If Steenbock

ration 2965 (Ration I) is consi de.red as a standard it may be seen
that at least two other modified rations gave equally good or better
results.

When the width of the metaphysis, the size of the bone,

the uniformity, location and general appearance of the line are
all considered Ration VI proved to be definitely superior while
Ration VII was equally as good as the Steenbock Ration (Ration i).
Other rations were considered unsatisfactory because of the inferior
lines produced in response to feeding vitamin D*
Little difference was observed in the ash content of femura
of the test animals.

Regardless of the ration fed the femura of the

negative control or basal animals (animals 8-10) all had a very low
ash content, indicating severe rickets.

On the other hand, the

total bone ash of all animals which had been supplemented either
•with vitamin D standard reference oil or with vitamin D milk was
increased slightly in each case,

ho comparisons could be made from,

the bone ash determinations alone in this experiment, as to which of
the rachitcgenic rations was superior for use in vitamin D assay.
Other factors must also be considered.

Table I
Growth Response of Younr Rats Fed Various Rachitogenic Rations
(All weights are averared from 10 animals)

Initial

Weekly weights

Yft. nained

Ration
3rd week

Grams
Steenbock Ration
2965

Grams

Grams

Grams

in 3 weeks

Grains

53

60

II. Same as I using
r-ranulated c o m

52

53

58

61

III.Same as II plus
l/o yeast

54

55

63

70

16

IV. Same as II plus
5/o linseed meal

52

55

70

18

Same as II plus
25/o oatmeal

55

77

66

24

19

V I • Same as V plus
1% yeast

52

60

73

85

33

VII.Same as V plus
linseed meal

53

60

71

81

28

Table II
Percentage of Bone Ash of Rats Fed Various Rachitogenic Rations

Rations
Vit. D Standard
Reference Oil
(3 rats)
I.

Steenbock Ration
2965

Supplements
Vitamin D Basal Ration Only
hi Ik
(4 rats)
(3 rats)

29.5

24.1

22.2

II. Same as I using
granulated

24.1

23.8

20.0

III.Same as II plus
lfo yeast

26.5

27.2

23.2

IV. Sarae as II plus
5c/
j linseed meal

23.4

24,9

21.7

25.2

22.9

20.5

VI. Same as V plus
1/a yeast

25.9

25.1

21.0

VII.Same as V plus
5/a linseed meal

23.0

26.4

21.6

V.

Same as II plus
25% oatmeal

Plate I.

Results of Line Tests

ANIMALS
1

2

3

4

5

RATIONS

t

6

7

*

VV

y t
ft

1,1 £ ? $

$ $ ♦ $

Animals:

m

•** 9 99v

1 ^ ^ ^

Rations:

10

* 3 «

IV

VII

9

*m

1 v f if
11 * 3 3
111 * 3 $
®
A,

8

V v V

y 9 9 4 fv v v
I, Steenbock ration #2965; II, Sane as I usinr granulated corn
III, Sane as II plus 1% yeast; IV, Sane as II plus 5% linseed
neal; V, Some as II plus 25,ci oatncal; VI, Sane as V plus ly>
yeast; VII, Sane as V plus
linseed neal.
1-3, supplemented with 2.7 U. S. P. units of vitamin D
reference oil each; 4-7, received 10,9 cc. of V.S.C.
vitamin D milk each -which contains 2.7 U. S. P. units of
vitamin D and 8-10, received basal rations only.

B.

VITAMIN D CONTENT OF SUN-CURED AND IRRADIATED CORN,
A PRELIMINARY

report

HISTORICAL AND INTRODUCTION

Since the irregularities reported by Harris and Bunker
(1931, 1934) in the production of experimental rickets, many
investigations have been conducted in various laboratories to find
the variable factor in the rachitogenic ration#

A brief review of

literature pertaining to this subject was included in Part I Section
A of this thesis*

In general, most of the investigators on this

problem were interested in the total P content, the partiti on of P
compounds or the CajP ratios of the rachitogenic ration but none
suspected the possibility of the variation of vitamin D content in
its yellow maize component.

Since c o m is relatively rich in

sterol content it can easily acquire antirachitic properties if
sufficient activation is given, either through exposure to sunshine
or through artificial irradiation.

It is a general practice in many

localities that during harvesting, the corn is husked in the field
and left there for a period of several days before it is taken indoors
for storage.

Under such conditions there is reason to believe that

such corn may receive enough ultraviolet irradiation from the sun
to contain an appreciable quantity of the antirachitic vitamin.
Coward (1933) irradiated two samples cf- dried yeast by
exposure to Indian sunshine for one hour (11:00 A.M. till noon on
October C, 1932) and found that one of the irradiated yeast samples

12

contained 5-20 I. U. of vitamin D per gram when tested biologically.
Bechtel (1955) observed that parts of the corn plant (tassels, silk
and the dry leaves on the lower part of the plant) that were sundried in the field at the time of ensiling were good sources of
vitamin D 5 while dried material prepared from the upper green leaves
of the corn plant was found to be devoid of the antirachitic factor.
He also demonstrated, by means of rat assay, that the sun-dried
material contained 1226-2449 U* S. P. units of vitamin D per pound,
whereas the dried naterial prepared from the upper green leaves
contained no vitamin D when so tested.
The purpose of this preliminary investigation was to
secure data on the vitamin D content of sun-cured corn under conditions
which are comparable to those in general farming practice.

EXPERIMENT TAL AHD RESULTS
I
Six bushels ofyellow corn were collected on October 12,
1956 from the Plant Breeding Farm of the Michigan Agricultural
Experiment Station.

It was husked indoors, and divided into three

equal batches of two bushels each.

One batch was exposed

on clear fair days between the hours of 11:00

to sunlight

A. M. and 5:00 P. M.

for a total of ten hours (October 15-27, 1956) while a second batch
was irradiated under a mercury vapor lamp for

one minute.

batch kept and dried indoors was used as control.

A third

IS

Corn meals made by using these various corns were employed
in preparing basal rs.tions according to the formula of M, S. C*
rachitogenic ration $2 which consists of: corn, 50; oats, 25; wheat
gluten, 20; CaCO3s 3; NaCl, 1 and yeast, 1*

Six standard young rats suitable for vitamin D assay were
placed on each of the three basal rations.
animals v/ere sacrified.

At the end of 30 days the

Results of the condition of metaphysis,

blood P, bone ash analysis, as well as, growth in each group are
presented in Table III#
Ro appreciable difference was obtained when the basal
rations were made up according to the formula of the Steenbock ration
2965 hence the results m i l not be included in this report*

Table

III

Vitamin D Content of Sun-cured and Irradiated Corns

Baso.l Ration,
L'.S.C. Rachito^enic ^2

ITo. of Initial Final ITeI rht Condition Blood
And.mol s Weight Weight Gained
of
P
Iletrohvsi s
Grans

Grams

mBone
Ash

Gr am s

Sun-curee
Corn

6

52

SI

39

Rachitic

3.10

26.6

Irradiated
Corn

C

55

101

48

formal

3.85

42.9

Control
Corn

6

55

89

56

RachitL c

2.01

22.4

15

DISCUSSION

Although no striking evidence was obtained in this
preliminary investigation, the results offer some support of the
view that under proper conditions of activation determined by season,
locality, the time of exposure etc. it is possible to increase vitamin
D content of c o m by the process of sun-curing.

The fact that rats

fed the sun-cured c o m had slightly higher blood P and bone ash
than those fed the control corn indicates clearly that the suncured corn was definitely less rachitogenic than that of the control
corn.

Rats fed the irradiated corn did not develop rickets at all,

as shown by the normal condition of the metaphysis, 3,85 blood P
and 42.9^ bone ash.

The results of the irradiated corn were con­

sidered significant because they indicate the abundance of pro-vitamin
D in the corn grain which can be easily activated.

Since all the

corn used for either sun-curing or uiltraviolet light Irradiation was
left on the cob and no efforts were made to turn the cobs over during
either process, the actual area of corn surface exposed was rather
small.

Considering the limited area of stirface exposed in each

corn grain, and the low intensity of ultraviolet rays in Michigan
sunshine in October, it is sur£>rising that even under such conditions
some differences were observed between the sun-cured corn and the
control c o m in their rachitogenic properties.

It is believed that

any exposure to the ultraviolet irradiation from a strong sunlight,
for an appreciable length of time, during the harvesting of corn may
alter its vitamin D content considerably.

If this is true the

1G

irregularities reported "by various investigators in the production
of experimental rickets may be partially explained.

Further work

on this problem, is being planned in order to obtain more conclusive
results•

PART II

STUDIES ON THE MODE OF ACTION OF VITAMIN D

A.

INTRODUCTION

Only a few references are available in the scientific
literature on the mode of action of the antirachitic vitamin since
its recognition by McCollum (1922).

It has been proven beyond doubt

that vitamin D is essential for normal Ca-P metabolism, for the
development of sound bone and teeth and for the prevention and cure
of rickets both in man and in animals, but the true mechanism in
which vitamin D acts in regulating these important biological
processes has never been made clear*

In 1926 Lewis and Zotterman first suggested that ultra­
violet light forms in the skin a histamine-like substance which,
presumably, would increase the gastric secretions*

The healing

action of vitamin D might be due to this increased gastric secretion.
This theory was disproved recently by Daly (1933) who Injected some
histamine into rachitic rats and produced a great increase of secre­
tion of gastric juice but no evidence of healing of the rachitic bones*
Bauer and Haddock (1931) were also unable to explain the healing
action of vitamin D on the basis of an increased formation of acid
in the gastro-intestinal tract*
proposed

Taylor, Brannon and Kay (1930)

that vitamin D functions by stimulating the parathyroid,

but Harris, (1932) after investigating clinical hypervitaminosis,
substantiated a theory that the characteristic mode of action of
vitamin D is to permit an increased "net absorption’* of Ca and / or
P.from the gut; tending to raise the level of the Ca and / or P, the
latter rise, automatically brings about increased calcification in

18

sites provided with the "bone" or calcifying enzyme, phosphatase*
Upon injection of a single large dose of viosterol (200,000 I. U.
of vitamin D) to rabbits, Eeymann (1936) found, that the rabbit
serum was rendered antirachitically active.
injections of
days.

0*6

Daily intramuscular

ml* of such serum cured rachitic rats in

8

to

10

The antirachitic substance (vitamin D) remained in the rabbit

blood for two to three months.

From the above evidence it is safe

to assume that vitamin D operates in the blood stream regulating the
’’net absorption” or ’’net retention1’ of Ca and / or P rather than at
the site of calcification.

The present investigation was planned

to obtain further evidence in support of this view.

B*

THE

effect

of

vitamin

d

on

fecal a lkalinity, fecal n

.

blood

p

and pH OF THE ALIMENTARY CANAL IN BOTH YOUNG AND ADULT RATS

HISTORICAL

As early as 1924 Zucker and Matzner observed that the
feces of rats, fed a rachitogenic diet, gave a pH of 7.4 to 8,0
when suspended in water.

When such rats were given a supplement of

cod liver oil, the reaction of the feces was lowered to about

6.2

while the feces of control rats, fed cotton seed oil, gave an
unchanged alkaline reaction.

Jephcott and Bacharach confirmed

Zucker and Matzner 1 s observation in 1926 and a few years later the
same authors (Bacharach and Jephcott, 1929) recommended a method for
vitamin D assay based on an examination of the fecal pH of rats on
a rachitogenic ration.

They found that the method was useful and

satisfactory provided certain experimental conditions were observed.
Redman (1929) found only a certain degree of correlation between pH
and % of Ca in the feces of rachitic children.

In studying the Ca-P

excretion in rabbits receiving diets containing varying Ca:P ratios
with and without irradiated ergosterol McGowan (1935) noticed a very
striking effect of irradiated ergosterol in causing a shift of the P
excretion from the feces to the urine in every case.

This evidently

may have an indirect effect on the total amount of Ca excreted through
the feces.

Nitzesou and co-workers (1935) found that treatment with

ultraviolet light reduced the fecal Ca and P in children.

Recently,

20

Friedman (1936) made a comparative study of the pH and bacterial
flora of the feces of normal and rachitic rats and of rachitic rats
healed with irradiated ergosterol or with vitamin D milk.

He

reported that during the development of rickets the pH of the feces
of rats on Steenbock ration 2965 rose from

6.6

healing the pH fell again to a normal level,

6

to 7.6.
.2-6.6 .

During
As far as

is known, no studies have been made trying to correlate the total
fecal alkalinity with the action of vitamin D.

The purpose of the

present experiment was to establish such a relationship.

EXPERIMENTAL

Eight different rations and both young and adult rats
were employed for this experiment.

The rations and their components

are as follows:
1.

Low Ca Ration - yellow corn, 39; ground oats, 39; wheat
gluten, 20; NaCl, 1; plain yeast, 1 and
CaCOg, none.

2.

With Vitamin D - same as 1 using irradiated yeast
instead of plain yeast

3.

I/o CaCOs Ration - Same as 1 including Vfo CaCOs

4.

With vitamin D - Same as 3 using irradiated yeast

5.

2/o CaCOg Ration - Same as 1 including 2% CaCOg

6

.

7.
8

.

With Vitamin D - Same as 5 using irradiated yeast
2>% CaCOg Ration - Same as 1 including

CaC0 3

With Vitamin D - Same as 7 using irradiated yeast

21

Owing to "tlie tremendous amount of time consumed in this
experiment, only two young white rats,

21

to 25 days of* age weighing

between 50 and 60 grams and one adult female rat weighing 200-350
grams were placed on each of the eight rations#

Weekly food consump­

tion and fecal excretion of each animal were recorded and the fecal
samples were dried and saved for analysis*

Care was taken to remove,

as much as possible, the

hairand food contaminations from the fecal

samples before they were

used for any of the determinations#

Determination of Fecal Alkalinity - For total alkalinity
determination, a simple titration method was employed#

One gram of

dried, ground feces was placed in a 500 ml# Erlymer flask to which
50 ml# of a 0#1N H 2 SO4 or HC1 had been added#

The flask was allowed

to stand for two hours at room temperature with occasional shaking#
Then the content was filtered through an ordinary filter paper and
25 ml# aliquots were titrated against 0.1N NaOH using phenolphthalein
as indicator#

The totalalkalinity" of the feces was expressed

as the

No. of ml# of 0.1N alkali per gram of dried feces#
Fecal N and Blood P_ Determinations - Fecal N was determined
by the Kjeldahl method while the blood inorganic F, by the Youngburg
method described in Hawk and Bergeimls ''Practical Physiological
Chemistry", tenth edition*
Determination of p H of Alimentary Canal -

The whole of the

gastro-intestinal tract was removed, and placed in physiolog. cal salt
solution immediately after the animal was killed by etherization.

22

The gastro—intestinal tract was 'then divided into seven portions as
follows:

The stomach, into the cardiac sac and the pylorus; the

small intestine, into three equal portions; the cecum and the colon
were taken as one portion each.

The content of each of the seven

divisions was emptied into a 50 ml. beaker containing 5 ml. of
distilled water and the mixture was thoroughly stirred.

Tito or three

ml* °f the supernatant fluid was taken for pH determination using the
quinhydrone electrode method.

All pH determinations were completed

within thirty minutes.

RESULTS AND DISCUSSION

Results on the growth, food consumption, fecal excretion
etc. and on the fecal h , fecal alkalinity, blood P and on the pH of
alimentary canal of young rats are presented in Tables IV-VII and
Graphs II-III inclusive.

Composite fecal samples from two rats within

each group were taken for the fecal U and focal alkalinity determina­
tions while the determinations for blood F and pH of alimentary canal
were made on individual rats*
Rats on the low Ca diet (Ration l) grew poorly during the
experimental period and seemed to have ]e ss ability to utilize their
food as indicated by the high feces/food ratios.

11/hen vitamin D

was added in the form of irradiated yeast (Ration 2), both growth
and food utilization \vere considerably improved.

In rations 5-6

23

the same general Improvement was also observed.

When the CaCO- level
O

In the diet was raised to Z% of the total weight (Ration 7) vitamin
D seemed to have little or no effect in improving the ability on
food utilization of the test animals.

This appeared to be due to

the artefact resulting from the unusual food mixture in which Z%
CaC 0 3 was incorporated.

The blood P picture in the various groups was as expected
and no significant changes were observed in any of the N determina­
tions.

pH values of the alimentary canal were in agreement with

those found by previous investigators.

These tests were included

merely to check the experimental error and to eliminate other possi­
bilities which might lead to wrong conclusions.
The total alkalinity of the feces of the test animals seemed
to

be directly proportional to the amount of CaCOg included in the

ration.

The feces from rats fed the low Ca ration had an average

alkalinity value of 5.5; on the 1% CaC0 3 ration, 7,8; 2% CaCOg ration,
13.8 and Z°/o CaCC>3 ration, 24.0.

When vitamin D was added to these

same rations the alkalinity was reduced in each case.

The average

values were: 4.5, 6.7, 11.4 and 21.3 respectively.
Only the total fecal alkalinity determinations were made
on

the adult rats.

the young rats.

These animals received the identical care as did

Their food consumption and feces excretion was

recorded weekly and the fecal samples were prepared in the same
manner for the alkalinity tests.

Weekly fecal alkalinity values

24

of the various groups are presented in Table VIII -while the average
valuers-: for the entire experimental period are given in Graph IV.

The results of the total fecal alkalinity tests were con­
sidered significant because they seem to confirm the "net absorption"
or "net retention" theory substantiated by Harris (1932).

In both

lovr Ca and high Ca rickets the ability of the animals to absorb Ca
and / or P from the gut or to retain the Ca and / or P at a normal
level in the blood stream appeared to be at a minimum.

One of the

functions of vitamin D is to remedy the above mentioned abnormal
conditions in the animal organism.

The characteristic action of

vitamin D then, as originally stated by Harris (1932), and further
confirmed by the present experiment, is to improve the "net absorption"
or "net retention" of Ca and / or P resulting in an Increased
calcification of the bones*

Table IY
Growth, Pood Consumption and Fecal Excretion of Younr Rats Fed
Various Rations with and without Vitamin D durinr a

Ration

Animal

Wei rht
rained
Grams

Food
consumed

Feces
excreted

Grams

Grains

4 -week

period

Feces / Food*
ratio

1

Low Ca
ration

a
b

23
26

156
153

18
16

.115
.105

Same plus
irrad. Y.

a
b

83
71

270
282

2b

27

.096
.095

3
1% CaCC>3
ration

a
b

62
60

237
259

23
24

.099
.093

4
Same olus
irrad. Y*

a
b

85
79

27 8
287

24
27

.086
.095

5
2% CaC 0 3
ration

a
b

82
64-

293
264

30
26

.104

Same plus
irrad. Y*

a
b

89
66

284
260

30
24

.105
.094

7
3'7o CaCOg
ration

a
b

75
53

290
260

32
28

.112

a
b

96
89

314
302

34
33

.110
.110

2

.100

6

.109

8

Same nlus
irrad. Y.

* Feces / Food Ratio • Total amount of feces excreted in 4 weeks /
Total amount of food consumed in 4 weeks*

Table V
Blood Inorganic P and Total Fecal F of Younr Rats Fed Various
Rations with and without Vitamin D

Ration

Blood*
P

.Animal

% Fecal IT **
week 3rd week 4th week

st week

2 nd

1

i

1

a
b

5.1
5.2

3
1< CaCOs
ration

a
b

4
Same plus
irrs.d. Y.

a
b

4.8
5.5

4.6

a
b

4.5
5.3

a
b

3 .C

3*6

4.6

4»o

4.1

4.1

5.8

3.8

4.8

3.8

5.6

5.8

6.3
5.9

4.5

4.0

3.8

5.6

a
b

5.3
3.8

4.0

o.5

3.5

a
b

5.3
5.1

4. 3

4.0

3*8

3.6

I
!

3.4

C\]

Same plus
irrad. Y.

4.0

CO

a
b

• to*
^ ^

Low Ca
ration

1

2

t-0

ca

!

j

0.5

3.6

5.6

!

to o|
* *■
^ ^i

... i

j

[
J

[
_

5
2% CaC0S
ration

4.5

6

Same plus
irrad. Y.
7

.

CaCOg
ration
8

Same plus
irrad. Y*
*
**

Uilli r-rans of inorganic P per 100 ml. of blood.
Values obtained by analyzinr composite fecal samples of both
rats within each rroup

Table VI
pH of Alimentary Comal of Young Rats Fed Various Rations ad.th­
an d without Vitamin D

Rati on

Animal

Cardiac Pylo­
sac
rus
1

3

2

Sma 1 1 intest ine
5
4

Cecum

Colon

1

Low Ca
ration

a
b

2.56
2.40

6.40
6.52

6.60
6.69

6.84
6.86

7.70
7.87

6.68
6 •60

6.43
6.52

Same plus
irrad. Y.

a
b

.2.55
2.29

6.26
5.84

6

.35
6.60

6.43
6.94

7 .45
7.20

6

.35
6.60

6.43

3
1% CaC0 3
ration

a
b

2.71
2.63

6.52
6.52

6.76
6 .43

7.03
6.43

7 .78
7 .53

7.10
7.10

7 .37

4
Same plus
irrad. Y.

a
b

2.29
2.38

6.52
6.78

6.43
6.78

7.03
6.94

7 .37
7.37

7 .11
7 .53

7.37
7 .20

5
2% CaC0 3
ration

a
b

2.29
3.05

6.35
7.53

7.11
7.03

6.94
6 •8 6

7.95
7.53

8.5* *
8.5 +

7.94
8.04

Same plus
irrad. Y.

a
b

3.30
3.30

6.86

C .69
6.52

6 •8 6
7.03

7.57
7.61

8.5 +
7.53

7 .70
7 .94

7
3f
,1 CaC0 3
ration

a
b

2.89
3.66

7.11

7.70
7 .62

7.53

6.86

8.21

7.87
8.46

8.5+
8.5 +

7 .78
8.04

a
b

3.83
3.47

7.30
7.03

7 .45
7.70

7 .37
7.62

8.29
8.50

8.5 +
8.38

7.53
7.45

2
6.10

6

6.69

8

Same plus
irrad. Y.

* All pH values higher t^an 8.50 are indicated as 8,5*.

Table VII
Toual Fecal Alkalinity of Young Rats Fed Various Rations with
and without Vitamin D
(No. of ml. of 0.1N alkali per gram of dried feces)

Ration

1

st week

2 nd

week

3rd week

4 th

week

Average

1

Low Ca
ration

5.3

5.7

5.2

6.0

5.4

4.0

4.0

4.6

4.7

4.3

3
Ifo CaCOs
ration

7.4

8.4

8.4

7 .2

7.8

4
Same plus
irrad. Y.

7.3

6.5

7.3

5.6

o.7

5
2% CaC0 3
ration

13.8

15.0

15.0

11.6

15.8

12.9

11.7

11.1

10.1

11.4

22.0

24.7

26.4

IS.8

22.9

2

Same plus
Irrad. Y.

6

Same plus
irrad. Y.

i
CT>
.
ca

7
rf, CaCOp
6
ration

24.0

8

Same plus
irrad. Y.

21

.o

21.1

21.5

Table VIII
Total Fecal Altai inity of1 Adult Rats Fed Various Ra.tions vdth
and mithout Vitamin D
(Fo. of ml. of

Ration

1

st meek

0

.11T alkali per pram of dried feces)

2 nd

meek

3rd meek

4th meek

Average

1

Lor/ Ca
ration

8.2

7.8

6.5

6.7

7 .3

7.8

7.1

6.5

6.3

6.9

3
1% CaCOp
ration

13.6

9.8

9.8

10.3

10.9

4
Same plus
irrad. Y.

13.2

10.6

11.5

10.9

11.5

5
2/p CaC 0 3
ration

18.1

17.1

17.7

16.3

17.3

18.1

17.1

16.4

16.3

17.0

17.7

26.2

28.6

30.2

25.6

22.5

23.1

26.0

22.3

2

Same plus
irrad. Y.

6

Same plus
irrad. Y.
7
3f' CaCOp
ration--

.

8

Same plus
irrad. Y.

17.7

C.

THE EFFECT OF RICKETS AND VITAMIN D ON
PLASMA PHOSPHATASE ACTIVITY IN RATS

HISTORICAL

In 193£ Kay published an extensive review on the subject,
Phosphatase in Growth and Disease of Bone 11 •

For our purpose only

the literature dealing directly with the effect of rickets and
vitamin D on plasma phosphatase activity will be given.

Working

with children Bodansky and Jaffe (1934) found that serum phosphatase
activity was an excellent criterion of the severity and rate of
healing of rickets.

Kinard and Chanutin (1933) concluded that

irradiated ergosterol, while causing a rise in phosphatase values
with growing rats, has no effect on adult rats.

Common (1936)

observed a marked'increase in blood phosphatase values in young
chicks but noted wide variations of the values of the enzyme in
laying hens as well as in cocks.

He also found that the development

of rickets in chicks produced a very marked increase in the phosphatase
activity level of the serum.

Mitchell (1936), studying the serum

phosphatase in 75 cases of fractures in adults, failed to find any
significant changes in serum phosphatase following fracture.

He

then concluded that the serum phosphatase value was not an index of
healing or of the rate of healing of the fractures.

Crinim and

34

Strayer (1936) found a reduction in blood phosphatase value in rats
only after the administration of toxic doses of vitamin D in the
form of viosterol.

There seems to be no comprehensive study

reported in the literature correlating vitamin D and rickets with
plasma phosphatase activity in rats*

The aim of the present

investigation was to see whether or not vitamin D had any definite
effect on the phosphatase activity in rats by studying the problem
extensively under various experimental conditions*

EXPERIMENTAL

Jenner and Kay*s method (1952) was followed throughout
this experiment.

The animals were killed by etherization, and blood

samples were obtained immediately by puncturing the heart*

The

phosphatase content of a sample of plasma Is defined as the number
of milligrams of P which would be liberated by 100 ml. of the plasma
under the conditions perscribed*
Since no consistant results were obtained in several
preliminary experiments on rats of varying ages fed both normal and
rachitogenic diets and rachitogenic diets supplemented with vitamin
D, it was decided to plan a more comprehensive study on this problem.
Three series of experiments were included in this study.
I, both young and adult normal rats were tested*

In Series

These animals were

taken from the breeding stock which had been fed a ration consisting

35

of*: maize, 30; oats, 30; milk powder, 30; linseed meal,
alfalfa, 5* yeast;, 3 and NaCI, 1*

6

, ground

Animals in Series II were given

a high Ca rachitogenic diet containing maize, 50; oats, 25; wheat
gluten, 20; CaCOg, 3 and NaCI, 1*

All animals in this group were

placed on the rachitogenic diet when they were approximately
25 days of age, weighing between 50 and 55 grams.

21-

Those receiving

the basal diet alone were killed at tne end of 31 days, the usual
testing period for rachitic rats.
form of irradiated yeast were added

Vitamin D supplements in the
21

days after the animals had

been on the rachitogenic diet. Animals were killed for plasma P
and phosphatase activity determinations at day intervals after
feeding the supplements.
in Series III.

A low Ca rachitogenic diet was employed

This was the same as the high Ca diet used in

Series I with the exception that CaCOs was omitted.
modifications in diet were made at the end of
animals were killed

10

21

Various

days and all

days later.

RESULTS AND DISCUSSION

Complete results are presented In Table IX.
No appreciable differences were observed in plasma phosohatase activity between high Ca and low Ca rickets, and between
rachitic animals and rachitic animals given a supplement of vitamin D.
In general, the phosphatase activity values were inconsistant and

36

duplicate or repeated determinations within any given group varied
considerably.

From these results it appeared clear that at least,

in rats, the plasma phosphatase activity can not be employed to
indicate the severity of rickets or the degree of healing of rickets*

It is generally believed that rickets experimentally pro­
duced in rats is of an entirely different nature as compared with
the rickets found in children*

In rats the rachitic condition is

produced not primarily by depriving the animal of vitamin D but
chiefly by upsetting the Ca:P ratio in the diet.

Besides, there

is a decided difference in the requirement of vitamin D between the
rat and the child.

In children, even under normal conditions a

certain amount of vitamin D must be available constantly in the
system, in order to assure proper bone development and to prevent
rickets while in rats, unless the Ca-P metabolism is greatly
disturbed such as in the production of experimental rickets, little
or no vitamin D is needed for normal calcification.

Under such

conditions it appears doubtful that any comparable results can be
obtained in the study of the plasma phosphatase activity of the
rat and that of the child.
Furthermore the phosphatase activity of the rat has been
found to be at a peak during the early stages of life at which time
bone development is proceeding at its greatest speed*

Kinard ana

Chanutin (1933) reported that the phosphatase activity of the whole
rat reaches a maximum between the 13th and the 20 oh day of age;

37

thereafter there is a decrease until, at the 60th day the concen­
tration of the enzyme remains constant.

Klarin (1935) obtained

the maximum values at the end of the first week, a decrease followed
in a few days and at the end of
constant*

6

to

8

weeks it was low and fairly

All this evidence points to the conclusion that at the

age when experimental rickets is produced in rats (6-7 weeks old)
the animals have passed the stage during which body phosphatase was
greatest, hence the enzyme is not readily affected either by the
production of rickets or by the action of vitamin D.

This may

help to explain why the results obtained in the study of plasma
phosphatase activity are of no particular significance or are at
variance with studies made on infants.

Table IX
Vitamin D and Plasma Phosphatase Activity in Rats
Animal and Experimental
Conditions

No. Animals Plasma
Used
P

Phosphatase
Activity
(Ave. Values)

SERIES I
9 week old young normal rats

6.0

39.42
32.94
24.84
19.38

4

8 .55
4.75

18.90
11.52

2

2.6

32.40

2

2.25

26.88

4
4

3.25
3.4

33.24
34.56

4
4
4
4

3.9
4.0
4.1
5.25

32.16
37.56
30.78
34.02

4

5.55

30.12

2
2

4.5
4.6

34.38
44.10

4

6.10

2

5.10

38.76
37 .50

4

4.9

40.20

4
4
4
4

6.1

4.7
3.4
3.3

41.82
27.90
23.82
35.94

2
2
2
2

Normal young rats starved
24 hours
Normal adult rats

2

6.8

6.7
5.8

SERIES II
High Ca rickets
High Ca rickets supplemented
with 0 . 0 1 gm irrad. yeast
Killed at 2 days
Killed at 4 days
High Ca rickets supplemented
with 0 . 1 gm irrad. yeast
Killed at 2 days
Killed at 4 days
Killed at 5 days
Killed at 10 days
High Ca rickets supplemented
with 0.5 gm irrad. yeast
Killed at 5 days
High Ca rickets supplemented
1 gm irrad. yeast
Killed at 2 days
Killed at 4 days

SERIES III
Low Ca rickets
Low Ca rickets supplemented
with 0 . 1 gm irrad. yeast
Low Ca rickets supplemented
with:
stock ration
1 % CaCOs ration
Z% CaCOg ration
irradiation, 1 min.

SUI.mRY AMD CONCLUSIONS

AND
BIBLIOGRAPHY

SUMMARY AND CONCLUSIONS
1*

Some studies have been made on the production of

rickets in rats with special reference to the rachitogenic diets*
By substituting granulated corn for ordinary corn in the
Steenbock ration 2965 with the addition of 25/£ oatmeal and 1%
dried yeast a very satisfactory rachitogenic ration was obtained.
This modified ration was found to give better results than the
Steenbock ration 2965 when employed for ordinary vitamin D assay
in the degree of rickets produced, the growth of the test animals
during the experimental period and the general appearance of the
lines in the line test.

2.

A preliminary study has been conducted in the vitamin

D content of irradiated and sun-cured corn.

Corn irradiated for

one minute tinder an ultra violet lamp was found to be highly
antirachitic, while the sun-cured corn (exposed to October sun­
light for

10

hours) contained a small but significant amount of

the antirachitic factor.

The e:xposure of corn to sunlight of greater

ultra violet intensity might easily result in the production of
sufficient vitamin D to seriously reduce the rachitogenic properties
of a diet.
3.

The mode of action of vitamin D has been studied

from the standpoint of its possible effect on the total fecal
alkalinity and the plasma phosphatase activity in rats.

The total

alkalinity of the feces of the test animals was found to be directly
proportional to the amount of CaCOg included in the ration.

40

When vitamin D was added to the ration the alkalinity was reduced
in each ease*

This finding seemed to confirm the "net absorption"

or "net retention" theory of Harris*

4.

Ho appreciable differences were observed in plasma

phosphatase activity between rachitic rats and rachitic rats receiv­
ing a supplement of vitamin D*

In rats, the plasma phosphatase

activity can not be employed to indicate the severity of or the
degree of healing of rickets*

BIBLIOGRAPHY

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Bauer, W. and Maddock, S. J., (1951), Am. J. Med. Sci., 181:399
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College, East Lansing, Mich.
Bodansky, A. and Jaffe, H. L., (1934), Am. J. Diseases Children,
48:1268
Common, R. H., (1936), J. Agri. Science, 26:492
Coward, K. H., (1933), Lancet, 225:920
Crinim, P. D. and Strayer, J. W . , (1936), J. Biol. Chem., 112:511
Daly, C., (1933), Proc. Soc, Exp. Biol, and Med., 31:.368
Friedman, H., (1936), J. Hutri., 12:165
Gy 8 rgy, P., Popoviciu, G, and Sano, T., (1933), Ztschr. f. Kinderheilk.
55:442
Harris, L. J., (1932), Lancet, 222:1031
Harris, R.

S. and Bunker, J.

W. M., (1931), Science, 73:95

Harris, R.

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W. M., (1934), J. Lab. and Clin.
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Holmes, A.

D. and Tripp, F.,

Jenner, H.

D. and Kay, K. D.,

(1933), Cereal Chem., 10:313
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Jephcott, I-I. and Bacharach, A. L., (1926), Biochem. J., 20:1351
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Kay, H. D., (1932), Physiol. Rev., 12:384
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