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Group Title: Bulletin - University of Florida. Agricultural Experiment Station ; no. 569
Title: Relation of calcium, phosphorus and protein deficiencies in the immature rat to defects in growth and skeletal development of the mature animal
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Permanent Link: http://ufdc.ufl.edu/UF00027121/00001
 Material Information
Title: Relation of calcium, phosphorus and protein deficiencies in the immature rat to defects in growth and skeletal development of the mature animal
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Physical Description: 36 p. : ill. ; 23 cm.
Language: English
Creator: French, R. B ( Rowland Barnes )
Abbott, O. D ( Ouida Davis ), b. 1892
Townsend, Ruth O
Publisher: University of Florida Agricultural Experiment Station
Place of Publication: Gainesville Fla
Publication Date: 1955
 Subjects
Subject: Calcium in animal nutrition   ( lcsh )
Proteins in animal nutrition   ( lcsh )
Nutritionally induced diseases in animals   ( lcsh )
Rats -- Diseases   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Bibliography: p. 36.
Statement of Responsibility: by R.B. French, O.D. Abbott and Ruth O. Townsend.
General Note: Cover title.
Funding: Bulletin (University of Florida. Agricultural Experiment Station) ;
 Record Information
Bibliographic ID: UF00027121
Volume ID: VID00001
Source Institution: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: aleph - 000926770
oclc - 12825220
notis - AEN7470

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Full Text

/ L-


Bulletin 569


JAN'O 2 01956

November 1955


UNIVERSITY OF FLORIDA
AGRICULTURAL EXPERIMENT STATIONS
J. R. BECKENBACH, Director
GAINESVILLE, FLORIDA










Relation of Calcium, Phosphorus and Protein
Deficiencies in the Immature Rat to Defects in
Growth and Skeletal Development of the
Mature Animal


By R. B. FRENCH, O. D. ABBOTT and RUTH O. TOWNSEND








TECHNICAL BULLETIN







Single copies free to Florida residents on request to
AGRICULTURAL EXPERIMENT STATION
GAINESVILLE, FLORIDA













Contents
Page

INTRODUCTION ... .. .. ... ...-. ... ... .............. ........... 3
BREAD FEEDING EXPERIMENTS ....... ....... .....-- ..--.. ... --.................. 4
Procedure .................................-.. ...................... ..... 4
Experim ental A nim als .. .. ....... ....-.... .. --................... 4
Diets ................... ..... ... ............4..---- -- ---- ----- -4
F feeding ........ ....... ......................
Roentgenogram s ............... ........ ....--- ------- -------- 5
R esu lts ..... .... .. ... .................... ........ ..- 5
Effect of Water Bread on Growth ......................... .. 5
Bone Density, Jcint Condition and Spinal Curvature ............- 8
Fem ur Length ....... ............ ............ .......- .... .. ..- .......- 8
D discussion ........ ................... .--------.-- .....- ------.......-- 8
LACTALBUMIN FEEDING EXPERIMENTS ....... ......-----..- ..... ----........... 11
Procedure ................... ....- ----- --...... ------- --- 11
Experim ental Anim als .. ....... .....-.....................--- ---- ... 11
D iets ....-- ....... ....... ................... ................--- .. .. .---- 11
Feeding ........... ............ ..---------- --------------- 13
Chemical M ethods .... ............................- --- --------- --------------------- 13
Roentgenogram s .......... ... .. ........----------------- .......... ........ 13
R results .......-..--- .... ...----------- .------ -------. ------ 13
Calcium Deficiency -..-. .... .... ... ...... --. .. ......... ...---. 13
Phosphorus Deficiency ...........................-.-.. --- 14
Calcium-Phosphorus Deficiency ................-.......- ------ -- 14
Effects of Realimentation ...................----.--------- 14
Effects of Deficiency During Immaturity upon Subsequent Growth 16
Roentgenograms of Deficient Weanlings ..-.......-----.--------..----- 19
Roentgenograms after Realimentation on Stock Colony Diet -..... 22
Femur Measurements from Roentgenograms -..........--- .....-...--------. 26
Pelvic Girdle Measures from Roentgenograms ............................ 29
Discussion ........... ---------------- ---------- 32
S UM M ARY -..- ----------------------..........- ...-.........--- ..------ -.... ............---- 34
LITERATURE CITED ..................-....................-- ---------------- --.. 36









Relation of Calcium, Phosphorus and Protein
Deficiencies in the Immature Rat to Defects in
Growth and Skeletal Development of the
Mature Animal

By R. B. FRENCH, O. D. ABBOTT
and RUTH O. TOWNSEND

Introduction
Many kinds of physical deformities are seen in any represen-
tative population sample. Some stem from genetic origin, others
from the impact of an unfavorable environment. Deformities
resulting from faults in a gene may be difficult to separate or
distinguish from abnormalities arising from change occurring
in cell organizers after conception. Only fairly recently has the
occurrence of defects in the child, such as goiter or rickets, been
associated with nutritional deficiency; or even more recently, de-
fects like Mongolism with maternal virus disease. Several years
ago this laboratory reported (1) 1 on a nutritional deficiency in
young rats that was associated with a deformity seen later in
the life of the animal.
Weanling rats in this early experiment (2) were fed a basal
bread diet for three months and then allowed to grow to
maturity on a complete diet. These animals were normal in
appearance, except for the fact that the femurs of the mature
animals were quite short when compared with the other long
bones of the skeleton. What was the cause of this defect? Was
it related to an early nutritional deficiency when the immature
rat was fed a basal diet of white bread made with water? Other
rats in the early experiment were fed bread made with milk and
bread made from whole wheat and none of these animals showed
the femur deformity. These facts suggested that a deficiency
in minerals, possibly in combination with a low level of protein
during the rapid growing period, caused the deformity. By
way of testing this hypothesis, diets were compounded that
provided normal and low protein levels in various combinations
with normal and deficient levels of phosphorus and (or) cal-

Italic figures in parentheses refer to Literature Cited.







Florida A,, I. ,It.. ,,, Experiment Stations


cium. These diets were fed to weanling rats until signs of
major deficiency appeared. At this point the rats were re-
alimented and kept upon a complete diet for the remainder of
their lives.
This paper reports experiments on the cause of the short
femur, on the signs and symptoms of bone mineral deficiencies
in the young rat, on the relation of deformities of the pelvic
girdle in the mature rat to a deficiency in the young rat, and on
the effect of a subsequent good diet on injury caused by a dietary
deficiency in the young rat. The data are presented under two
main sections titled "Bread Feeding Experiments" and "Lactal-
bumin Feeding Experiments."

Bread Feeding Experiments
Procedure
Experimental Animals.-A total of 48 weanling albino rats
was used in the bread feeding experiments. They were separ-
ated into groups of eight according to weight, sex and litter.
During the course of the experiment each animal was weighed
weekly and its physical condition was noted.

TABLE 1.-COMPOSITION OF WATER BREAD DIETS.
Plus
Plus Plus Calcium- Plus
Component Basal Calcium Phos- Phos- Vitamin
phorus phorus D
Gms. Gms. G ms. Gims. G ms. s.
Dry Bread .... 940 940 940 940 940
Ca lactate .... 26.5 26.5
KHPO, ......... 8.9 8.9
Vit. D. I. U.. : 300
Common to
all diets 60 grams Wesson oil: 1400 I. U. Vitamin A**
Viosterol (Squibb)
** Super A (Upjohnp

Diets.-Dried, coarsely-ground white bread made with water
was supplemented with either calcium lactate, potassium acid
phosphate or both (Table 1). Each diet then was completed by
the addition of 6 percent vegetable oil containing enough vitamin
A to bring the level of vitamin A to 140 I. U./100 gm. food. The
detailed method of making and the analysis of the bread







Relation of Food Deficiencies to Skeletal Development 5

were given in Florida Agricultural Experiment Station Bulletin
483 (2).
Feeding.-The weanling rats were allowed to feed ad lib for
13 weeks and then were realimented on the stock colony diet-
laboratory chow (Purina) and greens twice a week.
Roentgenograms.-Roentgenograms were made of all animals
at the end of the bread feeding period and again 14 weeks after
realimentation on stock colony diet. Skeletal observations and
measurements were made from these pictures.

Results
Effect of Water Bread Diet on Growth.-Data in Table 2 in-
dicates the effect of the bread diet with its supplements on
growth. Growth is figured as average percent of weight at-
tained as compared with that attained by control animals of the
same age.

TABLE 2.-PERCENT OF CONTROL WEIGHT ATTAINED BY ANIMALS AFTER
13 WEEKS' FEEDING ON BREAD DIETS AND THEN AFTER 14 WEEKS'
REALIMENTATION ON DIET FED CONTROL ANIMALS.

Group-weight Relationship at the End of the
Bread Feeding Period and after 14 Weeks of
Realimentation. Weights are Computed as Per-
cent of Control Animal Weights.
Diet When Weanling
Males Females
Bread Stock Bread Stock
Diet Diet Diet Diet
13 Weeks 14 Weeks*' 13 Weeks 14 Weeks**
Water bread (W. B.) .... 27 87 38 87
W. B. + calcium (Ca) 24 88 40 92
W. B. + phosphorus(P) 28 94 35 93
W. B. + Ca + P ....... 24 85 32 90
W. B. + vit. D........... 22 90 33 93

Control Weights .... 253* 340* 185" 230*

Numbers marked with asterisk are weignts in grams of control animals of the same
age fed stock colony diet all the time.
** At this point on the growth curves the period of rapid daily gain was over.

At the end of the bread feeding period of 13 weeks there was
little difference in amount of weight gain between groups fed
the basal bread diet, whether supplemented with minerals or
not. The effect of the deficient diet was more pronounced on







Florida Agricultural Experiment Stations


I














Fig. 1.-Roentgenogram of a rat fed a diet of water
bread for 13 weeks. Note skeletal demineralization, en-
larged knee joints and long femurs (115 percent as long
as control animal of same body length).






























































Fig. 2.-Roentgenogram of a weanling rat 21
days of age and weighing 40 grams. At this age
and size the weanlings were started on their period
of deficient feeding. Up to the time of weaning
they had had access to stock colony diet fed their
dams.






Florida Agricultural Experim-ent Stations


males than on females, since males at this age grow faster than
females and on this account have larger requirements. After
14 weeks realimentation on stock colony diet all groups had
gained rapidly in weight and were approaching the weight of
controls of the same age. In these animals the stunting effects
of the bread diet were small but sufficient to prevent them, when
placed on the stock diet, from attaining the weights of their
controls.
Bone Density, Joint Condition and Spinal Curvature.-Bone
density, joint condition and spinal curvature were assessed on
each picture as good, fair or poor at the end of 13 weeks on bread
diet and again 14 weeks after realimentation. All skeletal as-
sessments rated poor when the animals were still on the bread
diets (Figure 1) but after realimentation rated good for animals
that had received any of the mineral supplements.
The animals used in all experiments reported in the paper
started in on deficient diet feeding as weanlings. The weanling
rat pictured in Figure 2 weighed 40 grams and was 21 days
of age. Compare Figures 1 and 2 for size and bone mineraliza-
tion.
Femur Length.-Young rats eating the bread diets for 13
weeks grew femurs that averaged 110 percent (Figure 1) as
long as the controls of the same trunk length (Figure 8). Then,
after 14 weeks of feeding stock colony diet, the femurs of groups
that had been given supplementary calcium, phosphorus or cal-
cium-phosphorus were of normal length, while those that had
received no mineral supplements or only vitamin D were short.
Compare femur lengths in Figure 3 (length averages 59 percent
of control) and Figure 4 (a mature control animal).

Discussion
Deficiencies in a diet of water bread caused weight increase
in the weanling animal to stop quickly. But the femur kept on
growing after other growth had stopped and became dispropor-
tionally extended in relation to body length.
The amount of weight gained after realimentation suggested
that damage to growth control centers caused by the deficiencies
of any of the water bread diets could be largely repaired under
the influence of a good diet.
The basal diet of water bread contained 0.12 percent calcium
and 0.14 percent phosphorus. Supplementation of the bread diet
with salts containing either or both elements allowed normal






























































Fig. 3.-Roentgenogram of a rat that had been fed a water
bread diet for 13 weeks and then the stock colony diet for 14
weeks. Note disorganized joints, short, heavily mineralized
femurs with narrow marrow spaces (femurs are 57 and 61 per-
cent as long as control animal of same body length).






Florida Agricultural Experiment Stations


Fig. 4.-Roentgenogram of a mature control animal.







Relation of Food D, i..,, ,.'. to Skeletal Development 11

femur development. Possibly both the calcium and phosphorus
in the water bread diet were of low availability to the rat. Phytin
phosphorus of wheat is regarded as being low in availability and
the small calcium supply may have been rendered less available
by combination with the fatty acids of the corn oil. The condi-
tions essential to the development of short femurs were quite
critical, since at maturity an animal might show one short
femur and the other almost of normal length. Apparently an
"all or none" principle was involved in the development of the
short femur, since even in the same animal the femur either
failed to grow or grew normally.

Lactalbuiini Feeding Experiments
Procedure
Experimental Animals.-A group of eight or more weanling
albino rats equated as to size, sex and dam was started for each
feeding trial. A total of 128 rats was used in this experiment.
Each rat was weighed weekly during the period of its deficient
feeding and monthly thereafter. Records were kept of the con-
dition of the hair, eyes and skin, degree of activity, length
of life and cause of death. Development of skeletal structure
was recorded by a series of roentgenograms.
Diets.-A series of 10 diets was constituted employing puri-
fied lactalbumin2 at both a 20 and a 6 percent level of protein
and otherwise complete except for total deficiency in either
calcium or phosphorus or both. The composition and chemical
analysis of these diets are outlined in Table 3. Note that 0.15
percent choline has been added to all diets.3
SPurification of Lactalbumin.-Lactalbumin as purchased analyzed 8 per-
cent ash, 2.28 percent calcium and 0.38 percent phosphorus. Calcium and
phosphorus were removed by the following procedure:
One and two-tenths kilos of commercial lactalbumin were stirred with
125 ml. of concentrated hydrochloric acid in 16 liters of water for four
hours. After settling and decanting, it was neutralized to a pH of 4.8 with
potassium hydroxide, washed five times with water, five times with ethyl
alcohol (last washing with absolute) and finally with petroleum ether.
The dry, purified lactalbumin analyzed 0.07 percent ash, 0.000 percent
calcium and 0.038 percent phosphorus.
SCholine Deficiency in Diet.-The diets (Table 3) were first prepared
and fed without additional choline. Rats fed these diets usually died within
10 days. The only obvious gross pathology was hemorrhagic kidneys. At
the time of death they were gaining weight rapidly and appeared healthy.
Literature search (10) revealed that the methionine-choline content of lqc-
talbumin was borderline, so choline was added to the diet. With this addi-
tion the control diet using lactalbumin as the sole protein proved satisfac-
tory, as judged by the fact that rats fed this diet compared favorably
with the stock colony rats in regard to rate of gain in weight, maximum
weight obtained, reproduction and length of life.

















TABLE 3.-COMPOSITION AND ANALYSIS OF DIETS.


Component Control Low
SProtein
-C C


Composition
Salts
Lactalbumin
Vit. D.
U. S. P. units


Common to
all diets


Analysis
Calcium
Phosphorus
CaP ratio


Low
Calcium
1'r


oLo
Phosphorus


Low Calciun-
Phosphorus


Low Calcium-
Phosphorus
+ Vitamin D


4.55 4.55 2.11 2.11 4.20 4.20 1.15 1.15 1.15 1.15
20.0 6.0 20.0 6.0 20.0 0.0 20.0 6.0 20.0 6.0
30.0 ',0


10.0%7 vegetable oil, 200 units vitamin A; 3.0'/ alphacel; 0.1 puvule Becotin*;
0.15', choline; and enough corn starch to make each diet total 100'(


0.444 0.424
0 250 0.256
1.8 1.65


0.007
0.241
0.03


0.111 0.452 0.463
0.280 0.020 0.015
0.04 23.0 31.0


0.008 0.007 0.016
0.019 0.015 0.020
0.4 0.5 0.8


0.031
0.015 5
2.1
0J


* Vitamin B complex (Lilly).






1Relation of Food Deficien cies to Skeletal Development 13

The salt mixture used (Table 4) was basically that of Hub-
bell, Mendel and Wakemann (8). This mixture was modified ac-
cording to the requirements of each diet by elimination of
either calcium- or phosphorus-containing salts or both. In ad-
dition, calcium lactate was substituted for the carbonate and
iron citrate for the phosphate.

TABLE 4.-COMPOSITION OF SALT MIXTURES.
Low Low Low Calcium-
Ite;nm Control Caluium Phosphorus Phosphorus
Gms. Gms. Gins. Gn.

Ca lactate 72 ;72
KH.PO 212 212

MgCO.-25 gms.; MgSOi--1r gms.; NaC1-- gms.;
Common to KC1-112 gms.; Fe Citrate-10.2 gms.; KI-0.08 gms.;
all diets MnSO,-0.35 gms.: NaF--1.00 gnms.; AL(SOJ) KSO.
-0.17 gmns. CuSO.-0.90 ms.


Feeding.-Control animals were fed a complete diet contain-
ing lactalbumin at the 20 percent level all their lives, those at
the low protein level (6 percent) were fed for eight weeks and
then realimented on the stock colony diet and so continued for
the rest of their lives. The low calcium diets were fed for
eight weeks; the low phosphorus and low calcium-phosphorus
diets four weeks before realimentation for the remainder of their
lives on the stock colony diet. All feeding was ad lib. Distilled
water was furnished the rats while on the low mineral diets.
Chemical Methods.-Calcium (Table 3) was precipitated as the
oxalate and titrated with potassium permanganate (3). Phos-
phorus was determined by measuring the color absorption at
720 millimicrons produced by reduction of phosphomolybdate
(4, 12).
Roentgenograms.-Roentgenograms were made of lightly
etherized rats on high speed, non-screen film.

Results
Calcium Deficiency.-Weanling rats fed diets containing 20
percent protein and low in calcium showed signs of major de-
ficiency in eight weeks. The outstanding signs were loss of dorsal
hair, spastic abdomens, slight hind leg paralysis, priapism, pro-






Florida Agricultural Experiment Stations


truding, scarlet-tinged eyeballs, blood-incrusted nares and ali-
mentary hemorrhage as shown by discolored feces. All animals
were dizzy, apprehensive and squealed upon being touched, but
showed no tetanic symptoms.
Phosphorus Deficiency.-Weanlings on the 20 percent pro-
tein diet but deprived of phosphorus showed severe symptoms
and, unless realimented on the complete diet, died after three
to four weeks. These animals became cyanotic, hemorrhaged
from mouth, ears, nose and anus, and went into tetany when
handled. In contrast to the calcium-deficient rats that were
apathetic towards food, the ones deprived of phosphorus were
hungry all the time and, in spite of complete paralysis of the
hind legs, made frenzied efforts to reach the food.
Calcium-Phosphorus Deficiency.-Calcium-phosphorus deficient
animals on the 20 percent protein diet also hemorrhaged, showed
hind leg paralysis, incoordination and slight tetany. In size and
symptoms they could not be distinguished from the calcium-
deficient animals, except for slight tetany and the fact that the
symptoms appeared in half the time and were much more severe.
No change in deficiency symptoms was observed when vitamin
D was added to the calcium-phosphorus deficient diets.
Subcutaneous or intraperitoneal injection of the element that
had been omitted from the diet into several animals showing
severe deficiency symptoms was followed by death, possibly
caused by increased hemorrhage, particularly into the thoracic
cavity.
Signs of calcium-phosphorus deficiency in animals fed at the
6 percent protein level were not of the severity of those described
above. The only observable difference between control animals
and those on low protein diet was one of size.
Effects of Realimentation.-During the first days of realimen-
tation some rats that had showed major deficiency symptoms
died. Over two weeks elapsed before the animals on the 20
percent protein diet that had been calcium or calcium-phosphorus
deficient developed interest in or appetite for food and started
to gain weight.
One to two weeks after realimentation on stock colony diet,
hemorrhages had stopped, paralysis had disappeared and ab-
dominal spasticity and cyanosis were greatly improved. After a
month on the stock colony diet, several females, though still
in poor physical condition, were bred and produced litters. Evi-










TABLE 5.-EFFECT OF AN EARLY DEFICIENCY OF ErlHER BONE BUILDING i\AT'ERIALS, PROTEIN OR BOTH ON SUBSEQUENT
GROWTH OF RATS ON STOCK COLONY DIET AS SHOWN BY COrIPARArTIVE WEIGII INCREASE.

Group-Weight Relationships at the End of the Period of Deficient Feedinlg, and after Both
14 and 52 Weeks of Realimentation. Averaged Weights (omputed as Percent of (Control
Animal Weight.


Experimental Diets


Males


Females
I


Deficient Diet
4 Weeks 8 W eek~;


Stock Colony
14Weeks'* 52


Diet
Week


Defcielet Diet
4 Weeks 8 Weeks


Protein 20'; level
Less calcium (Ca) ..
Less phosphorus (P)
Less Ca-P .................
Less Ca-P (+ vit. D)

Protein -- (6; level .
Less Ca -.. ....
Less P .......
Less Ca-P .............
Less Ca-P (+ vit. D)

Stock Colony Diet .


S7 7 (;
84
5-1


90 84
87 (;
(il
(61
65
(:18


9:' 1 ,:
79
72

72

84 81
SO S0
75
79
(;9


10s 1 173: :04: :121: 12:: 95 147: 205: 2z: ,: 285:


NumlIers marked reprt'srtl weight in grains of control ;Iiils lif thi sinl( :t(.
At thi, point on Ih .r,,itlih cuiirves the iporiod of tr pi(d lily ;uiin \'l i,.


Stock
1 \Veeks


(olony D)iet
S52 Weeks
I






Florida Agricultural Experiment Stations


dently the drastic dietary treatment to which the dams were
subjected did not impair reproduction nor their ability to raise
young successfully. The young made standard weight gains,
showed normal skeletal development at maturity and lived a
normal life span.
Effect of Deficiency During Immaturity upon Subsequent
Growth.-Effects of the early dietary deficiencies on both imme-
diate and subsequent weights are detailed in Table 5. Average
weights of control animals are given in the last line of the table.
Above them are numbers valuing weight of the deficient animal
in terms of percent of the control animal weights of the same
age.
The percent listed under the headings four and eight weeks
suggest the immediate impact of deficiency upon weight or
growth, those under the heading 14 weeks the degree of recovery,
and those under the heading 52 weeks show the lasting effects of
the deficient diet in early life.
While on the deficient diets the gains of rats on the 6 percent
protein diets were less than those on 20 percent protein. For
both levels of protein, rats on phosphorus-deficient diets made
poorer gains than those on diets deficient in both phosphorus
and calcium. Calcium and phosphorus deficient diets cannot
be directly compared at this period because of the four-week
difference in feeding interval. However, one may safely as-
sume that the calcium-deficient animals at the end of the first
four weeks of this diet were in better condition and of heavier
weights than either the phosphorus or calcium-phosphorus de-
ficient animals. The faster growing males made slightly lower
percentage gains than the females on the deficient diets.
By the time the animals had been realimented on the stock
colony diet for 14 weeks, the period of rapid recovery of weight
decrement was over. At the end of this period several groups of
males, namely the low calcium, the low phosphorus, the low pro-
tein, and the low protein-calcium, had gained weight so that
they averaged only 10 percent below their controls. Shapes of
such animals were quite stocky when contrasted with the slender
and longer lines of control animals. Males that had been exposed
to calcium-phosphorus or low-protein-phosphorus deficiency suf-
fered greatest damage to their total growth potential and ex-
hibited upon realimentation only a slow rate of growth which
soon stopped. Female rats recovered normal growth patterns






Relation of Food Deficiencies to Skeletal Development 17


Fig. 5.-Roentgenogram after weanling
had been fed calcium-phosphorus deficient
diet for four weeks.





Florida Agricultural Experiment Stations


/ N











Fig. 6.-Roentgenogram after weanling had
been on phosphorus-deficient diet for four weeks.
Compare bone density with that of rat fed a low-
calcium diet for eight weeks (Fig. 7).






Relation of Food Deficiencies to Skeletal Development 19

to a greater degree after either calcium or protein deficiency and
to a lesser degree after exposure to any of the other deficiencies.
At the end of 52 weeks the stunting effect on male growth
originating in injury to the weanling's growth stimulus centers
was even more pronounced than at 14 weeks. The experimental
males, unlike their controls, made only small gains in weight
after reaching maturity. This smaller increase in weight after
maturity was found in varying degree in all groups, both male,
and female, that had been deficient when young. The trends in
results were the same as at 14 weeks: Maximum injury resulted
from low protein-phosphorus and low-calcium-phosphorus diets
and the injurious effect of the latter apparently was lessened by
lowering protein in the diet. Low protein alone effected a
weight depression that was of similar magnitude to that caused
by a deficiency of calcium or phosphorus at adequate protein
levels.
Stunting effect of early deficiencies on growth of the female
at the end of the year was less pronounced than on the male, but
was of similar nature and only less in degree.
Roentgenograms of Deficient Weanlings.-Roentgenograms of
weanlings that had eaten the calcium-phosphorus deficient diets
for four weeks (Figure 5) showed little bone detail, indicating
marked demineralization of the skeleton. Clearly outlined and
evidently containing the last reservoirs of bone mineral were the
enamel layers of the molars, the osseous labyrinth of the audi-
tory mechanism, the edges of fractures and the upper and lower
incisors.
Roentgenograms of animals fed the low-phosphorus diet for
four weeks (Figure 6) showed only faint skeletal outlines, while
those fed the low-calcium diet for eight weeks were well outlined
(Figure 7) and were much denser.
When the rats were fed any diet low in calcium, the knee
joint was much enlarged. In such cases the distal end of the
femur and occasionally even the body of the femur were enlarged.
In contrast, femurs and knee joints of the phosphorus-deficient
animals were apparently normal as to size and shape but were
lacking in mineralization.
In general, weanlings given protein at the 6 percent level
showed skeletal difficulties quite like their mineral-deficient cor-
respondents at the 20 percent protein level, but to a lesser de-
gree.






Florida Agricultural Experiment Stations
















































Fig. 7.-Roentgenogram after weanling had
been on calcium-lacking diet for eight weeks.
Note enlarged femur, swollen and disorganized
knee joints, poor thoracic contours, and pelvic
compression suggesting an X. Dense alimentary
material is solder gnawed from wire cage.






























































Fig. 8.-Roentgenogram of rat that had
been fed calcium-phosphorus deficient diet for
four weeks and then stock colony diet for two
years. Note short, cupped femurs (66 percent
of normal length), ankylosed knee joints, flar-
ing pelvis and disorganized thoracic vertebra.






Florida Agricultural Experiment Stations


The effect of adding vitamin D to the diet of animals on low
calcium-phosphorus intake was one of degree in amelioration
of calcium-phosphorus deficiency signs. Faint shadows on the
films indicated the addition prevented constriction in the chest
cavity and distortion in the spinal column. These and other ef-
fects became clear in the pictures of older animals.
Roentgenograms after Realimentation on Stock Colony Iiet.-
Roentgenograms were taken not only just before the rats were
realimented on the stock colony diet but also three months, one
year and two years later. Thus pictures were taken at the
stages when each rat was young and deficient, adolescent, ma-
ture and old. Comparison of these series of roentgenograms
suggested that certain skeletal defects originated at the time
of the single short dietary deficiency when the animal was young.
These deformities were not corrected by subsequent good dietary
practice and were apt to increase in apparent degree with age.
Roentgenograms of animals still eating the low calcium or low
calcium-phosphorus diets showed enlarged knee joints (Figures
7 and 5). Enlargement was followed in later pictures (Figure
8) by the appearance of eroded, flattened and even cupped artic-
ular surfaces. The articular sesamoids in some cases were not
identifiable because of excessive calcification of the joint.
Stiffened joints allowing only limited motion were often noted.
Many of these aged animals still squealed when handled, a habit
formed when they were weanlings on deficient diets and their
joints, then, were painful when touched.
A joint condition much less severe than that just described
accompanied the low calcium or low calcium-phosphorus diets
at the 6 percent level of protein. This less severe condition was
characterized by a slight enlargement in the knee joint in the
young; after realimentation on the stock colony diet no abnor-
mality was observed in the adolescent rat; but as the animal
aged, there was an increasing degradation in the knee joint.
Ratings on the aged animal were changed for both better and
worse when vitamin D had been added to this low-protein diet.
The effect of the addition of vitamin D was to drop knee joints
to the lowest rating and to raise to good the ratings for both
spinal alignment and contour of the thoracic cavity.
Joints in several of the animals exposed to lack of phosphorus
(Figure 9) appeared normal at all ages, although there were
some cases in which arthritic conditions developed in the knees
of aged animals.






Relation of Food Deficiencies to Skeletal Development 23


m F.











Fig. 9.-Roentgenogram of rat fed phosphorus-lack-
ing diet for one month and then stock colony diet for
two years. Compare with Fig. 4 and note lesser
flaring at either end of the innominates than is the
case with the control animal.






Florida Agricultural Experiment Stations


Fig. 10.-Roentgenogram of rat that had been
on a calcium-lacking diet for two months and then
fed stock colony diet for three months. Note sharp
vertebral break and joint disorganization.







Relation of Food Deficiencies to Skeletal Decelopment 25

Animals while on the low calcium or low calcium-phosphorus
diets occasionally sustained fractured bones. These fractures
were most common in the long bones, the pelvis, or in the
clavicle. After realimentation these animals on low-mineral diets
presented cases of both lateral and backward spinal curva-
ture. All the mature animals that had been fed at the high
protein level showed a severe thoracic kyphosis and many
showed a complete spinal column break, like that shown in the
roentgenogram of Figure 10.


FEMUR LENGTH MMS.


Fig. 11.-Relation betv


No. Part measured
1. Body of femur
2. Plus distal epiphysis
3. Plus growing neck
4. Whole femur


-een femur lengths and non-caudal vertebral length
of stock colony animals.


Length
mm1.
3-15
12-18
16-24
22-40


Regression
equation
y-3.56x- 17.77
y-4.02x = 3.88
y-4.42x = -2.95
y-3.76x= 12.20


Correlation
coefficients (r)
0.97
0.94
0.99
0.98


Standard
error(,rr)
.01
.02
.01
.01


The thoracic arch of the spine weakened by lack of mineral
in the diet overloaded easily. It would start to bend between the
3rd and 4th and 8th and 9th thoracic vertebrae and usually
snapped between the 5th and 6th. When this occurred the size
of the chamber available for operation of the heart and lungs
was reduced. markedly. These organs adapted their size to that
of the working space. Reduction in lung capacity was reflected






Florida Agricultural Experiment Stations


in the fact that these particular rats etherized with difficulty.
Too, at autopsy, the hearts and lungs of the animals were quite
small, roughly one-half normal size. Despite such handicaps,
these animals lived a normal life span.
Femur Measurements from Roentgenograms.-Measurements
of femur length on colony stock animals were used in construct-
ing a reference curve.
Femur length, which is measured from the head of the femur
to its distal end, is plotted along the abscissa (Figure 11) ; body
length, measured from the apex of the odontoid of the axis to and
including the last sacral vertebra, is plotted along the ordinate.
Four separate lines of regression with equations are given.
Each represents a stage of bone development. Line 1 starts
at a femur length of 3 mm. and measures shaft of the femur
only. The distal epiphysis is included in the measurement of
line 2. This epiphysis first appears when the body length is
52 mm. The neck of the femur begins to show at a body length
of 75 mm. (line 3) and extends and completes the upper epiphy-
sis when femur length reaches 22-23 mm. with a body length
of 95 mm. Line 4 expresses the relationship between body
length and whole femur length; this line shows practically the
same slope as the first equation.
Measurements of the individual experimental animal were
compared with data on this graph and computed as percent of
normal.
Table 6 summarizes the femur-body length ratios of the young
rats fed the deficient diets and the changes in ratios that occur as
the rats age. When the young rat was fed protein at the 6 per-
cent level or any of the low-mineral diets, femurs ranged from 105
to 123 percent as long as controls. Rats on the low protein-low
calcium diet grew femurs longer than any other group. Six of the
seven animals in this group gave values of about 120 percent.
In these animals more than half of the increase in length could
be accounted for in the body of the femur and the rest of the
increased length was divided about equally between the head and
the neck and the lower extremity of the femur.
When the rats were realimented on the stock colony diet, over-
all growth resumed and the proportionately long femur adjusted
to normal in length. This happened except when the rat had been
exposed to a diet lacking in both calcium and phosphorus. This
double deficiency injured centers directing growth of the femur
so that while the rest of the skeleton extended, the femur re-













TABLE .--EFFECT OF AN EARLY DEFICIENCY OF EITHER BONE BUILDING MATERIALS, PROTEIN OR BOTH ON SUBSEQUENT
CHANGES IN FEMUR-BODY LENGTH RATIOS AS RATS AGE.


Femur-Body Length Relationship at the End of the Period of Deficient Feedinj
14, 52 and 104 Weeks of Feeding the Stock Colony Diet.


Experimental
Diets



Protein 20', level
Less calcium (Ca)......
Less phosphorus (P)
Less Ca-P ................
Less Ca-P (+ vit. D)

Protein (;' level.......
Less Ca ..........- ...--
Less P ..... .. ........
Less Ca-P ..............
Less Ca-P (- vit. D)


Deficient Diet
4 or 8 Weeks
Number' Mean Range


110 102-
113 108
114 100-
105 100-


Stock Diet
S 14 Weeks
Number Mean IRange


Stock Diet
| 52 Weeks
Number Mean Range


100
97
77
92

101
95
100
90
94


Sto
S 104
Number M


93-103
92-101
(;5- 72
79- 96(


g, and After

ck Diet
Weeks
lean Range




! 93 -io0
83 79- 87
85 73- 93 i

99 97-101
!9 94-100
100 94-105
90 (66i- 97
93 89- 95

Z5
05

2T


I


I II





/1"


4116-- 111i'-
rfe 07'r
/* ^
C Y' r
*" *^'

2. L\


.' 7~,


i Iii, l *I






,, ((, I?! ^* '









Co.W ro -P .- Co -P
Fig. 12.-Bones of aged animals-controls and those exposed when
young to deficiencies of phosphorus, calcium, and calcium-phosphorus.
Note short femur of the latter and distortion of innominates of the former
two. Bones in rows: 1, mandible; 2, scapula; 3, humerus, clavicle; 4, ulna,
radius; 5, innominate, sacrum; 6, femur; 7, tibia, fibula.






Relation of Food Deficiencies to Skeletal Derelopment 29

tainted its youthful length. Shortness may be found in either
femur (Table 7) or in both (Figure 8). The short femur usually
grew broad and thick. In some cases the marrow spaces re-
mained quite narrow and did not enlarge. The percent of growth
values given for these groups depended largely on how much
the femur had grown during the deficiency and before realimen-
tation.

TABLE 7.-EXAMPLES OF BODY AND FEIUR EXTENSION IN RATS THAT HAD
BEEN EXPOSED AS WEANLINGS TO A SHORT CALCIUM-PHOSPHORUS DE-
FICIENCY.

Male No. 32 Female No. 191

Age Body Femur Length Body Femur Length
Length Right Left Length Right Left
Weeks Mm. Mmm Mm.m. M Mm.
5 76 17(106): 21(116) 73 20(116) 21(121)
33 135 25(77) 29(89) 126 23(77) 22(72)
54 138.5 26(78) 32(96) 127 23(77) 22(74)

I' Percent of femur't length a, compared against control.

To answer the question as to whether the other long bones
grew like the femur, measurements of the tibia, ulna, radius and
humerus were compared with similar measurements obtained
from Donaldson's data (6). In the mature animal the femur
was the only long bone showing the shortness phenomenon
(Figure 12). In the young animal all long bones with the pos-
sible exception of the humerus were long when compared with
stock colony animals of the same vertebral length. Measure-
ments on other long bones were not as precise as those on the
femur, since the femur was the only long bone definitely aligned
with the film.
The stunting effect of the low calcium-phosphorus diets on
femurs (Table 6) was lessened both by the addition of vitamin
D and by dropping the protein level from 20 to 6 percent.
Pelvic Girdle Measures from Roentgenograms.-Pelvic girdle
measurements on stock colony animals were used as a gauge
with which to compare the pelvic measurements of the individual
experimental animals. The measurements on stock colony ani-
mals were plotted in Figure 13.
Plotted on the abscissa were: Length of the ilium to the
ilio-cotyloid juncture, length of ischium from ilio-cotyloid junc-
ture, intercristal or between ilium crests, between innominates
at level of acetabulum, and between supertuberosities of the
























































PELVIC MEASURES-MM.

Fig. 13.-Relation between length of innominate and pelvic
of stock colony animals.
Regression Correlation
No. Part measured equation coefficient(r)
B. Length ilium y-1.64x = 1.31 0.99
C. Length ischium y-2.50x = -1.67 1.00
D. Between craniad ilia
Before puberty y-2.56x = -3.87 0.97
After puberty y-1.67x = 8.33 1.00
E. Between acetabula y-3.85x = -6.15 0.99
F. Between caudad ischia
Before puberty y-2.94x = -2.65 0.98
After puberty y-2.18x = 5.44 1.00


girdle measures

Standard
error (or)
.01
.01

.02
.01
.01

.02
.01






Relation of Food Deficiencies to Skeletal Derelopment 31

ischium. Plotted along the ordinate was the length of the
innominate bone. The length of innominate was used as a base
instead of the non-caudal vertebral length, since many of the
mineral-deficient animals showed bad spinal curvature, or even
spinal breaks. Curvature made vertebral measurements of little
value on these animals. Plots of vertebral length with innomi-
nate length of control animals graphed as a straight line, a fact
that indicated a constant relationship between the two.
Figure 14 summarizes the three averaged pelvic girdle meas-
ures of each group of experimental animals. At the left of each
diagram the number of animals measured is given and to the
right is found the percent variation from controls at three
heights across the pelvic girdle.
No attempt was made to diagram variation in length of the
ilium and ischium, since such variation was minor. The ratio
values of these bones averaged 100 and ranged from 96 to 105
percent, with a standard deviation of 0.3 percent. Exceptions
were found in the measurements of two groups of highly de-
ficient young animals; in these, the ranges were from 89 to 106
percent. Since these bones were barely definable on the roentgen-
ogram, no precise measurements were possible.
While the animals were still being fed any of the low-mineral
diets a compression between acetabulums occurred due to weak-
ness of girdle structure and stress of body weight. Such com-
pression allowed a flaring on either end of the innominate with
a formation suggesting an X, Figures 5, 6, 7.
The diagrams show that subsequent good diet was fairly suc-
cessful in repairing early compression of the pelvic girdle when
phosphorus or calcium had been the limiting factor. Repair after
phosphorus deficiency (Figure 9) left traces of the collapse at
either end of the innominate, while repair after calcium (Figure
10) was suggestive of the flaring next described. When both
calcium and phosphorus were limiting, all width measurements
became markedly increased (Figure 8). Note that these were
the deficiencies associated with the development of very short
femurs.
Pelvic shapes developed as aftermath of protein deficiency
resembled closely those resulting from severe phosphorus de-
ficiency (Figure 9), namely a smaller than normal flaring between
either end of the innominates. This lack of flare usually was
somewhat more between ischial than between ilial crests. The
effect of low protein on shape was enough to mask any addi-







32 Florida Agricultural Experiment Stations


tional abnormality that might be due to low calcium and phos-
phorus, except as may be noted in the young animals.


DEFICIENCY
WHEN YOUNG

CALCIUM (CA)


PHOSPHORUS(P)


YOUNG
00

5 -24
^ -11


ADOLESCENT

9 I


MATURE
100%

9)


12
12 > -23
12


7 \/ 0
-19


7 i
-5


-3 -
\() 13 -4


3) I12


4( _IO
20

22
S12


9/ -2
> 4 4) 5
\ -12 -12

S -13 -5
0 -2 -9


8 -5


-7
4 ( ) 3
-8


P -CA-P


PR-CA-P
(+VITAMIN D)

SCHEMATIC


o 6 -6 6 -3
-16 -8 -9 -13


1 8 -1O 6 5 3 4
*L :- *I 8* j > : 3( 2
-20 -4 -5 6
-_ INTRAILIA FIGURES=AV. %
NUMBER OF _INTRAACETABULA VARIATION FROM
ANIMALS L INTRAISCHIA J CONTROL


Fig. 14.-Summary of average pelvic shapes as molded by a deficient
diet and as repaired under good dietary practice while aging. To com-
pare diagrammed pelvic shapes with roentgenograms, see Figures 2, 5, 6
and 7 for those of young rats and 4, 8, 9 and 10 for those of the older rats.

Discussion

The symptoms of severe calcium deficiency reported here
confirm and extend those reported by Boelter and Greenberg (9).
The inquisitiveness and alertness of the phosphorus-deficient rats
contrasted sharply with the dullness and apathy of the calcium-
deficient rats. Also, the former never showed the squealing
reaction which in the latter became autonomic and was elicited
by approach of the hand, and, as noted, was still seen in the


CA-P


17 7 19
\33 19


3 5
CA-P 6 -25 19
(+VITAMIND) 13 25

PROTEIN (Pa) 6 5 7 4
-5 -4


PR-CA


PR-P


3 9 ) _5 9 -8
12 -7 9 3 9 2 4
X -7 -7 -13 -11






Relation of Food Delici( ocie to Skeletal Development 33

aged animals. None of the squealers carried the reaction pattern
beyond warning and tried to bite. They exhibited, however, an
appearance of frustrated, unhappy animals whenever a hand
neared to pick them up. Such signs suggest that the calcium-
deficient rats were under considerable thalamic control, possibly
effected by hemorrhages temporarily shorting out higher brain
centers.
Day and McCollum (5) reported work on diets extremely de-
ficient in phosphorus. In their work they employed a phosphorus
level of 0.017', which was about the same as that found in diets
in Table 2. Blood fibrin was used as the protein source. The
symptoms they reported for phosphorus deficiency resemble to
some degree the milder symptoms noted in this paper for animals
which expressed the double need for both protein and phos-
phorus. Also, pica, the abnormal craving for food, a character-
istic of phosphorus deficiency in the present paper, was never
observed. Moreover, these rats walked with only a "crippling
gait" and did not show complete hind limb paralysis. In addi-
tion, it took twice the time for the Day and McCollum diet to
produce deficiency symptoms.
The ability of the animals to live twice as long on the calcium-
lacking diet and even then show larger reserves of denser bony
material than on the phosphorus-low diets indicates an increased
rate of loss of bone material on the latter diet. Evidently both
calcium and phosphorus are lost by the body much faster on
a low-phosphorus than on a low-calcium diet. Such facts might
be of clinical interest, since they suggest a probable value in low-
phosphorus diets as a logical treatment in cases of lead or radia-
tion poisoning or, in any case where increase in rate of bone min-
eral turnover is desired.
Both growth and femur data showed that females were not
affected by early deficiencies as much as were males. It is sug-
gested in explanation that when both sexes were fed the same
deficient diet the larger growth requirements of the male allowed
development of severe deficiency and caused more injury.
Weanling rats undergoing vitamin A deficiency (7) showed ab-
normally long femurs like the calcium, phosphorus or protein de-
ficient animals of the text. Characteristic of all these diets was
interference with growth. When growth was slowed and stopped,
the femur, unlike the other long bones, continued growing for a
time. If the dietary lack during the period of deficient feeding
included both calcium and phosphorus, damage to epiphyseal cen-






Florida Agricultural Experiment Stations


ters, where bone extension initiates, was irreversible and the
femurs did not resume growth when the diet again became ade-
quate. Short femurs were associated with broad measures across
the pelvis. Thus, in effect, gangliness in the young rat was suc-
ceeded by the appearance of squattiness in the older rat.
A possible explanation for the formation of the two major
types of abnormal pelvic girdles in the mature rat is suggested
by noting the rate at which mineral nutrient became available
to centers of bone growth immediately after realimentation.
Since the groups that had been on the low-protein diets and in
particular the groups on the low-phosphorus diets began to eat
the complete diet avidly from the moment of realimentation,
minerals were supplied immediately to the bone-forming centers.
Girdle structure calcified before readjustment was complete and
the narrow pelvic girdles seen in Figure 14 resulted. Conversely,
animals on the low-calcium and particularly those on the low
calcium-phosphorus diets ate but small quantities of the new
food for more than two weeks after realimentation and minerals
for new bone became slowly available to the bone-forming cen-
ters. Adjustment in position were already overcorrected when
structure was fixed and the broad pelvic girdles in Figure 14
resulted.
None of the long bone or pelvic measurements made on stock
colony animals showed variability due to sex. Difference between
sexes in rats was found only in the relative amount of growth
on the same skeletal pattern. In connection with the same
problems in humans, Todd (11) wrote, "The pelvis is held by
many to be an infallible guide to sex. The only possible measure-
ments, namely intercristal, interspinous or some other form of
conjugate, have been attempted. In the living these are ob-
scured by the fat pads which give an illusory idea of sexual
pelvis differences There is no real or absolute sex difference in
mankind, even after puberty."

Summary
When weanling rats were fed a bread diet for 13 weeks and
then realimented upon a stock colony diet to maturity their
femur bones were short; they were as short as 60 percent of
normal mature length. Addition of either calcium or phos-
phorus-containing salts to the bread diet prevented development
of the short femur.
Other groups of weanling rats were fed diets containing 20 and







Relation of Food Deficiencies to Skeletal Development 35

6 percent levels of protein as lactalbumin but deficient in either
calcium or phosphorus or both; additional groups were fed diets
lacking calcium and phosphorus but carrying added vitamin D.
The rats were then realimented upon stock colony diet and the
carry-over effect of the early deficiency on later life pattern
was determined. A deficiency of both calcium and phosphorus
in the weanling's diet was associated with the development of
short femurs in the mature animal. When either calcium or
phosphorus-containing salts were added to the weanling's diet,
femurs of normal length were found in the mature animal.
While on the lactalbumin diets symptoms of severe calcium,
phosphorus or calcium-phosphorus deficiencies developed in the
weanling animals at both 20 and 6 percent levels of protein
intake. These symptoms have been described and contrasted.
Calcium-phosphorus deficiency resembled calcium deficiency in
most symptoms but resembled phosphorus deficiency in the more
rapid depletion of the body in bone mineral. Four weeks' feeding
of diets low in phosphorus depleted the body of bone mineral
more than did eight weeks of feeding diets low in calcium.
Young rats lost bone mineral faster on a low-phosphorus diet
than they did on a low-calcium diet. Mature rats reflected less
injury from their low-phosphorus weanling diet at the 20 per-
cent protein level than they did from any of the other deficient
diets.
Mineral and protein deficiencies in the weanling rats were
related to a. stunting of the animals in mature life. Weanling
diets deficient in calcium-phosphorus at both 20 and 6 percent
protein levels were associated with smaller mature animal
weights than were the other deficient diets. Weanling males
were injured more by the deficiencies than were females. Weight
trends appearing after a three months' realimentation period
continued throughout life.
Knee joint deterioration and vertebral distortion, particularly
in the thoracic region, were seen in mature animals that had
been exposed as weanlings to the deficiency of either calcium
or calcium-phosphorus. Degree of deformity found in the ma-
ture animal was less severe when the deficiency of low protein
was added to the mineral deficiencies in the diet of the weanling.
Addition of vitamin D to low calcium-phosphorus diets was asso-
ciated with increased stunting and joint injury but with de-
creased vertebral distortions and better thoracic contours.
When the young rats were on a mineral-deficient diet their
pelvic girdles were in the state of collapse, and while on the







Florida Agricultural Experiment Stations


subsequent good diet were repaired to varying degree. Vestiges
of the collapse were evident at either end of the innominates
when the animals had been on any of the low-protein diets or the
low-phosphorus diet. When the rats had been exposed to either
a calcium or a calcium-phosphorus deficiency as weanlings, re-
pair was marked by a wide flaring between the hip bones in the
mature animal.
Data on abnormalities in bone structure were demonstrated
by comparison with data on normal structure obtained from
curves that related femur/body lengths and pelvic girdle meas-
ures/innominate lengths in animals of the stock colony.

Literature Cited

1. ABBOTT, O. D., and R. B. FRENCH. Assessment of the nutritive value
of certain supplements when added to basal diets of enriched and
unenriched breads. Fla. Agr. Exp. Sta. Ann. Rep. p. 84. 1949.
2. ABBOTT, O. D., R. B. FRENCH and RUTH O. TOWNSEND. The nutritive
value of various breads and supplements in experiments with
white rats. Fla. Agr. Exp. Sta. Bul. 483. 1951.
3. Association of Official Agricultural Chemists. Official and Tentative
Methods of Analyses. Washington, D. C. 5th ed., p. 127. 1940.
4. CLAUSEN, DONALD F., and JOHN H. SHROYER. Molybdenum blue re-
action. Anal. Chem. 20: 925-6. 1948.
5. DAY, HARRY G., and E. V. McCOLLUM. Mineral metabolism, growth
and symptomatology of rats on a diet extremely deficient in phos-
phorus. Jour. Biol. Chem. 130: 269-83. 1939.
6. DONALDSON, HENRY H. The Rat. Data and Reference Tables for
the Albino Rat (Mus Norvegicus Albinus), and the Norway Rat
(Mus Norvegicus). Memoirs of the Wistar Institute of Anatomy
and Biology. No. 6, p. 185, 2nd ed. rev. 1924.
7. FRENCH, R. B. and 0. D. ABBOTT. Effect of carotene or vitamin A
deficiency in young rats on subsequent life pattern. Unpub-
lished data. 1954.
8. HUBBELL, REBECCA B., LAFAYETTE B. MENDEL and ALFRED J. WAKE-
MANN. New salt mixture for use in experimental diets. Jour.
Nutr. 14: 273-85. 1937.
'9. BOELTER, MURIEL O. D., and DAVID M. GREENBURG. Severe calcium
deficiency in growing rats. I. Symptoms and pathology. Jour.
Nutr. 21: 61-84. 1941.
10. SUPPLE, G. C., LORRAINE S. GALL and JEAN F. CAUL, with the co-
operation of L. C. Babcock and Ester G. Noble. The varying
requirement of proteins (casein, lactalbumin and soy protein)
with particular reference to the hepatico-rena] syndrome. Jour.
Dairy Sci. 28: 435-53. 1945.
11. TODD, T. W. Growth and development of the skeleton. White House
Conference, Pt. II, Anatomy and Physiology, pp. 98-99. 1953.
12. YOUNGBURG, GUY E., and MAMIE YOUNGBURG. I. A system of blood
phosphorus analysis. Jour. Lab. Clin. Med. 16: 158-66. 1930.




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