EPITHELIAL AND BONE TISSUE MAST CELL
POPULATIONS IN THE FEMALE RAT AS
INFLUENCED BY CALCIUM AND VITAMIN D
DEFICIENCIES, OVARIECTOMY, AND ESTROGEN
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
to my mother
who would have thoroughly enjoyed
observing this entire experience
My sincere appreciation is expressed to Dr. John P.
Feaster, chairman of the supervisory committee, for under-
taking that position, directing my academic program, and
giving his time and expertise whenever needed. Very deep
appreciation is also expressed to Dr. Morris Notelovitz,
who served as co-chairman of the supervisory committee
and directed the research with unfailing interest and
support. These two individuals made possible the under-
taking and completion of a very important goal.
Special thanks are also extended to Dr. Clarence
Ammerman, Dr. James Dinning, and Dr. W.C. Thomas for their
willing assistance in serving on the supervisory committee.
Thanks are given to Dr. Moreland, the veterinarians,
and staff at the Department of Animal Resources who helped
with the maintenance of the laboratory animals, especially
Terry who aided me in the most unpleasant aspect. The tech-
nical advice and use of equipment and facilities offered by
Dr. David Chen are also very much appreciated. Much thanks
goes to I.C. Song for teaching me the surgical and other
skills necessary in carrying out the experiments. Specially
remembered are Pat Ruzakowski and Liu for their assistance.
Terry Ansman and staff at the Department of Pathology
are thanked for their time and efforts in teaching me the
histology techniques required to carry out the work.
Sincere thanks are given to Dr. Hans Burchardt and
staff, Department of Orthopaedics, for technical guidance in
working with skeletal tissue. The assistance and expertise
Dr. Gary Miller has provided for the biomechanical tests is
Inestimable thanks are extended to John Funk, Ray Smith,
Pam Miles, John Easley, Dr. John Moore and others at the
Nutrition Lab for providing knowledge, assistance, equipment
and supplies, all necessary to complete the laboratory
analyses within the project. Closely felt is the friendship
of the graduate students there, especially Dawn, Oswaldo,
Roberto and Joshua.
Remembered also is Lynda McKenzie who assisted with the
densitometry and gave her time to listen and help. Bill
Noffsinger is thanked for all the statistical analysis
assistance. Pam Victor is thanked for the typing of this
dissertation. Her proficiency has helped immeasurably.
The cooperation and financial assistance provided by the
Department of Animal Science and The Center for Climacteric
Studies, University of Florida, in order to carry out the
research, are gratefully acknowledged. Sigma Xi, the
Scientific Research Society, has partially supported the
study by a Grant-In-Aid of Research. Appendix A contains
information relative to this grant.
Lastly, but of great importance, sincere gratitude is
also expressed to my children and husband for their patience,
interest and support, which were entirely necessary.
TABLE OF CONTENTS
ACKNOWLEDGEMENTS .................................. ... iv
LIST OF TABLES ........................................ ix
LIST OF FIGURES ....................................... xi
ABSTRACT ................................ .............. xii
I INTRODUCTION ................................. 1
II REVIEW OF THE LITERATURE ..................... 3
Description of the Mast Cells ................. 3
Mast Cells In Bone Marrow .................... 6
Effects of Diet ............................ 9
Mast Cells in Vaginal Tissue and Influence
of Gonadal Hormones .......................... 11
III MATERIALS AND METHODS ......................... 14
Animals and Treatments ........................ 14
Analytical Methods ........................... 16
Histology ..................................... 18
Data Analysis ............................... .. 19
IV RESULTS AND DISCUSSION ........................ 21
Body Weights .. ............................... 22
Serum Calcium ................................ 25
Bone Densitometry ............................ 28
Femur lengths ........................... 28
Bone mineral content .................... 31
Bone density ............. ............. .. 33
Biomechanical Tests ............................ 35
Torque ................................. 35
Deformation ............................. 38
Bone Ash ...................................... 38
Table of Contents continued
Mast Cells ..................................... 41
Bone Marrow Mast Cells ..................... 41
Normal diet group ....................... 44
Calcium-deficient group ................. 44
Vitamin D-deficient group ............... 45
Calcium- and vitamin D-deficient group .. 45
Vaginal Tissue Mast Cells .................. 47
Normal diet group ...................... 47
Calcium-deficient group ................. 49
Vitamin D-deficient group ............... 49
Calcium- and vitamin D-deficient group .. 49
V CONCLUSIONS ................................. 51
A SIGMA XI GRANT-IN-AID OF RESEARCH ............. 55
B LABORATORY ANIMAL USE ......................... 66
C EXPERIMENTAL ANIMAL BODY WEIGHTS ............. 70
D COMPOSITION OF EXPERIMENTAL DIETS ............. 74
E SERUM CALCIUM ANALYSIS ........................ 76
F DENSITOMETRIC BONE ANALYSIS ................... 83
G BIOMECHANICAL TESTING ......................... 96
H ASH ANALYSIS .................................... 103
I MAST CELLS ...................................... 113
REFERENCES ............. ............................... 162
BIOGRAPHICAL SKETCH .................................. 168
LIST OF TABLES
Study Design .................................
Body Weight Gain as Affected by
Diet and Treatment ...........................
Effect of Diet and Treatment on
Serum Caclium ................................
Effect of Diet and Treatment on Densitometric
Measurements of Rat Femurs ...................
Effect of Diet and Treatment on Biomechanical
Properties of Rat Femurs .....................
Effect of Diet and Treatment on Bone Ash ......
Mast Cell Populations as affected by Diet and
Treatment in the Female Rat ..................
Experimental Body Weights: Normal Diet ........
Experimental Body Weights: -Ca Diet ...........
Experimental Body Weights: -D Diet ............
Experimental Body Weights: -Ca, -D Diet .......
Normal Diet ......
-Ca Diet .........
-D Diet ..........
-Ca, -D Diet.....
Normal Diet ......
-Ca Diet .........
-D Diet ..........
-Ca, -D Diet .....
Bone Length ......
List of Tables continued
G.1 Biomechanical Properties: Normal Diet ......... 99
G.2 Biomechanical Properties: -Ca Diet ............ 100
G.3 Biomechanical Properties: -D Diet ............ 101
G.4 Biomechanical Properties: -Ca, -D Diet ........ 102
H.1 Bone Ashing Analysis Data: Normal Diet ........ 105
H.2 Bone Ashing Analysis Data: -Ca Diet ........... 107
H.3 Bone Ashing Analysis Data: -D Diet ............ 109
H.4 Bone Ashing Analysis Data: -Ca, -D Diet ....... 111
I.1 Mast Cell Count--Vaginal Tissue: Normal Diet .. 113
1.2 Mast Cell Count--Vaginal Tissue: -Ca Diet ..... 117
1.3 Mast Cell Count--Vaginal Tissue: -D Diet ...... 122
1.4 Mast Cell Count--Vaginal Tissue: -Ca, -D Diet 126
1.5 Mast Cell Count--Bone Marrow: Normal Diet ..... 129
1.6 Mast Cell Count--Bone Marrow: -Ca Diet ........ 133
1.7 Mast Cell Count--Bone Marrow: -D Diet ......... 138
1.8 Mast Cell Count--Bone Marrow: -Ca, -D Diet .... 142
LIST OF FIGURES
IV.1 Body Weight Gains in Female Rats .............. 24
IV.1 Femur Length by Photonabsorptiometry .......... 30
IV.3 Bone Mineral Content of Rat Femurs ............ 32
IV.4 Linear Bone Density of Rat Femurs ............ 34
IV.5 Torque Required for Fracture .................. 37
IV.6 Femoral Deformation Prior to Fracture ......... 40
IV.7 Bone Marrow Mast Cell Populations in
the Female Rat ............................... 43
IV.8 Vaginal Tissue Mast Cell Population in
the Female Rat .............................. 48
F.1 The Norland Digital Bone Densitometer ......... 86
F.2 Printout Display of Rat Femur Profile ......... 86
G.1 The Rapid Loading Torsional Testing Machine ... 98
G.2 Representative Torque-Deflection Curves ....... 98
I.1 Photomicrograph of Mast Cells--Bone Marrow:
Normal Diet .................................. 147
1.2 Photomicrograph of Mast Cells--Bone Marrow:
-Ca Diet ..................................... 149
1.3 Photomicrograph of Mast Cells--Bone Marrow:
-D Diet ...................................... 151
1.4 Photomicrograph of Mast Cells--Bone Marrow:
-Ca, -D Diet ................................ 153
1.5 Photomicrograph of Mast Cells--Vaginal Tissue:
Normal Diet ................................... 155
1.6 Photomicrograph of Mast Cells--Vaginal Tissue:
-Ca Diet ..................................... 157
1.7 Photomicrograph of Mast Cells--Vaginal Tissue:
-D Diet ...................................... 159
1.8 Photomicrograph of Mast Cells--Vaginal Tissue:
-Ca, -D Diet .............................. ... 161
Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
EPITHELIAL AND BONE TISSUE MAST CELL POPULATIONS IN THE
FEMALE RAT AS INFLUENCED BY CALCIUM AND VITAMIN D
DEFICIENCIES, OVARIECTOMY AND ESTROGEN
Chairman: Dr. J.P. Feaster
Cochairman: Dr. Morris Notelovitz
Major Department: Animal Science
Effects of dietary deficiencies on mast cell popula-
tions in the bone marrow and vaginal epithelial tissue of
rats were investigated. Additionally, effects of exogenous
estrogen, bilateral ovariectomy, and a combination of the
two treatments on mast cell populations were observed in
these two tissues in rats on normal and deficient diets.
Two-month old, female Sprague-Dawley rats were fed cal-
calcium- and vitamin D-deficient diets for five weeks. One
group of rats was given estradiol by injection (100 pg/.l ml)
three times a week for the duration of the experiment, another
group was ovariectomized, a third group received both treat-
ments, while a fourth, untreated group served as a control
Dietary-induced osteopenia was evidenced by densitomet-
ric measurements and bone ash of the rat femur. Significant
decreases in bone mineral content (P<.005) due to diet were
observed. Bone ash values were also significantly low due
to diet (P<.0005).
Femur length, measured by photonabsorptiometry, was
found to be decreased due to (a) dietary deficiencies of
calcium and vitamin D (P<.005); (b) estrogen administration
(P<.005); and (c) ovariectomy (P<.005).
Contrary to expectations, bone marrow mast cell popula-
tions were not altered by dietary deficiencies in untreated
rats. This result may be due to the age of the rat when
begun on the deficient diet (two months old) and to the
duration of the imposed diet deficiencies (five weeks).
Dietary deficiencies reduced marrow mast cell counts in
estrogen treated rats, however (P<.05). Ovariectomy induced
a reduction of mast cells in the bone marrow of calcium- and
vitamin D-deficient rats (P<.01), which suggests that bone
is an estrogen sensitive tissue even though estrogen recep-
tors are not present in bone.
Vaginal tissue mast cells were not significantly
altered in number by dietary deficiencies except when rats
were estrogen treated (P<.05). The most significant finding
was that of mast cell population increases in vaginal tissue
as a result of ovariectomy (P<.01) in all dietary groups.
The removal of endogenous estrogen by ovariectomy in
the female rat was found to affect both bone marrow and
vaginal tissue mast cell populations. The relevance of this
finding remains to be determined.
Rapid bone loss with resultant osteoporosis affects 25
to 30% of all postmenopausal women (Urist, 1971) and about
75% of women who have undergone a bilateral ovariectomy. The
significance of this condition is associated not only with
pathological fractures and potential invalidism but also with
the premature death of these women. The early diagnosis of
this condition and measurement of treatment response present
two problem areas in clinical management. To date there is
no universally applicable, noninvasive method for detecting
the early stages of rapid bone loss in humans.
Research has shown that mast cells, found in increased
numbers in the bone marrow of osteoporotic and calcium-defi-
cient subjects, accompany local loss of bone mass. The
present study seeks to determine whether vaginal epithelial
mast cell populations, as well as bone marrow populations are
affected by dietary deficiencies of calcium and vitamin D,
exogenous estrogen, and ovariectomy in the female rat.
Should such alterations be found in the female rat, similar
changes in populations might be expected in the osteoporotic
human female. Tissue biopsy of vaginal epithelium to deter-
mine mast cell populations could provide a relatively nonin-
vasive method for detecting osteomalacia and conditions
involving increased bone resorption such as osteoporosis.
The procedure might also be used to assess treatment
Therefore, two hypotheses were tested in this
(a) Dietary calcium- and vitamin D-deficiencies produce
changes in mast cell populations in female rat bone
marrow and vaginal epithelium.
(b) Exogenous estrogen, bilateral ovariectomy, or a
combination of the two treatments alters mast cell
populations in bone marrow and vaginal tissue of
calcium- and vitamin D-deficient female rats.
Other parameters considered and investigated in this
(a) weight changes of the laboratory animals during the
(b) serum calcium concentration
(c) bone mineral content, density, and length
(d) torsion and deformation of the rat femurs
(e) ash content of the femurs.
REVIEW OF THE LITERATURE
Addressed in the literature review are a description of
the mast cell and the activity of two constituents, histamine
and heparin. Research involving the mast cell in bone marrow
of the rat and human is reviewed. Effects of dietary defi-
ciencies, specifically those of calcium and vitamin D, on
the rat mast cell in bone tissue are examined. Also cited
are the few studies concerning mast cells in vaginal tissue
and those relating to the effects of gonadal hormones on
skeletal and vaginal tissues.
Description of the Mast Cell
Naming the specialized histiocytes as mast cells has
been credited to Paul Ehrlich when he suggested, in 1877,
that the cells arose from connective tissue cells which had
been well-fed, or mastedd' (Wilhelm et al., 1978). The German
masten (to feed) or mastzellen (mast cells), from which the
name originated, appropriately describes their usually full
appearance. This is due to a high content of cytoplasmic
Pathak and Goyal (1973) state that two separate types
of mast cells occur (1) large spindle shaped, fusiform or
cylindrical cells with or without elongated processes and
(2) small, round, or elliptical cells. Riley (1959) also
described differences in rat mast cells and distinguished
between two types based on granule maturation. Differences
also exist when mast cells are examined with electron micro-
scopy (Combs et al., 1965).
Rat peritoneal fluid, the most common source of mast
cells for laboratory investigation, exhibits round mast cells,
13.5 to 17 pm in diameter, each with a round or oval nucleus.
Cytoplasmic granules are approximately .7 pm in diameter
(Yong et al., 1975).
In an extensive quantitative analysis of rat mast cell
structure, Helander and Bloom (1974) report an average mast
cell diameter as 11 pm, granule diameter as .78 pm, with the
nucleus occupying 10.7% of the cell. The size, shape, stain-
ing properties, and distribution of mast cells vary with the
tissue and species of animal studied (Wilhelm et al., 1978).
Rat tissues abound in mast cells, whereas tissues of the
rabbit contain related basophils often referred to as blood
mast cells. Tissues in man and the guinea pig exhibit both
mast cells and basophils.
A well-known characteristic of both mast cell and baso-
phil leukocyte granules is an exhibition of metachromasia
upon treatment with certain basic dyes such as toluidine
blue, methylene blue, alcian blue, and azure A. These
stains are used to identify and demonstrate the mast cells
in tissues. Since many differences in mast cell reactivity
towards these dyes occur within and between species, an exten-
sive investigative interaction among histological studies,
identification of cell constituents, and physiological
functions of the mast cells exists. The constituents of the
mast cell granules, specifically the acidic mucopolysaccha-
rides, are responsible for the various staining properties
of the cells. However, partly because of the chemical diver-
sity of the granular contents, mast cell functions in health
or disease remain an enigma.
In 1937, the metachromatic component of the mast cell
was reported to be heparin (Jorpes et al., 1937); and since
then, the mast cell in hepatic tissue has been considered as
the only endogenous source of heparin production (Riley, 1962).
A protein matrix is thought to bind anionic heparin in the
mast cell granule, possibly by sulfate groups. Heparin, in
turn, binds histamine and other basic nitrogen-containing
compounds such as serotonin (Schubert, 1968). Histamine
levels in tissues correlate with the mast cell count and a
very large proportion of rat histamine formation takes place
in the bone marrow.
The close histological relationship of mast cells and
blood and lymph vessels is well-known. Small vessels are
the prime target of histamine-mediated inflammatory reactions.
Histamine is known to cause contraction of part of smooth
muscle (mainly the bronchioles), dilate blood capillaries,
and increase their permeability (Rahima and Soderwall, 1977).
In-vitro laboratory studies of the mast cells (in cer-
tain tissues and species) demonstrate that histamine is
released by liberators such as stilbamidine, 48/80, or pro-
tamine sulfate by displacing the heparin-bound histamine
Sudden degranulation of mast cells may cause adverse
reactions since large amounts of histamine are released into
the extracellular space. This release usually occurs in
response to a type I antigen-antibody reaction on the surface
of mast cells that have been previously sensitized by cell-
bound Ig E antibody (Coombs and Gell, 1975). Allergic rhin-
itis, allergic asthma, urticaria, angioedema, and mastocyto-
sis constitute some manifestations of extensive degranulation.
An editorial by Kaliner (1979) details this aspect of the mast
cell's varied activities.
Mast Cells in Bone Marrow
In addition to its role in immediate hypersensitivity
reactions, histamine has been reported to affect bone remodel-
ing and maturation (Norton et al., 1969). Systemic mastocyto-
sis in urticaria pigmentosa has been accompanied by marked
bone remodeling, bone hypertrophy (Sagher et al., 1956), and
osteosclerosis (Kruse et al., 1973).
In this same regard, the acid glycosaminoglycan, heparin,
in addition to its anticoagulant property, has been considered
as a bone resorbing and osteoporosis producing agent. This is
especially true with the use of high doses of the anticoagu-
lant for long periods of time (Goldhaber, 1965; Griffith et
al., 1965; Jaffe and Willis, 1965; Wise and Hall, 1980).
Heparin has been reported to stimulate bone collagenase activ-
ity in the rat (Asher and Nichols, 1965) and to potentiate the
action of parathyroid hormone (Goldhaber, 1965), suggesting
inducement of osteoporosis. A review of the relationship
between mast cells, heparin, and osteoporosis has been
provided by Hegsted (1969).
Many reports of heparin use with resulting skeletal
problems involved the use of heparin for control of blood
coagulation. It has been documented that heparin extracted
and purified from tissues rich in mast cells and reinjected
by the physician behaves differently from endogenous heparin
(Jaques et al., 1977). Only in the dog has the hepatic re-
lease of heparin been shown to have a rapid anticoagulant
effect in the circulation. Several species have no heparin
in the mast cell; its metachromasia is attributed to other
Osteoporosis has been viewed by some as a sequel to
diminished blood flow through the marrow (Burkhardt, 1973).
Contracted arterioles in mastocellular lesions of the bone
marrow in human osteoporotics provide evidence for this
concept (te Velde et al., 1978).
Increased numbers of bone marrow mast cells have been
reported in osteoporosis (Frame and Nixon, 1968; Kruse et
al., 1973; Peart and Ellis, 1975). Two theories have been
have been expressed (1) mast cells induce porosis (Frame
and Nixon, 1968) and (2) mast cells oppose the porosis
(Kruse et al., 1973).
Increased mast cell numbers have been associated with
bone resorption in regenerating parts of the marrow. Gillman
(1958) noted increased mast cells in long bone marrow of rats
fed sweet pea seeds containing a lathyrogenic agent. He
distinguished between newly formed and old femoral shafts,
with the increased number found in the marrow of the newly
formed shaft. Severson (1969) showed that mast cells secrete
a factor necessary for hydrolytic enzyme release in regions
of increasedresorption and remodeling. Walker (1970) reported
an eight-fold increase of mast numbers in regenerating rat
femoral marrow after mechanical disruption when compared to
the unoperated contralateral femur. Hypophysectomy resulted
in an even greater increase and longer effect.
Extensive studies in bone repair (Lindholm et al., 1967,
1969) demonstrated active involvement by mast cells, increased
mast cell numbers in callus formation, and mast cell provision
of alkaline phosphatase, phosphorylase, and other enzymes es-
sential for endochondral ossification.
Human alveolar bone resorption in chronic periodontal
disease is associated with increased mast cell counts in
gingival tissue (Shapiro et al., 1969; Riley, 1959; Sognnaes,
1965). Other investigators (Carranza and Cabrini, 1955;
Calonius, 1960; Dummet et al., 1961) failed to confirm this
In hyperparathyroidism, both the resorption and the for-
mation of bone are stimulated, but greater increases in bone
resorption occur (Bonucci et al., 1978). Mast cells have
been reported in fibrotic marrow spaces in human hyperpara-
thyroid patients. Other researchers (Rockoff and Armstrong,
1970) found that low doses of parathyroid hormone chronically
administered to rats produced mast cell hyperplasia in the
tibial metaphyseal marrow, without alterations of serum
calcium or phosphorous.
Secondary hyperparathyroidism is known to result from
lowered serum calcium levels and is thought to be a mechanism
whereby low dietary calcium intakes promote mast cell in-
creases in bone.
Effects of Diet
As early as 1922, increase of tissue basophils in the
immediate vicinity of the bone trabeculae and marked resorp-
tion of bone in rats on calcium deficient diets were reported
(Shipley and Park, 1922). Urist and McLean (1957) identified
those basophils as mast cells. They also maintained rats on
low calcium, low vitamin D, and high phosphorus diets which
produced rickets, osteoporosis, and osteitis fibrosa as well
as increased endosteal mast cells. Cass et al. (1958) con-
firmed the results of increased bone mast cells in rats fed
calcium-deficient diets and found an increase in bone marrow
content of histamine and 5-hydroxytryptamine, another mast
cell mucopolysaccharide. Rockoff and Armstrong (1970) also
administered a calcium-deficient but vitamin D-adequate diet
to a group of rats, with bone marrow mast cell hyperplasia
resulting in all test animals. In providing hypocalcemia-
inducing vitamin D-deficient and calcium- and vitamin D-
deficient diets to rats, Rasmussen (1972) observed signifi-
cant increases in tibial metaphyseal bone marrow mast cells.
Parathyroidectomy caused a significant reduction in mast
cells, again suggesting secondary hyperparathyroidism as a
mechanism for increased mast cell populations in bone. Other
rats given low calcium and high phosphorus diets with and
without vitamin D exhibited hypocalcemia, rachitic bone
changes, increased bone resorption and increased mast cells
in metaphyseal areas of long bone but not in the epiphyses or
caudal vertebrae (Feik and Storey, 1979); however, it was not
possible to relate the mast cell increases to specific areas
of bone formation or resorption, as had been planned.
In dietary calcium- and vitamin D-deficient rats with
induced fracture callus, mast cell counts in the callus
approximated 200 to 300 cells per mm2 which rose to 1,900
cell per mm2 until 35 days after fracture. These mast
cells were mostly degranulated. Normal rats exhibited
strongly granulated mast cells, 2,000 to 4,000 mm2 for the
first two-month period with remarkably decreased levels
thereafter. Mast cell numbers were correlated with mineral-
ization after fracture (Lindholm et al., 1972).
Accumulations of mast cells in healing sockets or
extracted mandibular first molars were found in rats fed
calcium-deficient diets, with control rats exhibiting only
an occasional mast cell (Smith, 1974). Besides the fact that
mast cell numbers were examined in different bones in the two
studies, the contradictory findings were not explained.
Other dietary deficiencies have also affected mast cell
populations in bone marrow. Belanger (1978) found a signif-
icant increase of bone marrow mast cells in rats on zinc
deficient diets, and also on magnesium-deficient diets (1977).
Concurrent decreases in skin mast cell numbers of the magnesium-
deficient rats agree with the findings of Bois (1962).
Mast Cells in Vaginal Tissue and Influences of Gonadal Hormones
Vaginal tissue mast cell population studies in the rat
are essentially nonexistent. Salvi (1952) found mast cells
more abundant and with greater metachromatic properties in
the mouse vagina than in the uterus. After daily estrogen
administration, adult mouse vaginal tissue revealed a con-
siderable increase in the number of mast cells (Arvy, 1955).
Westin and Odeblad (1956) also investigated the influence
of ovarian hormones on mast cells in the mouse vagina.
Darker metachromasia in the vagina than in the uterus and
difficulty in detecting granules were experienced. The con-
trol group had the highest number of vaginal tissue mast
cells and also the highest variation in number per field
examined. The estrogen treated groups had a significantly
reduced number; intermediate numbers were observed when
estrogen plus progesterone was administered. Mast cells
of the skin remained constant. The estrogen effect on the
mast cells was considered a local process within the repro-
ductive organs. Zwillenberg (1958) noted a variable occur-
rence of mast cells in the vaginal epithelium of human
Discrepancies in results among studies may be due, in
part, to differences in estrogen dosage. Iversen (1962)
notes that, while small doses of estradiol decreased the
number of uterine mast cells in the guinea pig, prolonged
treatment with large doses had an opposite effect.
Although there are mast cell studies which investigated
the effect of gonadal hormones in various tissue (Constan-
tinides and Rutherdale, 1954; Asboe-Hansen, 1956; Johansson
and Westin, 1958; Smith and Lewis, 1958; Schiff and Burn,
1961; Kameswaren et al., 1978), few reported on bone tissue.
Belanger (1977), in his study with magnesium deprived rats,
administered large doses of testosterone to males and estra-
diol to females. This treatment depressed the mast cell popu-
lation increase in the bone marrow and moderated skin mast
Similarly, there is no reference in the literature con-
cerning the effect of ovariectomy on mast cells in bone or
vaginal tissue. The mice in the study of Westin and Odeblad
(1956) were all spayed so that effect of ovariectomy could
not be compared to control groups. Two studies reported
that ovariectomy has no effect on uterine mast cells in
guinea pigs or hamsters (Iversen, 1962; Harvey, 1964).
Because of the differences in mast cell populations,
structure, function and activity in various species (and
in tissues within the same species), information cannot be
extrapolated from one species to another. Because of the
various mast cell constituents and their resulting diverse
functions and actions in tissues, inconsistencies in results
will continue to be reported. However, in the recent past,
much new information on the mast cell has been brought forth.
The particular role of the mast cell in the pathogenesis of
the osteoporoses and other demineralizing bone diseases re-
mains in need of further investigation.
MATERIALS AND METHODS
Animals and Treatments
To monitor the care, treatment, and use of laboratory
animals at the University of Florida, the All University
Committee on the Care and Use of Laboratory Animals requires
specific information pertaining to research involving labor-
atory animals. The application requesting use of laboratory
animals for this particular research project as submitted to
the Committee and its approval are found in Appendix B.
One hundred thirty-six female Sprague Dawley rats,l
9 weeks of age and weighing approximately 180 g at the
start, were used for the research.
The Health Center Animal Resources Department, Univer-
sity of Florida, provided housing for the animals. The rats
were kept in galvanized wire cages, two to a cage, in a room
maintained constantly at 240C and 60% humidity. The rats
were weighed at least once a week for the 4 to 5 week exper-
imental period. Appendix C contains data on the animal
The animals were divided into the following dietary
Outbred laboratory Sprague-Dawley rats were obtained from
Harlan-Sprague-Dawley, Madison, WI 53711.
I. Normal (control) rats (semipurified complete diet)
II. Calcium-deficient (-Ca) rats (calcium-deficient diet)
III. Vitamin D-deficient (-D) rats (rachitogenic diet;
calcium to phosphorus ratio of 4.2:1)
IV. Calcium- and vitamin D-deficient (-Ca, -D) rats
(custom formulated calcium- and vitamin D-deficient
Appendix D contains information on dietary formulations.
Each dietary group consisted of 32 rats, except for group
II, in which there were 40. The diets (pelleted) and dis-
tilled water were offered ad libitum. Group III rats were
borderline vitamin D-deficient at the beginning of the experi-
mental period. One rat in group IV was rejected because of a
Upon receipt of the animals and prior to further treat-
ment, each rat was tested by vaginal smear daily for an 8 day
period to determine presence of estrous cycling. Each rat
presented with at least once cycle, demonstrating reproductive
capability and ascertaining endogenous circulating estrogen.
Half the rats in each group were bilaterally ovariectom-
ized. Sham ovariectomies were performed on the remaining rats
in each group. The combination of ketamine and xylazine (Van
Pelt, 1977), at a concentration of 87 mg ketamine and 13 mg
xylazine per kg body weight of rat, was used to induce surgi-
cal anesthesia. One rat in group II died as a result of the
One-half of the ovariectomized and one-half of the sham-
operated rats in each group were injected three times per week
under the dorsal skin with .1 ml of a solution of estradiol
valerate (100 pg/.1 ml) in sesame oil (12 injections per rat
total). Concurrently, the remaining rats in each group were
injected with .1 ml of sesame oil as a control measure.
After the five to six week experimental period, all ani-
mals were killed by decapitation.
Table III.1 summarizes the research design described.
Normal -Ca -D -Ca, -D
(N) (N) (N) (N)
Ovariectomized + Estrogen 8 12 8 8
Ovariectomized 8 12 8 8
Estrogen 8 8 8 8
Normal (no treatment) 8 8 8 8
Blood was obtained at the time of decapitation by
exsanguination. Total calcium in serum was determined by
atomic absorption spectrophotometry (AAS). The procedure
followed was that of Fick et al. (1979). Data on serum
calcium values are found in Appendix E.
Immediately after the animals were killed, uterine and
vaginal tissues were removed, hind extremities were disarti-
culated at the acetabulum, and femurs were dissected free.
The femurs were cleaned of adherent tissues. The uterine and
vaginal tissues and femurs were fixed for 24 hours in 10%
Bone densitometric measurementsI using direct photon
absorptiometry were made on one femur from each animal.
Values of bone mineral content in grams per centimeter length
of bone, linear bone density in grams per square centimeter,
and bone length in centimeters were obtained. Appendix F
contains information on these paremeters.
The same femurs were subjected to torque and deformation
testing2 to determine the effect of treatment modality on
these biomechanical properties. The procedure followed was
that of Puhl et al. (1972). Explanation of this testing
procedure is found in Appendix G.
Ashing of these same femurs was done as described by
Fick et al. (1979). Appendix H contains data on ash
1Norland Digital Bone Densitometer, Model 278, Norland Cor-
poration, Ft. Atkinson, WI 53538.
2Rapid Load Torsional Testing Machine, Biomechanics Labora-
tory, Department of Mechanical Engineering, University of
Florida, Gainesville, FL 32611.
The alternate femurs of all animals were demineralized
in a 10% solution of di-sodium-ethylene-diamine-tetracetic
acid (EDTA) for a 7 to 14 day period. The solution was kept
at 50F with changes of solution every 2 to 3 days (Belanger
et al., 1965).
The demineralized femurs and vaginal tissues were dehy-
drated in 80% acetone for one-half hour and in 100% acetone
for another half-hour. Clearing was accomplished with two
changes of xylene (15 and 45 minutes); subsequently the tis-
sues were embedded in paraffin. Medial sagittal sections of
bone and cross sections of vaginal tissue were cut at 8 im
in a microtome-cryostat, floated on water, slipped onto
slides prepared with Haupt's solution, and air-dried.
Toluidine blue stain was chosen for mast cell quantita-
tion purposes (Pathak and Goyal, 1973). With this stain,
mast cells appear purple or reddish-purple against a general
blue background. The slides were subjected to the following
(1) 2 changes of xylene (4 minutes each)
(2) acetone (several dips)
(3) water rinse
(4) .2% toluidine blue
(5) water rinse
(6) acetone (several dips)
(7) 2 changes of xylene (4 minutes each)
Alternate slides were stained with .1% alcian blue in
3% acetic acid for thirty minutes followed by a water rinse
and .1% safranin in 1% acetic acid for five minutes in place
of step #4 (Spicer, 1960). Using this staining procedure,
the maturity of mast cells can be determined. Analysis of
these slides is planned for a future time.
After air-drying, cover slips were applied to the slides
with mounting medium.
Mast cells were counted in vaginal tissue and in the
distal part of the femoral metaphysis and in the bone marrow
of the diaphysis. Care was taken to avoid bone trabeculae
and sinuses. Counts were made over five fields in each of
five sections (25 fields per rat) for each type tissue at
a magnification of X400. Each field measured .458 mm in
diameter, representing a surface area of .165 mm and a
total surface area of 4.1 mm for each tissue per rat.
Counts were adjusted to 1 mm2 surface area. Appendix I
contains mast cell quantitation data.
The statistical evaluations for tests of significance
were carried out on the parameters using analysis of variance
and applying the t test (Steel and Torrie, 1960). The tables
list mean values and standard error of the mean (SE = s/Yn ),
and indicate level of significance.
1Labophot-Laboratory & Clinical Microscope, Nikon Instrument
Division, 623 Stewart Ave, Garden City, NY 11530.
Effects of diet were determined statistically by compar-
ing the means in each deficient diet group with means in the
normal diet group for each treatment.
Effects of treatment were determined statistically by
comparing means for each treatment with the mean of the
untreated rats within each diet group.
RESULTS AND DISCUSSION
The experimental animals used in this investigation
were at least three months old at termination of the experi-
ment and regarded as young adults. Reproductive capability
was determined prior to treatments by vaginal cell sampling.
One-half the population underwent ovariectomy, thereby re-
moving the source of estrogen production.
The diet used for the vitamin D-deficient group of rats
was also notably deficient in phosphorus, with a calcium to
phosphorus ratio of 4.2 to 1. Calcium to phosphorus ratios
of all the diets are given in Appendix D.
As a matter of information, the calcium content of the
complete diet (normal diet group) was 11.5 g/kg and the
phosphorus content was 10.1 g/kg. The calcium-deficient
diet contained 1.6 g/kg calcium and 26.6 g/kg phosphorus.
The rachitogenic vitamin D-deficient diet noted as being low
in phosphorus contained 12.4 g/kg calcium and 2.9 g/kg phos-
phorus. The calcium- and vitamin D-deficient diet contained
1.6 g/kg calcium and 26.6 g/kg phosphorus (identical to the
calcium-deficient diet but with omission of vitamin D3).
In addition, the protein in the vitamin D-deficient
diet provided by whole yellow maize and as gluten, was of
poor quality, lacking in essential amino acids. Therefore,
when evaluating effects of this diet, the deficiency in
protein and phosphorus must also be considered. Effects of
diet were not due solely to lack of vitamin D.
Body weight changes were observed in the rats. In
Table IV.1 is recorded the average weight gain for each
cell. The % weight gain is listed below the mean. The
untreated rats fed a normal diet, and used as a control
group, increased their body weight by 34% during the
experiment. Lower weight gains were observed as due to
calcium deficiency (P <.05). The vitamin D-deficient group
also had lower weight gains as an effect of diet (P<.01).
Administration of estrogen to both intact and ovariec-
tomized rats decreased body weight gains in the normal,
calcium-deficient, and the calcium- and vitamin D-deficient
groups (P<.05 to .005). Similar effects occurred with rats
on a normal diet (Cruess and Hong, 1979).
Ovariectomy increased the weight gains in the same
three dietary groups (P<.005) (Fig. IV.1).
This supports the findings of Cruess and Hong (1979)
and Lindgren and Lindholm (1979) in that removal of ovaries
subjected rats to high increases in body weight. This
effect has been associated with a higher food intake (Aitken
et al., 1972). However, significant decreases in body
weight of young castrated male rats have been observed
(Scow, 1952; Gumbreck, 1957; Saville, 1969; Wink and Felts,
IA 1D T-
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Results indicate a gain of weight in all cells of the
study. A calcium-deficient diet and a combination protein,
phosphorus, and vitamin D-deficient diet caused rats to gain
Estrogen inhibited the rate of weight gain, which is an
effect not well understood. This effect was seen with both
intact and ovariectomized rats. Ovariectomy clearly in-
creased weight gain and estrogen administration reduced that
gain to below the normal gain. The same pattern of this
hormonal effect was observed in the deficient diet groups.
Table IV.2 outlines the changes in serum calcium
observed in rats on four diet regimes and four treatments.
Untreated rats on a normal diet had a mean serum calcium of
9.63 mg/100 ml. Rats on deficient diets which had been
given estrogen had significantly lower serum calcium (P<.01
to .0005) than normally fed rats given estrogen. Rockoff
and Armstrong (1970) and Feik and Storey (1979) also
observed significant decreases in serum calcium with
untreated, calcium- and vitamin D-deficient rats (P<.1 to
Estrogen increased the serum calcium in the intact and
ovariectomized, normally fed rats (P<.01 to .001) on the
present study. Cruess and Hong (1979) found no consistent
change in serum calcium concentration when estrogen was
administered to intact, normally fed female rats over a 12
month period, but observed significant increases at one and
six months (P<.05).
CO tD0 1- 00
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Ovariectomy significantly increased serum calcium in
all dietary groups (P<.05 to .001) except in the vitamin
D-deficient group. Lindgren and Lindholm (1979) did not
observe an increase in serum calcium in normally fed,
ovariectomized rats. Others (Cruess and Hong, 1979) found
ovariectomy to significantly decrease the serum calcium
Results indicate that calcium- and vitamin D-deficient
diets including deficiencies in phosphorus and protein may
not have an effect on calcium concentration in the blood.
The rat may compensate for lack of dietary nutrients by bone
resorption of calcium and phosphorus to maintain normal
blood calcium levels. Evidence for bone resorption was
found in lower bone mineral content and lower ash values in
rats deficient in calcium and vitamin D in the present study
(Tables IV.3 and IV.5).
Estrogen increased serum calcium in normally fed intact
and ovariectomized rats. However, because of conflicting
findings in the several studies mentioned, no firm conclu-
sions can be made on the effect of estrogen on serum calcium
in normal rats.
Ovariectomy effects on serum calcium have also been
noted to vary among studies so that no conclusions can be
made. Unknown factors may be influencing these two treat-
ments, which causes findings to be inconsistent.
Femur lengths. Statistically significant differences
in femur lengths due to diet and to treatments were
observed. Values are found in Table IV.3.
The bone lengths of the normally fed rats were signifi-
cantly longer than those of calcium-deficient and calcium-
and vitamin D-deficient rats in all treatment groups (P<.05
Estrogen administered to intact and ovariectomized rats
reduced bone length in the normally fed group and the cal-
cium- and vitamin D-deficient group (P<.05 to .005).
Ovariectomy increased femur lengths in the normally fed
group (P<.05). Lindgren and Lindholm (1979) found femur
length unaffected by oophorectomy. The sensitive photon-
absorption method (Norland, 1980) used in the present study
may have been responsible for detecting length differences.
Deficient diets used in this study clearly bring about
a decrease in femur length in female rats during the growth
Estrogen tends to cause a shorter bone length, espe-
cially in normally fed rats and rats both calcium- and
vitamin D-deficient. It is thought that estrogen causes the
epiphyses to close prematurely which results in a shorter
bone. Use of estrogen in young human females has this
Confirming evidence of the preceding is observed in
Fig. IV.2. Ovariectomy, or absence of estrogen, caused bone
0 OO N
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cn r- -
(N (n\ m n
C- %0 N
+1 +1 +1
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+1 +1 +1
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r14 J N re
lengths to increase in normal rats. Addition of estrogen
depressed bone length to normal or below normal.
Bone mineral content. Means and standard errors of the
mean for mineral content of the femur as measured by the
photonabsorption method (Norland, 1980) are listed in Table
IV.3. Statistically significant differences in mineral
content of the femur due to dietary deficiencies and to
treatments were found. The calcium-deficient diet and the
vitamin D-deficient diet lowered bone mineral content
When administered estrogen, the normally fed intact
rats had decreased bone mineral content (P<.05) but other
dietary groups showed no effect from estrogen.
Ovariectomy caused a higher bone mineral content in
calcium-deficient rats (P<.01), and a lower bone mineral
content in vitamin D-deficient rats (P.<005); the latter
effect most probably was due to the multiple deficiencies of
When rats were ovariectomized and estrogen was added,
bone mineral content decreased in the normally fed group
(P<.01), and in both the vitamin D and calcium- and vitamin
D-deficient groups (P<.005).
The results indicate that diet does cause decreases in
bone mineral content in the female rat. When the rat is
depleted of certain nutrients, osteopenia results. That the
rat is a suitable model for this premise has been estab-
lished by this and other studies. Extrapolation of the
TOO Wa S .::.. :. .- ::
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I I I
d-IX ^l _
conclusion to the human female is difficult, however, the
corollary does exist.
Sanchez et al. (1981) measured bone mineral mass in
vivo in normally fed, untreated rats with a Norland-Cameron
model 178 bone mineral analyzer. They found highly signifi-
cant positive correlations between femoral mineral mass,
femoral ash weight, and body weights. Similar statistical
correlation tests are planned for the data in the present
Bone density. The bone density measurement is a ratio
of the bone mineral content and the femur width, so that
differences between bone mineral content and bone density
were due to bone width and did not vary with bone mineral
content, since the measurements were done simultaneously.
The means and standard errors of the means for bone
density values are found in Table IV.3.
Dietary deficiencies significantly decreased bone den-
sity in several treated and untreated groups (P<.05 to
.005). Calcium- and vitamin D-deficiency especially
affected density (P<.005) (Fig. IV.4). Only one intact
femoral bone from untreated calcium-deficient rats was
available; therefore it was not used for statistical
Estrogen treatment showed no effect on bone density,
whether given to intact or ovariectomized rats.
0 oo' '' s 2
SOO'"T-" n s ~
too-- w' a
0900 -" V S. :.'-...: .. :.:..:.... .:-:
900 TW"a "S
* I-- I -- I I -
L L` 1
Ovariectomy also did not affect bone density. However,
within the vitamin D deficient group, which also was defi-
cient in protein and phosphorus, bone density was decreased
by estrogen and by ovariectomy (P<.005).
Burkhart and Beresford (1978) castrated 1 1/2-year-old
male rats and reported decreased femoral density 3 to 6
months later. A Joyce-Loebel photodensitometer was used to
measure the density. Wink and Felts (1980) also reported
density decreases in male castrates (P<.01) and femoral oste-
oporosis four months after castration in year-old male rats.
Table IV.4 lists the mean and standard errors of the
torque required to fracture the femurs and of the deforma-
tion undergone by the bones at fracture.
Torque. In all treatments, the calcium-deficient group
and the vitamin D-deficient group required the least torque
(P<.01) for fracture to occur (Fig. IV.5).
Estrogen administered to intact rats significantly
lowered torque in the normally fed group (P<.05) and raised
it in the vitamin D-deficient group (P<.05).
Ovariectomy decreased the torque value significantly in
the calcium- and vitamin D-deficient group (P<.05).
When estrogen was given to ovariectomied rats, torque
values were reduced significantly in the normally fed group
(P<.05) and raised significantly in the calcium-deficient
o r- o r-
0 -- -
+1 +1 +1 +l
o r- C% -
+1 +1 +1
CO OD 0O
+1 +1 +1 +1
m CN o o
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+1 +1 +1
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8OT 'W' 'S
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I I I I I
Deformation. Dietary deficiencies had no effect on
deformation of the rat femurs.
When estrogen was administered to intact rats in each
group, the normally fed group and the calcium- and vitamin
D-deficient group showed significant decreases in deforma-
tion values (P<.005). This effect can be interpreted to
mean a harder bone with less bending ability resulting from
Ovariectomy did not affect deformation of the femurs in
Fig IV.6 represents the femoral deformation values for
the rats in the study.
Values for bone ash as % of dry, fat-free femoral bone
are listed as means SE in Table IV.5. Differences in bone
ash content between rats on deficient diets and those ade-
quately fed were statistically significant in all treatments
(P<.01), with highest values in the adequately fed group.
Decreases in skeletal ash weight of rats on calcium-defi-
cient diets have been previously observed (P<.001) (Rockoff
and Armstrong, 1970).
Estrogen administration did not alter bone ash in any
dietary group, however, ovariectomy decreased % ash content
in the calcium-deficient group. The effect of ovariectomy
and estrogen was an increase in bone ash in vitamin D-defi-
cient and calcium- and vitamin D-deficient groups (P<.05).
S < tO
(N v4 *<
+1 +1 +1
r C- o r-
+1 +1 +1
+1 +1 +1
co N -
U, '. '.
r- (N LAn
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1n 4 in
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*- 0 4.
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to v -* -
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0 D 0
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ir ( D) *n e
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8S "-T-' S S
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i i I I II
t's -T--w- a -S
Other normally fed animals have shown no alteration in
ash content of bones after estrogen administration, but
oophorectomy caused a significant decrease (P<.05) in ash
content which was reversed by estrogen (P<.05) (Cruess and
Hong, 1979). Other significant decreases in ash weight as %
of femur dry weight have also been reported with oophorec-
tomy (P<.01), even though the rats also had a high body
weight gain (P<.05) (Lindgren and Lindholm, 1979). By cas-
trating male rats and maintaining a normal diet, a decrease
in % ash was observed after 3 to 6 months (Burkhart and
Beresford, 1978; Wink and Felts, 1980). It is probable that
a longer period than one month must be observed post ovari-
ectomy in order to detect significant differences in bone
ash in rats on a normal diet.
Bone Marrow Mast Cells.
Means of mast cell counts in the distal metaphyseal and
diaphyseal portions of the femoral bone marrow are presented
in Table IV.6. Means for each group were obtained and
adjusted to an area of 1 mm2. The range of mast cell
counts is also given as counts per 1 mm2 surface area.
Appendix I contains photomicrographs of bone marrow mast
cells (Fig I.1-1.4). Figure IV.7 provides a visual repre-
sentation of bone marrow mast cell populations observed in
* o CO m
- N -- CN
+1 1 +1 1 +1I
LaN 0) Cn ci n
r o q *O 4 c-
o *- (0
+1 +1I 1
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*q, N (N C
CN r- en (a
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E'S 'WB' "S I
6 "S T^'' P8a S l
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C ,J >
Normal diet group. The mean number of mast cells per
mm2 in the bone marrow of these normal, untreated young
rats was 202.920.7. Belanger (1977) recorded mast cell
populations of 12316 per mm2 bone marrow in normally fed
Estrogen increased the count in normally fed, intact
rats (P<.10)(Fig. IV.7). Belanger (1977) found no change in
count when estrogen was used in rats on normal diets.
Ovariectomy produced a decrease in mast cell count
(P<.10). When estrogen was given to ovariectomized rats,
the count continued to remain below that of the untreated
Calcium-deficient group. The calcium-deficient, un-
treated rats in this study showed no change in bone marrow
mast cell count from that of the adequately fed rats. This
finding was somewhat surprising. The well-known study by
Urist and McLean (1957) describes extensive increases in
calcium-deficient rat bone marrow. However, no statistical
evidence was reported. Their rats were weaned at three
weeks to a calcium-deficient diet, whereas the rats on this
study began the deficient diet at two months of age. Great-
est increases in mast cell counts in their rats were re-
ported as occurring after six to 15 weeks. The rats on the
present study were fed a calcium-deficient diet for only six
weeks. Rockoff and Armstrong (1970) also experienced marked
mast cell hyperplasia in calcium-deficient rats. However,
mean number per field and distribution of cells in calcium-
deficient rats did not vary from the normally fed rats in
the study of Rasmussen (1972). It may well be that age of
rat and duration of calcium deficiency play an important
role in mast cell population changes, if alterations do, in
In contrast to normally fed animals, calcium-deficient
rats administered estrogen did not show altered counts.
However, calcium-deficient, estrogen treated rats did show
increased counts when compared to normally fed, estrogen
treated rats (P<.05).
Ovariectomy produced a significantly decreased mast
cell count in this group (P<.01).
Vitamin D-deficient group. A combined lack of protein,
phosphorus, and vitamin D produced no changes in bone marrow
mast cell count in the untreated animals. Estrogen signifi-
cantly increased the marrow count in the intact rats (P<.05)
and ovariectomy also increased the count (P<.10). Ovariec-
tomized rats also had an increased bone marrow mast cell
count when compared to ovariectomized, normally fed rats
(P<.05) indicating an effect of diet.
Contrary to the above finding, Rasmussen (1972) re-
ported marrow mast cells in a vitamin D-deficient group to
be higher than those of normally fed rats. The statistical
significance level was not given.
Calcium- and vitamin D-deficient group. Effect of diet
in untreated rats in this group was not observed. Rasmussen
(1972) reported significantly higher numbers of marrow mast
cells per field in a calcium- and vitamin D-deficient group
when compared with a normally fed group.
In the present study, when rats in this dietary group
were treated with estrogen, the mast cell count decreased
from that of the estrogen treated, normally fed group
(P<.05) and from the untreated rats in the same calcium- and
vitamin D-deficient group (P<.10) indicating an effect of
diet and treatment.
Ovariectomy also caused a significant decrease in mast
cell marrow count (P<.05).
It has been suggested that mast cell increases in bone
marrow are due to secondary hyperparathyroidism caused by
hypocalcemia (Rasmussen, 1972). As was mentioned previously
in the serum calcium section, dietary deficiencies did not
consistently cause hypocalcemia in the present study nor in
other studies. As stated, diet did not affect serum calcium
or bone marrow mast cell numbers in the present study.
The rats given estrogen were made hypocalcemic in all
deficient diet groups, but did not exhibit mast cell
increases. The reverse was found with normally fed rats,
i.e., an increased mast cell count was observed in estrogen
treated rats with normal serum calcium levels. Hormone
treatment may have interfered with the theory mentioned
above, even though estrogen receptors are not known to
occur in bone.
A consistent decrease in marrow mast cell number was
observed in calcium- and vitamin D-depleted, estrogen defi-
cient rats (ovariectomized). With the addition of estrogen,
marrow mast cell numbers were returned to the normal range.
This finding clearly indicates the presence of hormonal
activity in bone.
Vaginal Tissue Mast Cells
The mean number of mast cells per mm2 vaginal tissue
in the different groups of rats is given in Table IV.5 and
is illustrated graphically in Fig. IV.8. Photomicrographs
of vaginal tissue mast cells observed in this study are con-
tained in Appendix I (Fig. I.5-1.8).
Normal diet group. Vaginal mast cells in the untreated
control group numbered 27.22.6 with a range of 14.5 to
39.3. Estrogen given to intact rats in this group did not
alter the count. However, ovariectomy did increase the
count significantly (P<.05). Estrogen given to ovariecto-
mized rats, however, did not return the count to a normal
Estrogen given to intact mice increased vaginal mast
cells substantially (Westin and Odeblad, 1956) again sug-
gesting species difference; however, Johannson and Westin
(1959) report estrogen as suppressing true mast cell numbers
in mouse vaginal tissue.
z t ST a s "': """::
" Z"1 [W W S.
".'. ".' '...':'::.:' -,*' '
I I i i ,
6IT- -:.:. ...,- ..
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."T w "s :".. ".,'; ":'A", .'
I I I
Calcium-deficient group. Calcium deficiency produced a
decrease in the vaginal tissue mast cell count at the P<.10
level, indicating a very weak, almost non-existent effect.
Calcium deficiency also produced a decrease (P<.01) in
estrogen treated rats. Likewise, the deficient diet showed
an effect of decreasing the mast cell count when these rats
were ovariectomized and given estrogen (compared with nor-
mally fed rats of similar treatment (P<.05).
Within the group, when compared with untreated rats,
ovariectomized rats exhibited an increased vaginal tissue
mast cell count (P<.01). With the addition of estrogen, the
cell count was reduced to the normal range of a calcium-
Vitamin D-deficient group. Untreated rats in this
group had higher vaginal tissue mast cell counts than nor-
mally fed rats (P<.05). Treated rats did not display an
effect of diet deficiencies on mast cell numbers.
Estrogen did not alter mast cell counts; but lack of
estrogen caused a reduction in number (P<.10). These rats
were given a diet deficient in protein and phosphorus as
well as vitamin D; results would possibly be affected by the
lack of those nutrients.
Calcium- and vitamin D-deficient group. Diet had no
effect on mast cell count in this group.
Within the group, estrogen given to intact rats pro-
duced no changes in vaginal tissue mast cell counts. Ovari-
ectomy, however, caused an increase in cell numbers (P<.01).
Estrogen added to the ovariectomized rats maintained a count
higher than normal for this group (P<.10). Overall, mast
cell counts in rat vaginal tissue were much less variable
within groups than in bone marrow.
Summarizing the influence of diet and treatment on
vaginal tissue mast cell populations, one observes that
depletion of bone in the rat by dietary calcium deficiency,
as evidenced by densitometric bone analysis and bone ash
levels, has no effect on the vaginal tissue mast cell
number. This is a finding which has not been supported by
the literature since studies of vaginal tissue mast cells
do not exist.
It is important to examine the pattern of hormonal
effects. Estrogen given to intact rats did not affect mast
cells in the vaginal epithelium, an estrogen sensitive
tissue. But when the rats were deprived of estrogen by
ovariectomy, mast cells increased above normal, irrespective
of diet. When estrogen was added, the increased populations
were maintained above normal, but a hormonal effect of
reducing the numbers may be a possibility.
The vitamin D-deficient group does not fit this pat-
tern, most probably because of the effects of additional
dietary deficiencies. As an effect of this diet, however,
mast cells did increase in vaginal tissue. With ovariectomy
a decrease was observed.
Change (reduction) in mast cell numbers in rats made
osteopenic by dietary deficiencies of calcium and vitamin D
as evaluated by bone densitometry and bone ash content was
observed in vaginal tissue as an effect of a calcium-defi-
cient diet at the P<.10 level of significance. Because of
the weak evidence of mast cell reduction occurring, the
first hypothesis is not proven. This finding is not docu-
mented in the literature as studies concerning mast cell
populations in rat vaginal tissue do not exist.
Bone marrow mast cell populations did not vary as a
result of dietary deficiencies. That significant changes in
mast cell populations did not occur in the bone marrow was
surprising because other studies have indicated a substan-
tial increase in bone marrow mast cells with calcium defi-
ciency and osteoporosis (Urist and McLean, 1957; Frame and
Nixon, 1968; Rockoff and Armstrong, 1970; te Velde et al.,
Two possible explanations for lack of change in marrow
mast cell numbers are suggested. The rats in the present
study were at least two months old before being fed defi-
cient diets. It was desirable to have rats with estrous
cycles in order to observe the effect of removing the circu-
lating estrogen. The rats in the aforementioned studies
In addition, the duration of dietary deficiencies may
have been too short to overcome the effect of age, even
though osteopenia was indicated. The rats in the present
study were kept on deficient diets for a period of five
weeks which approximated .05% of their life span. With
dietary deficiencies extended over a longer period of time,
significant alterations in marrow mast cell numbers may have
The effects of exogenous estrogen were seen to reduce
bone marrow mast cell numbers in rats both calcium and vita-
min D deficient, which supports the second hypothesis of
this study, in part. An opposite effect of estrogen admini-
stration was seen in normally fed rats: the bone marrow
increased significantly in mast cells. This inconsistency
is not understood. However, these results strongly suggest
that bone is an estrogen sensitive tissue, even though
estrogen receptors have not been found in bone.
Bilateral ovariectomy significantly affects both
vaginal tissue and bone marrow with respect to mast cell
populations. Removal of estrogen production in the rat
reduces bone marrow mast cells in rats normally fed and also
in those deficient in calcium and vitamin D. An opposite
effect of estrogen removal is seen in the vaginal tissue.
Both observations support the hypothesis that ovariectomy
alters mast cell populations in bone marrow and vaginal
tissue of calcium- and vitamin D-deficient rats.
In absolute terms, ovariectomized rats resupplied with
estrogen demonstrated increases in vaginal tissue mast cells
and a decrease in marrow mast cells. These are changes
similar to those stated as an effect of ovariectomy alone.
However, upon observing Figs. IV.7 and IV.8, one can detect
that replacement of estrogen may be causing a reversal of
mast cell population change due to ovariectomy. Statistical
analyses between ovariectomized groups with and without
estrogen will be performed in the near future to determine
whether added estrogen changes mast cell populations in
It is important to consider again the possibility that
both age of the rat when begun on a deficient diet and
duration of feeding a deficient diet may greatly influence
mast cell populations in the two tissues examined in this
Ovariectomy, the procedure which removes the endogenous
gonadal hormone supply has known consequences relating to
bone loss in the human female. Whether mast cell changes
similar to those in the rat occur in the human is not known.
Considering the results of the present study, a quantitative
investigation of mast cell populations in vaginal tissue of
ovariectomized women may provide information useful in the
study of osteopenia and bone resorption.
It has also become apparent that qualitative investiga-
tion of the mast cell populations, including histologic bone
evaluations, needs to be considered from data obtained in
the present study. The need for further examination of
correlations between bone densitometric measurements, bone
ash, serum calcium, biomechanical tests and the mast cell
populations is also immediate. Statistical analysis is
scheduled for these parameters.
SIGMA XI GRANT-IN-AID OF RESEARCH
rS, 1586 ,I
THE SCIENTIFIC RESEARCH SOCIETY OF NORTH AMERICA
For the Encouragement of Scientific Research
Grants-in-Aid of Research
Grants-in-Aid of Research are supported by voluntary contributions to 'he Researi
Program from the membership of SIGMA XI. Awards are normally made in amounts rngin.
from 5100 (or less) to a maximum of S 1.000.
Research awards may be made to support scientific investigation in any field. Each
award is made payable to the individual recipient. No part of a irant may be used for
the payment of any indirect costs to the recipient's institution-al, of the funds must be
expended directly in support of the proposed investigation. All equipment purchased shall
be the property of the institution. Grants normally are rot made for expenses of publication.
salary or tuition, travel to meetings. or usual and routine institutional obligations. Priority is
usually given to applicants who are in an early stage of their scientific careers.
The Committee on Awards meets on or about the first of March. June. and December
of each year and applicants are notified of the Committee's Jecisions within si.\ weeks.
In order to be considered, applications must be received by February I for the .'la-ch
meeting. May 1 for the June meetiiin, and November 1 for the December eetring at Sigma
Xi National Headquarters. 345 Whitney Avenue. New Haven. Connec::cut 06511. Attention:
Committee on Awards.
Franklyr. S. VL.n licuHoe. C:;
APPLICANT: Please filU i0n these thee item only.
APPLICANT: Tesar Rogene E.
APPLICANT: ............................... .......
LAST NAME FIRST NAME MIDDLE NAME
FIELD: ..Nutrition and Osteoporosis
TITLE OF STUDY: .The RelatiO.n h~Between
Epithelial and Connective Tissue Mast Cell
Populations in the Female Rat
....... .................... ...................
FOR COMMITTEE USE ONLY
Full ($ )
Partial ($ )
Conditional ($ )
Date of mailing award:
Date of receipt of
COMMENTS AND RECOMMENDATIONS
DATE OF MEETING
Please type or print all information
APPLICATION FOR GRANT-IN-AID OF RESEARCH
ame Tesar Rogene E.
LAST NAME FIRST NAME MIDDLE NAME
Addess 6916 N.W. 20th Place, Gainesville, Florida 32605
Peent iion and int on Graduate Assistant, Department of
Obstetrics and Gynecology, University of Florida
Degrees, it un coneing them, daetu B.Sc. (Home Economics).
Kansas State University, 1962; B.Sc. (Food Science) University of
Florida, 1977; M.Ag. (Human Nutrition) University of Florida, 1979;
Ph.D. (in progress) University of Florida
MembeAzhip in SIGMA XI ..........onmember...................................
Please attach a. ti.t o6 titezS o6 articlu published dwZing the tast
jive yeau, with names o6 peAiodicaZc and dates: Lis;t of tit.es
Tt.e. o ppod on: The Relationship Between Epithelial
Title of proposed investigation: .....................................
and Connective Tissue Mast Cell Populations in the Female Rat
Proposed investigation, desucibed in non-technica language:
Previous studies suggest a relationship between bone marrow mast cell
(MC) activity and local bone loss. There is also evidence that changes
in skin MC activity may be indicative of bone loss. The proposed study
is designed: 1) to determine whether a correlation exists in female
rats between MC activity in bone marrow and vaginal tissue and 2) to
examine the effects of calcium- and vitamin D-deficient diets, exogenous
estrogens and removal of the ovaries on this relationship.
The following hypotheses will be tested: 1) dietary calcium and
vitamin D deficiencies produce an increase in bone marrow MCs and a
decrease in vaginal epithelial MCs in female rats; 2) administration
of exogenous estrogens alters the bone and vaginal tissue MC activity
in the osteoporotic female rat; and 3) removal of the ovaries (removal
of primary source of endogenous estrogens) produces changes in bone and
vaginal tissue MC activity in the female rat.
Should the correlation be shown to exist, a similar correlation in
the pre-osteoporotic and osteoporotic human female could be suggested.
At present there is no universally applicable, non-invasive method for
evaluation of bone resorption and formation. Evaluation of MC activity
in vaginal tissue may prove useful as a non-invasive means of detecting
increased bone resorption (indicative of osteoporosis associated with
endogenous or exogenous excess of corticosteroids, hyperthyroidism,
hyperparathyroidism and osteomalacia). In a similar manner, the method
could be used to assess treatment efficacy.
Locations wheAe problem wii be studied: Department of Animal
Resources, J. Hillis Miller Health Center, University of Florida;
Department of Animal Science, College of Agriculture, University of
Nature o6 assistance desired and amount o6 grant needed, itemized:
Purchase of 128 Sprague-Dawley female rats $544.00
......... o ......................
Feed and bedding for above rats for 5 wk. period 394.25
Institutiona support 6or study o problem: Remaining necessary support
to carry out entire research project: anesthesia, estrogen, stains
and chemicals, microscope slides and use of all equipment.
Previous gtrant-s received fom SIGMA XI and other: .none..............
OtheA applications pending: .none
Attach a list giving name od each assistant oa co-wodkeA, i6 any,
engaged in the. investigation:
List attached X Number o6 co-workker 7
Names and addrAesse o0 at least two specialists* in thki6 ield who
will be ASKED BY THE APPLICANT to send toSigma Xi National
Headquattea statements indicating (7) the importance o6 the proposed
investigation and (2) the qualijications o6 the investigator.
Morris Notelovitz, M.D., Ph.D., Dept. of Obstetrics and Gynecology,
Box J-294 JHMHC, College of Medicine, U of F, Gainesville, FL 32610
J.P. Feaster, Ph.D., Dept. of Animal Science, 20 Nutrition Lab, IFAS
College of Agriculture, U of F, Gainesville, FL 32610
*I1 applicant is a degree candidate, one must be that facuLty or
research sta6j membeA su.peAvising hi research.
Applicant's Signatuwe .--
Date October 28, 1980
Title: The Relationship Between Epithelial and Connective Tissue Mast
Cell Populations in the Female Rat
Investigator: Rogene Tesar
Assistants and Co-workers Engaged in Investigation:
Morris Notelovitz, M.D., Ph.D.
J.P. Feaster, Ph.D.
A.F. Moreland, D.V.M.
Laboratory Technician of Dr. Moreland
Lynda McKenzie, R.N.
Advisor Endocrine Functions
The University of Florida College of Medicine
Department of Obstetrics and Gynecology
iIdf-/ ?November 26, 1980
BOX J-294. JHMHC
GAINESVILLE. FLORIDA 32610
Committee on Awards
345 Whitney Avenue
New Haven, CT 06511
RE: Application by Rogene Tesar for grant to investigate
the relationship between epithelial and connective
tissue mast cell population in the female rat.
The above application relates to the potential role
that mast cell activity may have in the development of
osteoporosis, a condition that affects some 25% of meno-
pausal women and about 75% of women who have undergone a
surgical menopause. Osteoporosis is a significant disease
since it is not only associated with pathologic fractures
and potential invalidism, but can frequently result in the
premature death of elderly women. One of the problems in
the clinical management of this condition is the difficulty
of its diagnosis and measurement of its response to treat-
ment. Mrs. Tesar's research could provide the answer to
.I have been acquainted with Mrs. Tesar for approxi-
mately two years and am currently an advisor to her for
her Ph.D. requirements. She is a highly competent
research worker, and I am confident that she will be able
to accomplish all the goals of her research project. I
have no hesitation in supporting her request.
Warm kind regards. ince
\ urs sincrely ,*
Morris Note ovitz, M.D.
(RAND), Ph.D., F.R.C.O.G.,
Director of the Center for
EQUAL. EMPLOYMENT OPPORTUNITIES FFIRMATIVE ACTION EMPLOYER
SIGMA X., THE SCIENTIFIC RESEARCH SOCIETY
345 WHITNEY AVENUE
OFFICE OF THE NEW HAVEN. CONNECTICUT 06511
COMMITTEE ON AWARDS 20 April 1981 (203 624.93
Ms. Rogene E. Tesar
6916 N.W. 20th Place
Gainesville, FL 32605
Dear Ms. Tesar:
I am happy to inform you that at a recent meeting of the Committee on Awards a
Grant-in-Aid of Research of $250.00 was given you to further the work in your
application: The Relationship Between Epithelial and Connective Tissue Mast
Cell Populations in the Female Rat. Please complete the enclosed acceptance
form so that we may write and forward your check.
This award is one of eight made possible this year from the income of a
specific gift to the Research Fund by Mrs. Daisy Yen Wu in memory of her
husband, Dr. Hsien Wu.
It is understood that in accepting this award you will at the close of the
academic year (1981-82) submit a report of the work done to the Committee on
Awards, Sigma Xi, The Scientific Research Society, 345 Whitney Avenue, New
Haven, Conn. 06511. This should be a short one or two-page summary of the work
accomplished with your Sigma Xi grant.
It is further understood that all published reports of your work will contain a
statement that the research was aided by a Grant-in-Aid of Research from Sigma
Xi, The Scientific Research Society. Also, any equipment purchased with the
funds which have been made available is to be considered the property of the
institution where the research is being carried on. It is also to be under-
stood that no indirect costs are to be paid to your institution from this
It is a great pleasure to express the Committee's hope for your continued
success in scientific research.
'7. i. V'1 1_^A*-
Franklyn B. Van Houten
Date April 27, 1981
Committee on Awards
Sigma Xi, The Scientific Research Society
345 Whitney Avenue
New Haven, CT 06511
I have received your letter stating that a grant has been
awarded to me by the Committee on Awards.
I. (X ) I accept the award in the amount of $250.
() I cannot accept the award because
)I shall let you know by whether
or not I can accept it.
II. The name of the President or Chancellor or Chief
Executive Officer of my institution is:
President: Robert Q. Marston
III. The name of the Head or Chairman of my Department
Animal Science: H.D. Wallace, Chairman
Center for Climacteric Studies:
M. Notelovitz, M.D., Ph.D.
IV. Will you please have Sigma Xi, the Scientific
Research Society, forward to me a check made
payable for the amount of the award. I under-
stand that it will be sent to my institutional
address only, and I have made arrangements for
its being forwarded if necessary.
NAME (Please Print): Rogene Tesar, R.D., M.Ag.
ADDRESS ONLY: Center for Climacteric Studies
Univ. of Fla., 901 N.W. 20 P1.
Suite B-l Gainesville, FL 32601
SIGMA XI, THE SCIENTIFIC RESEARCH SOCIETY
345 WHITNEY AVENUE
OFFICE OF THE NEW HAVEN, CONNECTI(UTokW I
EXECUTIVE DIRECTOR 10 June 1981 i20Ml 624-~W
Ms. Rogene E. Tesar
University of Florida
Center for Climacteric Studies
901 N.W. 20th Place
Gainesville, FL 32601
Dear Ms. Tesar:
Enclosed please find our check in the amount of $250.00,
which represents the Grant-in-Aid of Research award made
to you by the Sigma Xi Committee on Grants-in-Aid at their
This award is one of eight made possible this year from
the income of a specific gift to the Research Fund by Mrs.
Daisy Yen Wu in memory of her husband, Dr. Hsien Wu.
Upon completion of your research a report of your findings
should be forwarded to the Committee on Grants-in-Aid, 345
Whitney Avenue, New Haven, CT 06511.
May I take this opportunity to wish you every success with
Thomas T. Holme
cc: Office of the President
LABORATORY ANIMAL USE
All University Cornitcee on the Care and Use
of Laboratory Animals
In order to comply with DHEW policy and all federal, state and local rules
and regulations concerning the care, treatment and use of laboratory animals,
tne following information is necessary for grants to be processed by the
Division of Sponsored Research. Instructional programs and research projects
supported internally using laboratory animals must also complete this question-
naire and return to the Comrnittee completing the appropriate parts.
1. Principal investigator: Rogene E. Tesar
Department Obstetrics and Gynecology
College College of Medicine
2. Proposal submitted to:
Starting date: December 15, 1980
Telephone 392-3184 Date Nov. 17, 1980
345 Whitney Ave., New Haven,
Aaoress Conn. 06511
; conclusion: August 30, 1981
4. Proposal title:
The Relationships Between Epithelial and Connective Tissue Mast Cell
Populations in the Female Rat
5. Animal species: Rat
Age 3 weeks and 2 months
Size 50 gram and 120 gram
6. Is the animal-model appropriate?
Explain Yes. The female rat has been used in many experiments involving
the mast cell. The metabolism of the rat closely relates to that of the human
and will produce results due to the treatment given which can be implicated
as occurring in the human.
7. Abstract of animal use:
(Use continuation sheet if needed)
137 rats will be utilized to observe differences in population and activity of the
mast cell in bone and vaginal tissue. Of these rats, 12 will be utilized for a
preliminary trial to define whether mature rats or weanlings are to b used for
treatment (a) castration. Treatments are as follows: a) castration (7 rats)
at beginning of study b) estroeen (LV.) (67 rats) 3 X week for 5 weeks. c) normal
diet (32 rats). d) calcium deficient diet (32 rats) e) vitamin D deficient diet
(32 rats), f) calcium and vitamin D deficient diet t32 rats), (continued)
8. Care and location of animals:
The rats will be maintained at the Animal Resources Department, JHMHC, University
of Florida in individual cages with daily care and feeding.
9. Is the study designed to avoid inflicting needless pain and/or suffering
including the appropriate use of tranquilizers, analgesics and/or
anesthetics? If not, explain: Yes;
Anesthesia will be used for surgery purposes.
10. Method(s) of euthanasia to be used:
At the end of a maximum 5 week feeding
Pri6 rpal Investigator
Rogene Tesar, M.A., R.D.
period, the rats will be killed by
Eduard G. Friedrich, Jr., M.D.
All University Committee on the Care and Use
of Laboratory Animals
7. h) photon absorptiometry of the femur and tibia during the 5 week diet period.
Castration (removal of ovaries) will be performed under anesthetization. At
the termination of the 5 week diet period the rats will be killed by decapitation.
-Bone and vaginal tissue specimens will be obtained for histological purposes.
University of Florida
All-University Committee for
The Care and Use of
1. Acknowledge receipt of your form for the care and use
of laboratory animals entitled:
"The Relationship Between Epithelial and Connective
Tissue Mast Cell Populations in the Female Rat"
2. Review Results
3. For questions, please call: Dr. Halliwell at (904)
EXPERIMENTAL ANIMAL BODY WEIGHTS
ID OW 1W 2W 3W 4W 5W
Weights expressed in grams
ID OW 1W 2W 3W 4W 5W 6W
1 161 179 176 179 177 169 171 176 176 175 181
2 187 204 197 197 196 195 196 202 196 195 205
3 181 192 189 190 197 193 198 196 198 197 195
4 183 202 201 197 196 196 193 199 199 208 209
5 175 175 176 187 190 188 196 200 201 202 200
6 175 187 185 185 184 181 188 188 195 195 197
7 177 194 192 182 173 185 187 189 188 186 190
8 183 198 205 210 210 210 215 213 214 212 215
la 211 211 211 215 216 222
2a 197 201 213 212 213 211
3a 182 187 187 194 200 194
4a 188 196 203 203 209 211
9 181 191 210 225 239 247 255 261 275 274 290
10 167 174 190 205 220 226 236 244 250 259 265
11 190 204 222 248 262 275 286 295 301 305 310
12 204 210 233 264 282 295 310 320 328 336 342
13 170 178 203 221 238 242 253 259 268 275 285
14 168 184 200 228 247 257 270 276 279 290 294
15 194 212 226 241 255 260 262 263 258 265 265
16 185 199 217 242 258 274 291 299 303 310 317
5a 191 192 251 275 292 298
6a 177 177 214 253 269 278
7a 186 196 243 283 295 306
8a 183 191 239 275 291 299
17 184 207 192 195 194 200 203 213 208 215 218
18 196 195 201 205 205 209 215 224 223 233 228
19 187 201 204 215 219 229 233 233 234 242 235
20 202 217 213 220 229 236 233 239 239 234 244
22 167 186 184 192 190 191 191 201 203 207 207
23 183 198 197 206 207 215 230 215 215 214 204
24 200 216 217 225 219 230 216 235 232 234 220
25 181 189 189 199 199 202 205 210 209 210 210
26 184 198 210 220 225 228 239 246 246 245 249
27 187 195 203 214 219 224 230 233 236 242 241
28 181 188 194 201 206 210 212 216 219 222 223
29 173 190 203 208 216 217 223 228 228 235 235
30 177 198 211 223 225 228 234 239 242 245 247
31 185 204 210 219 226 231 242 247 239 242 245
32 191 204 213 221 228 232 241 248 242 245 249
Weights expressed in grams
ID Ow 1W2W 3W 4W
Weights expressed in grams
-Ca, -D DIET
ID i W 1W 2W 3W 4W 5W
COMPOSITION OF EXPERIMENTAL DIETS
AIN-76 Semipurified Diet (Ca:P
Corn Oil 5.0%
AIN Mineral mix 3.5%
Calcium Phosphate, Dibasic (CaHPO4)
Sodium Chloride (NaCl)
Potassium Citrate, Monohydrate
Potassium Sulfate (K2SO4)
Magnesium Oxide (MgO)
Manganous Carbonate (43-48% Mn)
Ferric Citrate (16-17% Fe)
Zinc Carbonate (70% ZnO)
Cupric Carbonate (53-55% Cu)
Potassium lodate (KIO3)
Sodium Selenite (Na2SeO3 5H20)
Chromium Postassium Sulfate (CrK
Sucrose, finely powdered
All Vitamin mix 1.0%
(Vit. E), Pre-mix
Cholecalciferol (Vit. D3)
Menaquinone (Vit. K)
Sucrose, finely powdered
Choline bitrate 0.2%
per kg Mixture
200 myl Acetate
*Obtained from Nutritional Biochemicals, Cleveland, OH
Calcium Deficient Diet (Ca:P .07:1)*
Casein (purified) 24.0%
Corn Oil 5.0%
Calcium Free Salt Mixture 3.0%
Plus Special ICN Vitamin Diet Fortification
Vitamin A Concentrate (200,000 units/gm)
Vitamin D Concentrate (400,000 units/gm)
Rachitogenic U.S.P. No. 2 Diet
Ground Whole Yellow Maize
Custom Vitamin D and Calcium Deficient Diet
Based on the Calcium-Deficient Diet
with omission of Vitamin D Concentrate
* Obtained from Nutritional Biochemicals, Cleveland, OH
** Formulated and obtained from Nutritional Biochemicals,
SERUM CALCIUM ANALYSIS
Sample Collection and Procedurel
Blood was obtained at decapitation of the laboratory
rats by exsanguanation. The blood was collected in tubes,
allowed to clot and centrifuged. Serum was drawn off using
Pasteur glass pipettes, transferred to clean tubes, labeled,
and frozen for future determination.
A protein-free filtrate was required for analysis of
calcium. After thawing the serum samples at room temperature
for one half-hour, precipitation of serum protein was accom-
plished as follows:
(a) 9 ml 10% (w/v) trichloracetic acid (TCA) were deliv-
ered into labeled test tubes.
(b) 1 ml serum from each well-mixed sample was pipetted
into the TCA.
(c) the solutions were mixed on a vortex mixer, allowed
to stand for 10 minutes, and centrifuged 10 minutes
at 2,500 rpm. This filtrate represented a 1410
dilution of the serum samples.
(d) 1 ml of the supernatant was diluted to 5 ml with
1% lanthanum. The dilution factor was 10 x 5 = 50.
1Fick et al., 1979
This procedure provided the appropriate serum calcium
concentration for the reading of absorbance by the Perkin-
Elmer 306 atomic absorption spectrophotometer (AAS), which
has a linear working range of 7 ppm for calcium. Confirma-
tion of the above is shown by the following calculation:
(ppm calcium expected
calcium concentration = for original sample)ample)
calcium concentration =(100 ppm) x (1 ml)
calcium concentration = 2 ppm.
Calcium standards of 0, 2, 3, 4, 5, and 7 vg/ml were
prepared in 100 ml volumetric flasks. The 1,000 ppm stock
standard calcium solution was first diluted to 100 ppm. Each
standard was made to contain 18 ml of 10% TCA to match the
final dilution of serum and 16 ml of 5% lanthanum. Table E.1
lists the concentration and absorbance of the standards, as
read by AAS.
SERUM CALCIUM STANDARDS
Standard Readout Calculated
pg/ml Absorbance (A) Slope (a)
0 .000 .000
2 .074 .037
3 .120 .040
4 .164 .041
5 .184 .037
7 .268 .038
Calcium in the sample solutions was then measured for
absorbance by AAS and concentration was calculated as mg/100
ml serum as follows:
sample ppm = absorbancee) x (dilution factors)
(slope) x (sample weight)
This equation is derived from Beer's Law, which states that
A = abc
A = absorbance (optical density)
a = absorptivity or slope of the standard
b = length of the light path (always constant)
c = concentration
The absorbance reading was obtained from the machine.
The slope was determined as an average of the slopes of
the standards (a = A/c from Beer's Law). The slope used
in calculations for samples was .0386, obtained from
Tables E.2 through E.5 contain serum calcium concen-
tration values of the laboratory rats.
SERUM CALCIUM ANALYSIS DATA
Rat Diet Normal
Total Dilution 50
Technician R. Tesar
ID Absorbance I ppm mg/100 ml
SERUM CALCIUM ANALYSIS DATA
Rat Diet -Ca
Total Dilution 50
ID Absorbance jppm mg/100 ml
SERUM CALCIUM ANALYSIS DATA
Rat Diet -D
Total Dilution 50
ID Absorbance I ppm mg/100 ml
SERUM CALCIUM ANALYSIS DATA
Rat Diet -Ca, -D
Total Dilution 50
ID Absorbance I ppm mg/100 ml
DENSITOMETRIC BONE ANALYSIS
Instrumentation and Procedure
A Norland Digital Bone Densitometer Model 278 (Norland
Instruments, Ft. Atkinson, WI) was used to measure bone
mineral content, bone width, and bone length in the excised,
cleaned rat femur by direct photon absorptiometry. This
technique measures the attenuation of a beam of gamma radi-
ation by calcified tissue and is based on the concept that
the mass of bone mineral present is directly proportional
to the attenuation by bone (Sanchez et al., 1980).
The densitometer consisted of a scanner module and a
computer module (Fig. F.1). The scanner transported a
highly collimated beam of monoenergetic gamma rays from a
radioactive sealed source of I25 across the bone being
measured. A 1/16" diameter detector collimator and a thresh-
old setting of 85% were used to enhance accuracy. The compu-
ter module calculated the bone mineral content (BMC) and
bone width (BW) values from the resulting absorption curve.
These values and the bone profiles were displayed on the
computer module screen. See Fig. F.2.
BMC and BW measurements were made at six distinct scan
sites of the femoral bone, beginning at the edge of the
lesser trochanter proximall end) and progressing to the
widening of the distal end. Scans were made perpendicular
to the bone axis.
The quantity measurement of BMC is a linear mass den-
sity of g/cm length of bone, an average linear density over
the approximately 4 mm wide scan path. In terms of another
explanation, BMC is the grams of mineral which would be
obtained if a 1 cm thick crosswise slice were cut out of
the bone and this slice were heated in a crucible to burn
away all non-mineral material (Norland Corp., 1980).
BW represents the distance in cm from one longitudinal
edge of the bone to the other.
The value of BMC/BW, calculated by the computer module,
provides a measurement of linear bone density in cm The
entire depth of the bone, i.e., the distance from top sur-
face to bottom surface of a bone lying flat, is included in
this measurement. The BMC, BW, and BMC/BW values are re-
corded in Tables F.1 through F.4.
Bone lengths were measured by placing each of the bones
in a longitudinal position along the scan path of the scanner
module. Data on these lengths are recorded in Table F.5.
Fig. F.1 The Norland Digital Bone Densitometer,
Model 278, with computer module at left. The densitometer
scanner module transports a highly collimated beam of
monenergetic gamma rays from a radioactive sealed source
(125I) across the bone to be measured. The computer
module calculates the values from the resulting absorption
curve and displays the results on the screen. A calibra-
tion standard is shown on the scanner deck.
Fig. F.2 Printout display of rat femur profile.
Measurement results are BMC (bone mineral content-mass)
expressed in gr ms per cm, BW (bone width) in cm, BMC/BW
in grams per cm .
._ .. m m-- Elm