EPITHELIAL AND BONE TISSUE MAST CELL
POPULATIONS IN THE FEMALE RAT AS
INFLUENCED BY CALCIUM AND VITAMIN D
DEFICIENCIES, OVARIECTOMY, AND ESTROGEN

BY
ROGENE TESAR

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
1982

Copyright 1982

by

Rogene Tesar

to my mother
who would have thoroughly enjoyed
observing this entire experience

ACKNOWLEDGEMENTS

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.e. 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.

IV

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
also appreciated.

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

PAGE

ACKNOWLEDGEMENTS iv

LIST OF TABLES ix

LIST OF FIGURES xi

ABSTRACT xii

CHAPTER

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 3 5

Deformation 38

Bone Ash 38

Table of Contents continued

CHAPTER PAGE

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

APPENDICES

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 16 2

BIOGRAPHICAL SKETCH 168

Vlll

TABLE

III.1

IV. 1

IV. 2

IV. 3

IV. 4

IV. 6

IV. 6

C.I

C.2

C.3

C.4

E.I

E.2

E.3

E.4

E.5

F.I

F.2

F.3

F.4

F.5

LIST OF TABLES

PAGE

Study Design 15

Body Weight Gain as Affected by

Diet and Treatment 23

Effect of Diet and Treatment on

Serum Caclium 26

Effect of Diet and Treatment on Densitometric

Measurements of Rat Femurs 29

Effect of Diet and Treatment on Biomechanical

Properties of Rat Femurs 36

Effect of Diet and Treatment on Bone Ash 39

Mast Cell Populations as affected by Diet and

Treatment in the Female Rat 42

Experimental Body Weights: Normal Diet 70

Experimental Body Weights: -Ca Diet 71

Experimental Body Weights: -D Diet 72

Experimental Body Weights: -Ca, -D Diet 73

Serum Calcium Standards 77

Serum Calcium Analysis Data: Normal Diet 79

Serum Calcium Analysis Data: -Ca Diet 80

Serum Calcium Analysis Data: -D Diet 81

Serum Calcium Analysis Data: -Ca, -D Diet 82

Densitometric Bone Analysis: Normal Diet 87

Densitometric Bone Analysis: -Ca Diet 89

Densitometric Bone Analysis: -D Diet 91

Densitometric Bone Analysis: -Ca, -D Diet 93

Densitometric Bone Analysis: Bone Length 95

List of Tables continued

TABLE PAGE

G.I 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.I 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

1.1 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

FIGURE PAGE

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.I The Norland Digital Bone Densitometer 86

F.2 Printout Display of Rat Femur Profile 86

G.I The Rapid Loading Torsional Testing Machine 98

G.2 Representative Torque-Deflection Curves 98

1.1 Photomicrograph of Mast Cells — Bone Marrow:

Normal Diet 1 47

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 I53

1.5 Photomicrograph of Mast Cells — Vaginal Tissue:

Normal Diet I55

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

xi

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

By

Rogene Tesar

May 1982

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 Mg/.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
group.

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.

xxii

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.

CHAPTER I
INTRODUCTION

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
efficacy.

Therefore, two hypotheses were tested in this
investigation:

(a) Dietary calcium- and vitamin D-def iciencies 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
study were:

(a) weight changes of the laboratory animals during the
experimental period

(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.

CHAPTER II
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 'masted' (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
granules.

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 ym in diameter, each with a round or oval nucleus.
Cytoplasmic granules are approximately .7 jam 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 ym, granule diameter as .78 ym, 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
(Schubert, 1968).

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 Cell, 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
sulfated mucopolysaccharides.

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 increased resorption and remodeling. Walker (1970) reported
an eight-fold increase of mast niimbers 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
finding.

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 calciijm, 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 (19 70) 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

10

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 mm^ which rose to 1,900
cell per mm^ until 35 days after fracture. These mast
cells were mostly degranulated. Normal rats exhibited
strongly granulated mast cells, 2,000 to 4,000 mm^ 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

11

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. iMast 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
subjects.

Discrepancies in results among studies may be due, in
part, to differences in estrogen dosage. Iversen (1962)

12

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, 19 56; 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
cell loss.

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.

13

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.

CHAPTER III
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,
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 24°C 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
weights.

The animals were divided into the following dietary
groups :

Outbred laboratory Sprague-Dawley rats were obtained from
Harlan- Sprague-Dawley, Madison, WI 53 711.

14

15

I. Normal (control) rats (semipurif led 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
diet) .
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
malfunctioning eye.

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
surgery.

16

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 yg/. 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.

TABLE III.1

STUDY DESIGN

Diet Group

Normal
(N)

-Ca -D
(N) (N)

-Ca, -D
(N)

8

12

8

8

8

12

8

8

8

8

8

8

8

8

8

8

Treatment
Ovariectomized + Estrogen
Ovariectomized
Estrogen
Normal (no treatment)

Analytical Methods
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 Pick et al . (1979). Data on serum
calcium values are found in Appendix E.

17

Inmiediately 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%
aqueous formalin.

Bone densitometric measurements^ 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
testing 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
analysis .

^Norland Digital Bone Densitometer, Model 278, Norland Cor-
poration, Ft. Atkinson, WI 53538.

2

Rapid Load Torsional Testing Machine, Biomechanics Labora-
tory, Department of Mechanical Engineering, University of
Florida, Gainesville, FL 32611.

Histology

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 5°F with changes of solution every 2 to 3 days (Belanger
et al. , 1965) .

The demineralized femurs and vaginal tissues were dehy-
drated in 8 0% 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 ym
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
staining procedure:

(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)

19

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

2
diameter, representing a surface area of .165 mm and a

2
total surface area of 4.1 mm for each tissue per rat.

2

Counts were adjusted to 1 mm surface area. Appendix I

contains mast cell quantitation data.

Data Analysis
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/Vn ) ,
and indicate level of significance.

Labophot-Laboratory & Clinical Microscope, Nikon Instrument
Division, 623 Stewart Ave, Garden City, NY 11530.

20

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.

CHAPTER IV
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,

21

22

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 Weights

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 occured 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,
1980).

23

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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
less weight.

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.

Serum Calcium
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
.01).

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).

26

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27

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
(P<.05).

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.

28

Bone Densitometry

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
to .005).

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
period.

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
effect.

Confirming evidence of the preceding is observed in
Fig. IV. 2. Ovariectomy, or absence of estrogen, caused bone

29

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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
(P<.005) .

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
the diet.

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

32

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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
study.

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 signficantly 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
comparisons.

Estrogen treatment showed no effect on bone density,
whether given to intact or ovariectomized rats.

34

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35

Ovariectomy also did not affect hone 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.

Biomechanical Tests

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
group (P<.05).

36

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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
estrogen administration.

Ovariectomy did not affect deformation of the femurs in
any group.

Fig IV. 6 represents the femoral deformation values for
the rats in the study.

Bone Ash

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).

39

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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.

Mast Cells
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 mm^. The range of mast cell
counts is also given as counts per 1 mm^ 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
this study.

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44

Normal diet group. The mean number of mast cells per

2
mm-' m the bone marrow of these normal, untreated young

rats was 202.9±20.7. Belanger (1977) recorded mast cell
populations of 123±16 per mm^ bone marrow in normally fed
rats.

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
rat (P<.10).

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,

45

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
fact, occur.

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

46

(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.

47

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 mm^ 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 27.2±2.6 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
range.

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.

48

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49

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-
deficient animal.

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).

50

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.

CHAPTER V
CONCLUSIONS

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 <. 1 0 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.,
1978) .

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

51

52

cycles in order to observe the effect of removing the circu-
lating estrogen. The rats in the aforementioned studies
were weanlings.

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
been seen.

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

53

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 betveen ovariectomized groups with and without
estrogen will be performed in the near future to determine
whether added estrogen changes mast cell populations in
ovariectomized rats.

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
study.

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.

54

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.

APPENDIX A
SIGMA XI GRANT-IN-AID OF RESEARCH

<> 1386 1/.)

SIGMA XI

THE SCIENTIFIC RESEARCH SOCIETY OF NO?iTH AMERICA

For the Encouraoement of Scientific Research

Granfs-In-AId of Researcn

Grants-in-Aid of Research are supported by voluntur/ coniribacioas :o :he Resdjr.n
Program from the membership of SIGMA XI. Awards ure normally naJ< m amounts nin^ir.i:
from S 100 (or less) to a maximum of SI. 000.

Research awards may be made to support scientific investiiiation in any Held, tach
award is made payable to the individuaJ recipient. .No part of a irant may be used !or
the payment of any indirect costs to the recipient's institution- ai' of the funds must be
e.xpended directly in support of the proposed investigation. All equipment purchased shall
be the property of the institution. Grants normally are not made for expenses of publicatioa.
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 scientitic careers.

The Committee on Awards meets on or about the firs: of March. June, a.nd December
of each year and applicants are notified of the Committee's decisions within six weei<s.
In order to be considered, applications must be received by February- 1 tor the March
meeting. .\iay 1 for the June meeting, and November 1 for the December meeting at Sigma
Xi National Headquarters. 345 Whitney Avenue. New Haven. Connecticut 065 1 1. Attention:
Committee on Awards.

Franklyr. 3. '-'.i.-. Hcuten. CV.-;.

56

57

APPLICANT: PloMz iUl. hi tku^ thn.ze, Itmi, only.

FOR COMMITTEE USE ONLY

APPLICANT: .. ^^^^^ Rogene E. Date Received

LAST NAME FIRST NAME MIDDLE NAME Amount Requested:

Nutrition and Osteoporosis

Action:
TITLE OF STUDY: .T^? .^f?-?*--'-?^?^^? Between Not Granted

Epithelial _ and _ConnectiYe_ Tissue Mast Cell Granted:

Populations in the Female Rat Pull ($ )

Partial ($ )

Conditional ($ )

Date of mailing award;

Date of receipt of
final report :

COMMENTS AND RECOMMENDATIONS

COMMITTEE ACTION

DATE OF MEETING

ADDITIONAL REMARKS

58

Please type or print all information
APPLICATION FOR GRANT-IN-AID OF RESEARCH

wo/ne Tf!?y ?????? ?:

LAST NAME FIRST NAME MIDDLE NAME

Add/iU.i> ^^^^ ^'^' ^^^^ Place, Gainesville, Florida 32605

42

Age

r,^^, J. .^- J - ,^-j. v.- Graduate Assistant, Department of
Pn.u e.nt po6-vtcon and -cyutyUtijutcon .\

Obstetrics and Gynecology, University of Florida

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

Membm^p In SIGMA XI .^e^Tl^fT^fy.

P£ea4e cuttach a tl&t o^^ tLtt<^^ oi (Wtiditi, puJoLUhzd duJung tkz lcu,t
^-cue yzffJiA, MAXk namu o^ pznJjodlzaLi> and datzi>: llit o^ tiXLu
appzndzd

-r-j.n , ^ , J ■ ,+- J. • The Relationship Between Epithelial
T-utcz ofj pfwpo6zd ^nvzi>ti.gatLon-

and Connective Tissue Mast Cell Populations in the Female Rat

Ffiopo^zd A^nvutx-gaXion, ducAibzd Zn no n- tzchnlcal tanguagz:

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 deficiences 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.

59

At present there is no universally applicable, non- invasive method for
evaluation o£ 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.

60

Locations whoAt pKoblm mIU be ^tadlzd: ???fy??f?5.?f.^?f!"f}

Resources, J. Hillis Miller Health Center, University of Florida;

Department of Animal Science, College of Agriculture, University of

Florida.

WotuAe o{i (Ui-<j>tance, du^Azd and amouyvt o{^ gfuxYvt m<ide.d, Itomlzzd:

Purchase of 128 Sprague-Dawley female rats $544.00

Feed and bedding for above rats for 5 wk. period 594. 25

TOTAL $938.25

to carry out entire research project: anesthesia, estrogen, stains
and chemicals, microscope slides and use of all equipment.

F^zvloui, QfuxYvU n.zc2l\)Qjd {^fiom SIGMA XI a.n.d athoJUi : .????

OthM. appLicaXloyn, pQ.ndlng : . ??"?

Attach a LLit g-ivAjig ymmz o^ zack aiii-Utarvt on co-mn.k2A, t{, any,
Q.ngag2.d i.n tko.. ■invojityigati.on:

LU>t attackzd X Wombe/i Oj^ zo-wofikzAM 1

WojTie^ and addAizA^e^ o{, cut l2xut tU)o ipucUjatliti* i.n tka> {^loZd iA)ko
M-OZ be ASKEV BY THE APPLICANT to 6znd to Sigma U HatlonjcUL
HejadqujO^eA^ itatmznts tndlavting (J) the, mpoKtanco. o;^ thz pfiopoi,zd
tnvutigation and (2) the, quatl^lcation^ o{, thz InvutlgatoM..
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

*I^ applicant -U a degA.ee candidatt, one. mvu>t be that {^acuity ok
fieAzcvtch iita{^{^ memboA iitxpoAVAJitng kl6 fieAeuAch.

Applicant 'i, S-cgnata/ie^^v^^r^jVv^-,^ , s^_L^yi^.-L^^

p^ October 28, 1980 - /

61

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. Advisor - Endocrine Functions

J. P. Feaster, Ph.D. Advisor - Dietary

A.F. Moreland, D.V.M. Laboratory Assistance

Laboratory Technician of Dr. Moreland Laboratory Assistance

Marsha Ware Laboratory Assistance

Lynda McKenzie, R.N. Laboratory Assistance

Cindy Soroski Statistical Assistance

62

The University of Florida College of Medicine

Department of Obstetrics and Gynecology

November 25, 1980

BOX J.294. JHMHC
GAINgSVILUE. FLORIDA 32610
TELEPHONE: 904392-2893

Convmittee on Awards
Sigma Xi

National Headquarters
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.

Sirs,

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
this problem.

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.

urs sincerely.

"aM^

Morris Notelovitz , M.D.
(RAND), Ph.D., F.R.C.O.G.,
F.A.C.O.G.

Director of the Center for
Climacteric Studies

63

Sigma Xi, The Scientific Research Society

345 WHITNEY AVENUE
NEW HAVEN. CONNECTICUT 0*511

20 April 1981 (2031624988.1

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. Hslen 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
grant.

It is a great pleasure to express the Comnittee's hope for your continued
success in scientific research.

Sincerely yours,

Franklyn B. Van Houten
Chairman

FVH/ia
Enclosure

64

Date April 27, 1981

Committee on Awards

Sigma Xi , The Scientific Research Society

345 Whitney Avenue

New Haven, CT 06511

Gentlemen :

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

is :

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, forv/ard 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.

INSTITUTIONAL

ADDRESS ONLY: Center for Climacteric Studies

Univ. of Fla. , 901 N.W. 20 PI.
Suite B-1 Gainesville, FL 32601

Signature

65

Sigma Xi, The Scientific Research Society

:J4S WiirTNEY AVFNLT
NFW HAVEN. CONNECTKXn

10 June 1981 ,m:i,82..*w3

Ms. Rogene E. Tesar

University of Florida

Center for Climacteric Studies

901 N.W, 20th Place

Suite B-1

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
March meeting.

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 Uu.

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
your research.

Sincerely,

^f^Lt^^^k,^

Thomas T. Holme
Executive Director

TTH/ia
Enclosure

cc: Office of the President
Department Chairman

APPENDIX B
LABORATORY ANIMAL USE

All University Co/ralizas on cJie Cars ana Use
of Laboratory Animals

In order to comoly witJi OHEW policy and all federal, state and local rules
and regulations concerning the care, trsat.-nent and use of laboratory animals,
tne ,ol lowing Inroniiation is necessary for grants to be processed by the
Division of Sponsored Research. Instructional proarams and research projects
supported intamally using laboratory animals ,-nust'also corrolete this question-
naire and return to the Coimiittee completing the approp'riate parts.

1. Principal investigator: Rogene e. Tesar

pepart.T:ent Obstetrics and Gynecology Telephone 392-318A Date Nov. 17 1980

College College of Medicine ' *'**"

2. Proposal submitted to: qicm^ n i/c ,n.^.

• aigma Xl ^^ 3A5 i'/hitnev Ave.. New Haven.

'^^"'S AaarSSS Conn. 06511

3. Starting date: December is. 1980 _; conclusion: August 30. 1981

4. Proposal title:

pe Relationships Between Epithelial and Connective Tissue Mast Cell
Populations in the Female Rat v,cj.j. _

5. Animal species: Rat

Strains Sprague-Oawley

Numbers 137

Sex Female

Age 3 weeks and 2 months

SiZS 50 gram and 120 gram

5. Is t.^s an i.-na.l' model appropriate?

EjtpUin 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

mast cell ir

preliminary

treatment (a

at beeinning of study b) estrogen (LV.) (67 ratsj-^'x w4ek"for"5*weeksr crnormal

°^" {32 rats), d) calcium deficient diet (32 rats), e) vitamin D deficient diet

U^ 'cacs) , f) calcium and vitamin D deficient diet (32 rats), (continued)

a. (.are and location of animals:

The rats will be maintained at the Animal Resources Department, JHMHC. University
of Florida in Individual cages with""Ha"iiy care and feeding.

^' lL?M-^^''tl '^"^■^ned to avoid inflicting needless pain and/or suffering
I^„^H^^ '^^ appropriate use of tranquilizers, anaiaesics and/or
anesthetics? If not, explain: yes. ' "^'wur

Anesthesia will be used for surgery purposes.

10. Method(s) of eut.hanasia to be used:

At the end of a maximum 5 week feeding period, the rats will be killed bv
decapitation ^_^ ^>'. ^

rn r,cipai I'^vesti gator ' ' ^UeparT^ent Chaiman ^

Rogene Tesar. M.A. . R.D. gduard G. Friedrich? Jr., M.D.

67

68

All University Committee on the Care and Use

of Laboratory Animals
Continuation Sheet

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.

69

University of Florida
All-University Committee for
The Care and Use of
Laboratory Animals

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"

submitted 11/17/80.

2. Review Results
Approval: XX

Disapproval :

Incomplete-please provide

3. For questions, please call: Dr. Halliwell at (904)
392-4751.

APPENDIX C
EXPERIMENTAL ANIMAL BODY WEIGHTS

TABLE C. 1
NORMAL DIET

Sample
ID

OW

1W

2W

3W

4W

5W

1

192

197

195

191

195

196

2

175

184

189

197

200

204

3

184

198

202

208

213

212

4

180

186

187

190

192

193

5

185

198

198

201

204

209

6

183

183

189

196

199

200

7

186

187

195

197

197

198

8

197

204

237

231

234

237

9

195

195

227

259

276

280

10

189

196

251

299

321

332

11

185

196

240

264

282

294

12

13

212

205

248

291

308

315

14

195

198

253

282

297

309

15

177

175

222

257

270

278

16

184

195

259

298

325

337

17

177

182

206

217

219

217

18

183

187

197

205

209

206

19

183

202

199

228

231

230

20

197

191

215

209

214

216

21

191

196

206

214

204

206

22

23

184

190

190

208

204

209

24

201

209

254

242

241

245

25

194

203

225

234

244

250

26

206

204

230

238

255

264

27

196

209

228

247

251

255

28

200

209

242

267

275

285

29

171

188

201

217

226

228

30

180

191

231

250

256

263

31

209

214

241

267

273

275

32

194

199

226

245

252

259

Weights expressed in grams

70

71

TABLE C.2

-Ca

DIET

Sample

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

21 1

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

21

172

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

23 2

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

21 1

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

72

TABLE C.3
-D DIET

Sample
ID

OW

1W

2W

3W

4W

1

144

161

165

164

172

168

2

157

166

167

167

170

176

3

146

173

171

167

174

174

4

158

181

179

186

196

198

5

146

165

166

165

176

180

6

151

173

177

171

175

174

7

154

171

169

168

173

177

8

154

180

175

172

171

172

9

156

180

187

207

223

232

10

175

190

193

208

224

234

11

161

173

174

189

204

214

12

153

165

172

182

200

216

13

203

192

196

213

231

250

14

184

183

184

203

210

219

15

173

166

164

179

201

213

16

177

176

179

203

220

230

17

202

209

199

196

210

218

18

134

145

146

149

161

163

19

136

151

157

158

170

176

20

142

165

169

169

173

177

21

148

174

177

181

195

193

22

161

189

190

189

20 3

206

23

139

157

158

163

169

172

24

152

177

181

183

190

194

25

133

149

158

168

173

188

26

154

176

182

188

201

210

27

149

172

171

185

197

208

28

155

181

184

203

210

217

29

150

169

174

182

191

199

30

175

171

174

182

193

197

31

187

180

179

195

21 1

219

32

184

183

185

191

191

203

Weights expressed in grams

73

TABLE C.4
-Ca, -D DIET

Sample
ID

OW

1W

2W

3W

4W

5W

1

204

217

211

223

229

228

2

204

215

210

224

225

217

3

199

207

199

213

224

231

4

176

184

180

187

194

196

5

194

202

190

197

207

206

6

187

192

190

201

203

202

7

218

225

216

230

239

242

8

184

185

186

194

196

197

9

205

205

234

269

294

315

10

171

183

214

251

272

287

1 1

212

194

258

303

329

343

12

191

200

238

278

305

318

13

187

198

223

262

280

300

14

197

214

239

283

306

323

15

212

223

224

240

248

258

16

197

208

204

224

228

242

17

196

205

209

246

222

177

18

196

209

224

214

252

253

19

221

229

234

244

258

246

20

195

197

209

224

234

215

21

198

210

209

212

200

190

22

201

212

216

230

232

231

23

210

212

215

234

245

234

24

200

201

202

220

229

198

25

211

224

248

246

268

268

26

27

217

225

237

243

259

266

28

205

213

220

231

259

257

29

214

229

243

257

270

277

30

194

201

219

225

253

247

31

194

203

221

229

241

247

32

207

208

242

225

268

280

Weights expressed in grams

APPENDIX D
COMPOSITION OF EXPERIMENTAL DIETS

AIN-76 Semipurified Diet (Ca:P 1.14;1)*

Casein 20.0%

DL-Methionine 0.3%

Cornstarch 15.0%

Sucrose 50.0%

Fiber 5.0%

Corn Oil 5.0%

AIN Mineral mix 3.5% g/kg Mixture

Calcium Phosphate, Dibasic (CaHP04) 500.0

Sodium Chloride (NaCl) 74.0
Potassium Citrate, Monohydrate

(HOC(COOK)CH4COOK)4 • H2O 220.0

Potassium Sulfate (K2SO4) 52.0

Magnesium Oxide (MgO) 24.0

Manganous Carbonate (43-48% Mn) 3.5

Ferric Citrate (16-17% Fe) 6.0

Zinc Carbonate (70% ZnO) 1.6

Cupric Carbonate (53-55% Cu) 0.3

Potassium lodate (KIO3) 0.01

Sodium Selenite (Na2Se03 • 5H2O) 0.01
Chromium Postassium Sulfate (CrK

(304)2 ' 12H2O) 0.55

Sucrose, finely powdered 118.0

All Vitamin mix 1.0% per kg Mixture

Thiamine HCl 600 mg

Riboflavin 600 mg

Pyridoxine HCl 700 mg

Nicotinic Acid 3 mg

D-Calcium Pantothenate 1.6 mg

Folic Acid 200 myl Acetate

(Vit. E), Pre-mix 20 g

Cholecalciferol (Vit. D3 ) 2.5 mg

Menaquinone (Vit. K) 5.O mg
Sucrose, finely powdered 972.9 g

Choline bitrate 0.2%

>^Obtained from Nutritional Biochemicals, Cleveland, OH

74

75

Calcium Deficient Diet (Ca;P .07; 1)*

Casein (purified) 24.0%

Sucrose 68.0%

Corn Oil 5.0%

Calcium Free Salt Mixture 3.0%

Dipotassium Phosphate 52.873%

Monosodium Phosphate 10.313%

Magnesium Sulfate 8.188%

Sodium Chloride 23.125%

Ferric Citrate 4.500%

Potassium Iodine 0.130%

Manganese Sulfate 0.741%

Zinc Chloride 0.080%

Copper Sulfate 0.050%

Plus Special ICN Vitamin Diet Fortification Mixture:

gm/kg

Vitamin A Concentrate (200,000 units/gm) .1

Vitamin D Concentrate (400,000 units/gm) .006

Alpha Tocopherol . i

Ascorbic Acid 1.0

Inositol . 1

Choline Chloride 1.7

Menadione .05

-Aminobenzoic Acid .1

Niacin . i

Riboflavin .02

Pyridoxine Hydrochloride .02

Thiamine Hydrochloride .02

Calcium Pantothenate .007

mgs/kg

Biotin .4

Folic Acid 2.0

Vitamin B-12 .03

Rachitogenic U.S. P. No. 2 Diet
(Ca:P 4.23;1)*

Ground Gluten 20%

Ground Whole Yellow Maize 76%

Calcium Carbonate 3%

Sodium Chloride 1%

Custom Vitamin D and Calcium Deficient Diet
(Ca;P .07:1)*

Based on the Calcium-Deficient Diet
with omission of Vitamin D Concentrate

* Obtained from Nutritional Biochemicals, Cleveland, OH

** Formulated and obtained from Nutritional Biochemicals,
Cleveland, OH

APPENDIX E
SERUM CALCIUM ANALYSIS

Sample Collection and Procedure
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 1-»10
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.

Fick et al. , 1979

76

77

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 , , ^ ,
5^ . . , '^ , V X (ml sample)
„ -, ■ J. ^- ' for original sample) ^

calcium concentration = ^^ ? — W^ — r-.

sample dilution

calcium concentration = dOO Ppm) x (1 ml) .

50 ml

calcium concentration = 2 ppm.

Calcium standards of 0, 2, 3, 4, 5, and 7 lag/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.l
lists the concentration and absorbance of the standards, as
read by AAS.

TABLE E.l
SERUM CALCIUM STANDARDS

Standard Readout Calculated

yig/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 = (absorbance) x (dilution factors)
(slope) X (sample weight)

This equation is derived from Beer's Law, which states that
A = abc

where

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
Table E.l.

Tables E.2 through E.5 contain serum calcium concen-
tration values of the laboratory rats.

79

TABLE E.2
SERUM CALCIUM ANALYSIS DATA

Rat Diet

Total Dilution

Normal

50

Date

Technician

2/16/82

Tesar

Sample
ID

Readout

ppm

mg/100 ml

Absorbance

1

.100

2

.099

3

.096

4

.097

5

.097

6

.102

7

.102

8

.082

9

.103

10

.067

11

.096

12

13

.084

14

15

.088

16

.092

17

.082

18

.088

19

.091

20

.092

21

.098

22

23

.088

24

.093

25

.062

26

.074

27

.092

28

.070

29

.068

30

.068

31

.073

32

.087

129.5

13.0

128.2

12.8

124.4

12.4

125.6

12.6

125.6

12.6

132.1

13.2

132.1

13.2

106.2

10.6

133.4

13.3

86.8

8.7

124.4

12.4

108.8

10.9

114.0

11.4

119.2

11.9

106.2

10.6

114.0

11.4

117.9

11 .8

119.2

1 1.9

126.9

12.7

114.0

11 .4

120.5

12. 1

80.3

8.0

95.9

9.6

119.2

11 .9

90.7

9.1

88.1

8.8

88.1

8.8

94.6

9.5

1 12.7

11.3

80

TABLE E.3
SERUM CALCIUM ANALYSIS DATA

-Ca

Rat Diet

Total Dilution

50

Date

Technician

2/16/82

R. Tesar

Sample
ID

Readout

ppm

mg/100 ml

Absorbance

1

.074

95.9

9.6

2

.064

82.9

8.3

3

.068

88.1

8.8

4

.060

77.7

7.8

5

.074

95.9

9.6

6

.074

95.9

9.6

7

.064

82.9

8.3

8

.074

95.9

9.6

la

.069

89.4

8.9

2a

.071

92.0

9.2

3a

.064

82.9

8.3

4a

.064

84.2

8.4

9

.082

106.2

10.6

10

.082

106.2

10.6

11

.076

98.4

9.8

12

.093

120.5

12.1

13

.076

98.4

9.8

14

.073

94.6

9.5

15

.076

98.4

9.8

16

.095

123.1

12.3

5a

.074

95.9

9.6

6a

.056

72.5

7.3

7a

.084

108.8

10.9

8a

.082

106.2

10.6

17

.077

99.7

9.9

18

.072

93.3

9.3

19

.071

92.0

9.2

20

.071

92.0

9.2

21

22

.071

92.0

9.2

23

.068

88.1

8.8

24

.078

101.0

10.1

25

.071

92.0

9.2

26

.073

94.6

9.5

27

.065

84.2

8.4

28

.070

90.7

9.1

29

.073

94.6

9.5

30

.070

90.7

9.1

31

.070

90.7

9.1

32

.068

88. 1

8.8

-D

Rat Diet

Total Dilution

TABLE E.4
SERUM CALCIUM ANALYSIS DATA

50

Date

Technician

2/16/82

R. Tesar

Sample
ID

Readout

Absorbance

PP"^

mg/100 ml

1

.080

103.6

10.4

2

.059

76.4

7.6

3

.065

84.2

8.4

4

.065

84.2

8.4

5

.067

86.8

8.7

6

.077

99.7

10.0

7

.070

90.7

9.1

8

.068

88.1

8.8

9

.057

73.8

7.4

10

.047

60.9

6. 1

11

.078

101.0

10.1

12

.076

98.4

9.8

13

.082

106.2

10.6

14

.078

101.0

10.1

15

.079

102.3

10.2

16

.090

116.6

11.7

17

.079

102.3

10.2

18

.077

99.7

10.0

19

.066

85.5

8.6

20

.064

82.9

8.3

21

.073

94.6

9.5

22

.073

94.6

9.5

23

.061

79.0

7.9

24

.065

84.2

8.4

25

.082

106.2

10.6

26

.068

88.1

8.8

27

.079

102.3

10.2

28

.067

86.8

8.7

29

.064

82.9

8.3

30

.081

104.9

10.5

31

.072

93.3

9.3

32

.066

85.5

8.6

82

TABLE E.5
SERUM CALCIUM ANALYSIS DATA

Rat Diet

Total Dilution

-Ca, -D

50

Date

Technician

2/16/82

R. Tesar

Sample
ID

Readout

Absorbance

PPi"

mg/100 ml

1

.059

2

.070

3

.071

4

.058

5

.057

6

.053

7

.072

8

.057

9

.053

10

.063

11

.079

12

.062

13

.057

14

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15

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16

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17

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18

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19

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20

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21

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22

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23

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24

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25

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26

27

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28

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29

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30

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31

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32

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76.4

7.6

90.7

9.1

92.0

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75.1

7.5

73.8

7.4

68.7

6.9

93.3

9.3

73.8

7.4

68.7

6.9

81.6

8.2

102.3

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80.3

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73.8

7.4

89.4

8.9

84.2

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95.9

9.6

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8.7

55.7

5.6

55.7

5.6

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77.7

7.8

46.6

4.7

32.4

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49.2

4.9

29.8

3.0

40.2

4.0

22.0

2.2

29.8

3.0

62.2

6.2

APPENDIX F
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.l). The scanner transported a

highly collimated beam of monoenergetic gamma rays from a

125
radioactive sealed source of I 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

84

lesser trochanter (proximal 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,

2

provxdes 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.l 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.I 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
(■••^^I) 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 grams per cm, BW (bone width) in cm, BMC/BW
in grams per cm-^.

86

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TABLE F.5
DENSITOMETRIC BONE ANALYSIS
BONE LENGTH (cm)

Bone Rat Femur

Collimator 1/16"

Threshold 85%

Date 12/10/81

Technician R. Tesar

Sample
ID

Normal

-Ca

-Ca, -D

1
2

3
4
5
6
7
8
la
2a
3a
4a
9
10
11
12
13
14
15
16
5a
6a
7a
8a
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32

3.081

3.113

3.098

3.143

3.237

3.018

3.169

3.209

3.203

3. 142

3.118

3.207

3.097

3.118

2.994

3.244

2.994

3.119

3.235

2.970

3.207

3.047

2.974

3.122

3.207

3.453

3.210
3.308
3.076
2.914
3.220
3.370

3.165

3.457

3.097

3.205

3.301

3.197

3.487

3.118

3.563

3.234

3.292

3.581

3.316

3.297

3.518

3.130

3.123

3.364

3.609

3.260
3.197

3.423

3.235

3.148

3.039

3.207

3.220

3.085

3.010

3.148

3.199

3.197

3.083

3.169

3.254

3. 100

3.201

3.337

3.082

3.138

3.109

3.159

3.160

3.301

2.928

3.039

3.101

3.222

3.163

3.489

3.010

3.373

3.342

3.163

3.413

3.092

3.216

3.426

3.082

3.405

3.417

3.139

3.159

3.292

3.342

3.134

3.147

3.222

3.471

3.184

3.204

APPENDIX G
BIOMECHANICAL TESTING

One femur from each rat was used in a biomechanical test-
ing procedure (Puhl et al. , 1972) to determine torque and de-
formation values at time of fracture. This testing procedure
is based on a dynamic test, with load being applied very
rapidly. More reliability of results is expected to be ob-
tained than by use of static methods which would allow creep
to occur in the molded ends of the bones.

In preparation, continually keeping the bones moist, both
ends of each femur were embedded in soft, pliable methyl metha-
crylate, placed in a special mold, and allowed to harden.
After the bones were removed from the mold, they were individ-
ually mounted in the torsional testing machine shown in Fig.
G.l. The specimens were loaded to fracture in external torsion
about their longitudinal axis. A torsional pendulum provided
the load by engaging the bone midway in its free fall.

A coupled oscilloscope traced out a torque-deflection
diagram as each bone was twisted to fracture. A permanent
record of each torque-deflection trace was obtained by an
electronically triggered camera. The photographs provided
a means for determining the value of torque (kg-cm) and
deformation, or twist angle (degrees) , as shown by repre-
sentations in Fig. G.2. Values are recorded in Tables G.l
through G. 4 .

96

Fig. G.I The Rapid Loading Torsional Testing
Machine, with the recording oscilloscope at left. The
bone specimen is loaded in dynamic torsion between the
headstock and tailstock of the machine, indicated by the
arrow. Biomechanics Laboratory, University of Florida.

1. Normal Diet 2. -Ca

-D 4. -Ca, -D

Fig. G.2 Representative torque - deflection curves.
The horizontal scale is 10 degrees/division (deformation)
the vertical scale is 1 kg force-cm/division (torque).

98

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99

TABLE G. 1
BIOMECHANICAL PROPERTIES

NORMAL DIET

Sample
ID

Deformation
(degrees )

Torque
(kg-cm)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

22

6
13,

15
17,

13,
12,
16,
14.
17.

15.5
15.0
17.5

14.0
14.0
11 .0
13.0

12.5
14.0
15.5
15.0
15.0
16.0
17.0
16.5
19.0

3.6
1.8
4.5
3.0
4.2

4.5
4.6
4.6
5.0
5.0

5.2
3.6
4.3

3.5
3.7
3.5
4.4

4.2
4.2
4.8
5.1
3.8
3.7
4.2
5.1
4.8

100

TABLE G.2
BIOMECHANICAL PROPERTIES

-CA DIET

Sample
ID

Deformation
(degrees)

Torque
(kg-cm)

1

2
3

4
5
6
7
8
la
2a
3a
4a
9
10
11
12
13
14
15
16
5a
6a
7a
8a
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32

15.0
13.5
12.0
17.0
12.5
21 .0
17.5
16.0

10

19

16

20

22

18

16

14

16.0

23.5

13.5

21.5

15.0
19.0
23.0
12.5

17.5

2.4
3.0
2.4
2.4
2.2
3.0
2.2
2.6
2.7
2.5
2.4
2.1
2.5
1.8
2.1
2.4
3.3
2.6
3.0
2.4

2.3
2.5
2.2
2.4

3.2

17.0

1.7

101

TABLE G.3
BIOMECHANICAL PROPERTIES

-D DIET

Sample
ID

Deformation
(degrees )

Torque
(kg-cm)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

12,
15,
21,
13,
15.
15,
23.
20.

14.0
17.5

17.0
16.0
25.0
16.0
15.0

17,
14.
16,
19.
14.
16.
17.

17.5
23.0
21 .0
10.5
15.5
16.5
13.5
16.0

2.2
2.6
2.8
3.1
2.7
1.5
3.0
3.0
3.2
4.0

2.1
3.6
3.2
2.4
3.0
3.6
2.7
2.8
3.8
2.9
2.9
2.6

2.6
2.6
2.4
2.4
3.0
3.2

1.7

102

TABLE G.4
BIOMECHANICAL PROPERTIES

-CA, -D DIET

Sample
ID

Deformation
(degrees)

Torque
(kg-cm)

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

13.0
19.0
10.0
9.0
15.5
18.5

16.0

12,
12,
15,
16,
17.

16.5

12,

14

14,

13,

14,

13.0
14.0

17.5

17.0
15.0
18.0
14.5
17.0

3.3
4.0
2.6
2.4
3.0
3.0

3.1

2.0
3.0
2.6
2.7
2.8
3.2

3.2
3.6
3.0
3.3
2.8
3.5
3.3

3.5

2.8
3.6
2.6
3.0
3.4

APPENDIX H
BONE ASH ANALYSIS

Ashing Procedure^

One femur from each rat was dried at lOO'C for 24 hours.
After cooling, each bone was wrapped in cloth and labeled.
Fat was removed by the ether extraction in a Soxhlet extractor
for 36 hours. After extraction, the bones were placed under a
hood until the odor of ether was no longer detectable. The
bone samples were then placed in an oven to dry at lOO'C for
24 hours.

Clean, demineralized crucibles were placed in a drying
oven (100°C) for two hours. The crucibles were removed from
the oven and cooled in a dessicator for two hours. The cru-
cibles were then weighed to four decimal places on a digital
anlytical balance. While on the balance, a bone sample was
weighed into each crucible.

Crucibles containing the dry samples were placed in a
muffle furnace and the temperature (200 °C to begin) was rasied
100° every hour until SSO'C was reached. Ashing proceeded
overnight. The crucibles were removed from the furnace after
they cooled partially to an oven at 100°C. After one hour.

Vick et al. , 1979

103

104

they were removed to dessicators to cool for two hours. The
crucibles and bone ash were then weighed. The % ash of dry,
fat-free bone was calculated for each sample. Recorded
weights and % ash are found in Tables H.I through H.4.

105

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APPENDIX I
MAST CELLS

TABLE I . 1
MAST CELL COUNT — VAGINAL TISSUE

NORMAL

DIET

Rat
ID

Section

1

2

1 3 1

4 1

5 1

1

6

5

14

19

2

1

4

3

8

2

3

5

5

2

3

6

3

2

6

9

2

4

5

6

10

2

5

11

10

7

8

12

10

6

2

4

5

5

4

6

2

4

8

2

6

4

4

6

6

9

2

3

4

6

5

4

3

5

5

7

8

3

8

14

8

3

2

6

2

16

11

3

21

17

3

21

13

4

6

3

1

3

5

8

2

5

2

2

4

3

8

3

2

4

9

4

3

6

4

7

2

4

4

5

6

9

3

9

12

2

3

4

18

5

6

9

13

16

9

6

12

9

7

8

28

11

2

10

24

6

2

4

11

3

6

3

4

2

3

1

1

7

5

4

8

11

6

3

5

3

2

7

5

4

2

113

Table 1.1 continued

114

Rat
ID

Section

1 1

2

1 3 1

4

5

7

6

4

9

5

13

10

5

7

3

7

10

9

5

12

12

5

5

5

5

12

3

9

2

9

7

8

5

7

6

4

5

6

5

6

8

7

12

8

8

3

8

11

5

4

7

5

6

3

7

7

6

17

16

6

8

10

11

9

7

5

4

8

9

13

6

11

13

4

5

8

8

19

3

13

8

22

10

13

8

9

6

10

16

6

9

14

7

5

11

8

20

5

7

5

4

6

7

8

9

2

7

11

11

8

8

5

7

8

11

5

9

6

6

3

16

13

3

4

5

7

8

6

8

11

5

3

11

9

12

13

3

4

6

11

8

8

5

10

19

18

3

6

7

9

12

5

9

8

12

12

6

6

6

12

19

14

2

0

2

2

0

9

3

0

0

0

0

4

2

0

0

5

0

0

0

0

2

1

0

0

0

15

16

5

5

1

11

13

2

6

3

9

11

2

3

3

5

5

4

1

3

6

6

5

4

5

8

14

Table 1.1 continued

115

Rat
ID

Section

1 1

2

1 3 1

4 1

5

17

5

4

4

5

9

2

1

7

0

0

3

3

4

2

1

7

4

3

3

1

6

4

4

2

2

18

7

6

5

9

6

0

2

0

3

4

6

2

2

0

2

1

3

0

6

2

8

6

1

6

1

19

12

4

4

6

4

1

1

4

3

0

0

6

5

1

6

5

5

3

1

5

3

2

1

4

1

20

8

9

2

6

5

2

4

6

3

2

5

4

1

1

2

5

5

7

12

0

10

5

1

24

5

21

6

4

2

4

10

2

2

1

9

6

3

4

4

1

4

3

5

3

3

1

1

9

5

4

2

22

23

6

12

4

4

3

2

7

2

1

. 6

1

7

3

4

3

7

4

2

3

2

3

4

4

2

2

24

5

8

3

9

6

7

7

4

2

8

11

6

5

6

3

8

7

4

5

3

8

2

7

2

3

25

8

6

15

3

12

0

4

8

0

4

0

0

4

6

2

3

4

2

0

7

2

2

2

8

3

Table 1.1 continued

116

Rat
ID

Section

1 1

2

1 3 r

4 1

5

26

11

4

2

2

6

4

5

4

4

0

5

5

3

1

1

3

0

11

1

2

9

1

0

4

5

27

8

11

3

4

6

0

1

3

4

9

2

1

1

0

4

3

3

1

2

7

12

6

1

1

4

28

4

4

2

8

5

4

0

2

1

1

1

3

1

10

1

2

2

3

0

1

8

6

2

5

5

29

4

9

8

4

6

0

0

10

11

3

1

2

10

0

0

2

1

3

1

6

4

0

3

2

7

30

1

2

3

4

2

0

2

3

0

0

0

0

2

0

0

2

3

4

1

4

1

1

5

2

1

31

4

9

3

10

16

4

12

2

19

3

1

5

0

8

11

7

3

4

9

0

7

5

1

3

3

32

11

9

6

8

6

2

7

2

6

2

4

1

7

2

5

2

2

7

1

6

5

3

2

10

9

117

TABLE 1.2
MAST CELL COUNT — VAGINAL TISSUE

-CA DIET

Rat
ID

Section |

1 1

2

1 3 1

4 1

5 1

5

4

11

5

3

0

1

0

1

0

0

0

1

3

3

5

0

0

0

1

2

1

1

6

3

13

0

3

3

3

0

10

11

3

3

4

5

2

5

4

2

2

5

22

17

1

21

1

10

4

1

3

1

3

1

1

2

1

5

0

3

0

2

2

0

0

3

1

1

4

1

3

2

2

15

6

7

7

8

2

3

10

3

4

3

8

3

1

3

10

9

3

3

6

5

5

4

3

6

4

3

3

2

2

4

0

4

2

2

3

0

3

3

0

8

2

22

• 5

5

2

5

13

8

27

1

5

3

6

5

2

2

1

3

8

3

8

1

6

2

5

2

8

3

5

10

7

2

6

4

2

2

6

5

2

4

1

5

6

10

1

7

4

6

10

8

4

11

5

6

7

8

6

7

6

4

0

2

0

1

0

2

2

3

8

2

5

2

3

1

1

1

3

2

6

4

0

0

6

4

2

Table 1.2 continued

118

Rat
ID

Section

1 1

2

1 3 1

4

5

la

7

3

2

2

2

10

8

7

2

4

4

10

1

7

1

3

3

12

2

1

3

8

14

6

4

2a

6

4

2

1

4

0

2

4

3

3

5

0

2

3

4

1

0

1

1

1

11

2

5

1

6

3a

5

7

7

2

4

1

7

13

4

7

7

3

5

1

1

8

4

4

3

4

7

4

3

3

3

4a

12

6

2

5

4

5

7

9

4

3

6

4

2

8

1

3

6

8

3

5

9

3

11

6

4

9

14

5

8

4

4

4

8

10

2

4

3

7

8

2

3

7

11

8

7

3

5

3

6

4

2

10

5

6

4

7

5

6

5

8

12

12

5

3

6

4

4

4

6

4

3

5

5

2

2

2

2

11

5

10

7

5

8

8

5

6

4

12

7

5

3

5

10

9

10

3

5

6

5

4

2

4

4

12

9

6

7

4

19

9

3

10

6

11

7

12

8

13

28

5

9

6

13

3

2

8

19

14

11

Table 1.2 continued

119

Rat
ID

Section

1 1

2

1 3 1

4

5

13

7

3

4

8

5

6

5

3

6

8

5

7

4

7

7

4

4

3

9

0

7

12

10

8

11

14

9

3

9

4

5

13

12

13

2

22

9

16

12

9

9

6

■ 7

10

19

10

3

11

6

10

10

15

5

5

6

7

9

5

7

13

10

3

4

5

3

3

4

4

7

6

8

7

6

5

9

5

4

16

5

5

8

7

7

3

7

10

6

10

6

12

10

4

10

11

4

8

10

16

6

4

9

3

13

5a

11

4

11

9

11

5

5

5

6

8

12

5

6

5

4

8

6

8

10

7

10

9

28

12

6

6a

15

9

9

6

5

14

7

5

5

6

6

5

11

7

7

5

3

7

9

14

9

6

8

4

4

7a

18

61

14

13

16

15

37

5

19

10

19

25

6

8

27

16

36

33

13

15

26

28

34

11

8

8a

8

5

4

33

7

5

7

4

18

3

3

3

3

8

8

6

6

10

6

10

28

6

17

5

18

Table 1.2 continued

120

Rat
ID

Section

1 1

2

1 3 1

4 1

5

17

5

6

7

2

6

3

1

2

2

2

4

3

3

6

4

0

4

4

2

3

2

2

1

3

4

18

9

7

2

4

12

6

1

0

3

5

3

4

0

2

2

2

2

0

9

5

3

3

1

3

3

19

5

4

4

3

1

0

0

1

2

0

3

0

1

1

2

1

2

2

0

2

0

0

1

0

4

20

6

7

4

6

3

2

0

2

1

0

3

1

2

3

2

1

2

4

0

3

2

0

3

2

0

21

4

2

6

1

5

5

3

1

6

1

1

1

6

5

5

3

3

2

0

4

6

2

5

4

2

22

23

17

5

5

5

6

0

2

0

5

7

3

5

2

1

5

0

7

4

1

1

1

6

5

1

4

24

1

4

7

5

4

3

2

3

1

4

1

2

1

2

4

4

2

3

3

1

3

1

2

1

2

Table 1.2 continued

121

Rat
ID

Section

1 1

2

1 3 r

4 1

5

25

5

6

1

3

1

1

0

7

6

3

2

2

0

3

4

1

6

4

0

1

1

3

3

2

2

26

5

4

6

6

6

4

2

2

2

2

2

1

1

6

3

0

0

5

5

2

3

1

0

0

4

27

28

4

14

17

4

5

4

0

0

1

3

3

8

5

2

3

3

2

0

6

2

6

3

2

4

5

29

7

3

5

6

3

4

5

4

1

4

7

7

0

1

2

5

3

5

5

6

3

5

2

3

6

30

5

5

2

1

8

0

1

2

1

1

0

1

0

0

8

1

2

4

0

2

1

1

1

0

1

31

5

4

7

2

8

5

3

3

2

1

1

5

3

1

0

0

1

12

0

2

2

0

5

2

1

32

6

2

3

9

5

1

0

0

1

5

1

4

3

0

4

6

2

5

2

5

3

2

2

0

2

122

TABLE 1.3
MAST CELL COUNT—VAGINAL TISSUE

-D DIET

Rat
ID

Section

10

4

6

5

6

5

14

7

4

4

10

5

6

7

6

14

6

7

4

4

7

6

7

8

3

8

6

19

6

16

17

4

4

21

13

17

5

4

12

35

21

6

33

4

4

16

4

32

20

5

3

5

5

6

4

3

8

5

6

5

3

2

3

6

3

4

5

2

4

6

5

6

4

0

8

8

5

3

3

9

5

10

2

3

10

4

6

13

4

3

6

3

2

8

5

4

4

1

7

5

5

6

13

24

16

10

11

7

9

9

8

9

5

4

5

5

4

13

2

7

9

6

3

13

3

6

7

11

12

10

6

n

10

10

5

6

6

15

5

2

8

15

1

5

26

6

13

1

5

2

4

9

7

5

6

6

2

10

2

5

2

4

5

5

7

2

6

4

11

3

3

7

4

5

2

7

8

7

5

4

13

2

3

7

5

6

6

3

3

5

5

6

2

5

2

6

2

3

4

4

Table 1.3 continued

123

Rat
ID

Section

1 1

2

1 3 1

4 1

5

9

42

8

4

5

8

9

5

5

10

2

4

4

4

17

9

6

5

5

6

22

4

12

3

7

21

10

4

4

5

5

6

3

6

8

4

4

12

10

8

6

4

4

8

5

8

3

4

4

5

8

7

11

3

3

6

6

4

3

3

8

7

4

4

2

4

5

7

1

2

8

2

5

4

7

5

9

2

12

6

4

2

4

5

2

3

5

17

5

2

4

4

5

9

3

3

4

3

3

4

3

5

3

2

13

3

3

3

13

9

2

4

6

6

7

3

3

7

8

8

3

3

7

10

5

2

3

4

6

4

14

25

5

5

31

7

16

2

2

3

4

8

1

2

9

11

3

2

4

2

5

3

1

1

5

2

15

2

5

3

5

10

8

3

4

8

3

7

4

5

1

4

10

5

3

4

6

3

4

3

4

6

16

6

6

11

17

5

6

5

6

11

6

9

4

7

5

4

5

5

7

5

3

4

10

8

6

3

Table 1.3 continued

124

Rat
ID

Section

1 1

2

1 3 1

4 1

5

17

5

10

2

4

5

0

2

2

2

0

2

3

3

6

7

3

7

5

5

3

1

5

5

4

4

18

10

5

3

9

4

5

0

1

7

3

0

8

3

3

9

1

3

8

11

1

4

3

1

18

2

19

11

10

3

6

3

9

8

3

5

4

4

4

4

6

4

7

3

8

5

0

5

11

3

4

5

20

11

2

12

7

3

5

3

0

13

6

4

1

6

6

5

3

0

2

1

4

3

8

8

45

3

21

4

6

8

4

6

4

2

7

5

7

3

5

5

0

3

3

4

8

4

5

7

2

2

1

2

22

42

5

18

21

11

8

1

5

8

6

6

7

1

52

6

42

17

1

24

24

40

25

4

81

8

23

3

9

5

4

16

0

0

2

7

6

5

0

3

3

5

0

3

5

2

7

3

2

0

3

1

24

1

4

5

12

5

1

3

0

7

5

14

1

4

0

6

7

3

6

1

3

3

2

2

3

0

Table 1.3 continued

125

Rat
ID

Section

1 1

2

1 3 1

4 1

5

25

11

3

0

9

6

2

11

0

12

8

1

0

5

4

1

5

5

2

2

1

6

10

2

4

3

26

13

7

27

15

12

66

7

15

2

11

16

25

26

4

10

37

44

6

7

4

16

34

13

9

15

27

10

43

13

12

5

7

11

7

13

16

5

14

9

13

29

3

26

5

16

9

8

23

21

15

11

28

4

5

3

8

7

5

3

4

7

10

2

3

7

10

4

9

3

2

9

8

13

7

7

7

5

29

19

19

12

8

4

6

3

3

6

14

9

4

7

4

12

6

7

1

3

8

3

2

0

10

5

30

12

2

11

3

3

3

9

4

6

5

1

4

7

6

9

4

7

8

7

12

9

8

14

3

1

31

7

11

6

8

13

4

6

5

2

1

4

13

6

4

12

10

6

7

8

21

2

11

8

5

9

32

16

17

11

4

3

9

7

18

5

11

6

3

5

5

4

1

2

6

8

8

9

25

7

10

2

126

TABLE 1.4
MAST CELL COUNT — VAGINAL TISSUE

-CA, -D DIET

Rat
ID

Section |

1 1

2

1 3 1

4

5 1

1

1

5

3

5

8

4

6

4

6

7

7

4

8

3

4

2

2

3

5

7

10

10

2

4

6

2

4

6

7

12

5

6

4

8

5

4

10

11

7

7

2

9

3

6

4

3

5

5

10

3

3

3

4

5

2

6

6

7

3

10

3

4

6

5

6

2

7

5

5

10

3

6

7

7

5

2

8

4

3

2

4

7

6

6

4

1

7

3

2

5

5

4

3

4

6

5

3

2

5

2

3

10

5

5

4

5

6

11

6

11

4

7

9

5

7

6

3

10

12

23

6

18

5

2

12

3

4

7

12

6

3

9

7

2

2

5

3

2

17

16

13

2

8

6

12

11

9

3

4

6

8

13

12

16

6

7

8

3

5

9

6

6

3

7

2

3

12

1

7

20

3

5

1

4

6

15

5

7

5

7

4

8

9

7

5

4

7

3

11

3

3

7

6

8

3

38

6

10

12

8

28

8

3

5

4

5

5

Table 1.4 continued

127

Rat
ID

Section

1 1

2

1 3 1

4 1

5

9

6

6

4

7

9

12

3

6

5

11

5

3

15

5

6

10

9

3

4

9

10

7

8

10

8

10

6

5

6

6

14

3

4

5

5

5

7

9

3

9

2

5

14

3

14

2

6

12

9

6

9

11

13

17

9

7

14

24

21

6

15

8

28

24

6

6

5

8

5

21

5

14

5

9

4

11

7

12

10

6

4

4

8

7

4

12

10

1 1

5

7

13

7

6

5

6

5

7

4

7

7

5

10

12

13

5

8

15

18

10

8

6

5

3

7

5

3

7

6

4

3

4

2

8

14

4

5

9

10

4

14

19

3

6

4

40

4

9

11

4

36

5

4

42

8

15

9

11

17

3

8

4

6

31

13

20

15

15

11

15

12

17

39

14

6

11

22

33

6

24

10

20

7

8

14

4

12

10

12

5

8

4

16

5

4

14

7

6

4

5

12

5

7

8

4

13

5

8

12

2

3

6

6

12

6

9

5

7

Table 1.4 continued

127

Rat
ID

Section

1 1

2

1 3 1

4 1

5

17

7

5

6

3

4

7

4

5

3

0

5

6

4

2

1

8

4

8

0

4

5

5

6

9

1

18

4

3

11

4

7

16

4

15

6

3

11

14

8

11

0

8

16

8

10

13

8

4

4

2

4

19

2

4

5

4

1

6

3

1

0

3

2

1

4

0

2

7

2

4

0

2

2

3

2

5

2

20

4

5

12

1

2

1

1

2

4

3

2

3

9

0

7

1

2

4

6

1

3

5

2

0

1

21

12

7

26

13

6

2

0

21

0

1

3

1

2

0

2

1

0

0

1

2

0

4

3

3

2

22

2

6

1

4

1

2

0

1

3

5

1

2

2

4

1

6

1

4

5

0

2

2

6

2

2

23

5

3

4

5

3

4

2

2

0

0

1

1

6

0

3

5

2

2

1

3

1

0

1

1

3

24

4

7

12

14

9

2

5

4

11

8

1

0

5

3

6

8

7

8

1 1

8

3

2

2

7

5

Table 1.4 continued

128

Rat
ID

Section

1 1

2

1 3 1

4 1

5

25

4

7

10

11

7

4

12

6

6

5

2

3

0

4

5

1

5

2

5

4

6

4

3

7

5

26

27

3

13

6

6

3

5

8

5

4

4

7

2

9

4

1

0

10

1

5

2

2

5

0

3

3

28

3

9

4

11

2

3

5

3

1

9

2

2

2

2

4

1

4

7

1

8

1

8

3

1

7

29

27

4

4

7

0

5

1

2

1

0

2

1

3

2

0

2

1

0

0

1

1

2

2

4

1

30

12

6

9

11

8

8

8

9

3

5

3

7

4

5

5

17

4

9

7

2

16

7

2

0

4

31

1

5

3

3

5

4

1

1

2

3

0

1

1

4

7

3

4

9

3

1

5

1

4

4

2

32

4

6

11

15

4

7

7

8

6

5

5

8

3

2

3

6

13

1

5

5

9

4

2

4

1

129

TABLE 1.5
MAST CELL COUNT — BONE MARROW

NORMAL

DIET

Rat
ID

Section

1

2

1 3 1

4

5

1

24

1

1

6

0

29

0

1

1

0

3

0

0

0

0

6

0

0

0

1

0

33

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

3

21

22

22

35

92

7

5

10

42

62

11

32

17

57

103

17

14

40

49

38

31

19

16

52

25

4

0

0

0

3

2

0

0

0

0

5

0

0

0

4

4

0

2

0

0

0

0

0

4

0

0

5

36

31

23

26

34

39

27

24

31

32

42

22

22

32

17

28

20

6

31

34

39

23

0

34

15

6

10

3

0

32

36

39

8

2

10

11

6

12

0

21

2

8

7

18

28

18

0

19

46

38

17

7

0

5

15

15

4

0

8

2

9

5

0

11

21

8

5

0

12

14

12

5

26

5

17

0

12

8

109

27

49

82

33

64

58

29

101

13

56

33

9

107

16

78

18

11

92

15

73

21

32

47

7

Table 1.5 continued

130

Rat
ID

Section

1 1

2

1 3 1

4

5

9

5

0

0

0

1

3

4

0

0

2

0

6

0

0

0

2

4

4

0

0

2

3

2

0

0

10

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

11

41

54

15

32

31

18

48

15

22

23

37

101

41

17

29

10

52

30

14

34

14

37

26

33

15

12

13

0

0

8

20

26

0

7

10

14

13

9

2

11

4

15

0

3

4

9

9

0

2

1

21

6

14

64

88

40

42

33

36

67

27

32

58

53

49

24

48

60

40

53

31

36

25

66

76

42

55

45

15

25

14

10

16

6

9

10

14

5

11

1 1

3

8

9

5

23

9

9

20

16

17

10

5

18

11

16

2

11

2

3

5

3

4

4

9

13

5

8

9

14

10

2

8

4

0

32

2

12

0

5

18

Table 1.5 continued

131

Rat
ID

Section

1

2

1 3 T

4

1 5 1

17

48

75

26

27

91

41

80

16

33

62

57

65

26

26

75

84

56

24

20

83

102

66

37

29

28

18

32

31

20

33

68

21

21

21

32

24

35

23

13

25

63

21

27

19

26

70

15

27

34

34

57

19

32

53

52

104

61

55

111

83

126

85

68

101

70

50

23

47

54

100

78

62

93

47

96

142

54

20

52

29

59

43

41

62

39

80

34

38

32

56

50

22

30

29

76

20

17

34

20

63

50

39

42

21

45

37

13

52

51

60

49

21

59

66

43

33

37

43

29

64

32

20

38

48

38

38

38

37

55

22

23

84

117

103

112

168

105

137

90

77

89

97

142

99

71

132

91

102

117

90

90

62

120

118

109

52

24

64

31

39

35

45

41

34

41

29

43

53

26

28

47

38

36

48

32

17

39

21

52

35

14

31

Table 1.5 continued

132

Rat
ID

Section

1 1

2

1 3 1

4

5

25

47

35

25

32

66

42

24

37

39

48

36

27

23

22

58

25

33

33

17

68

72

33

19

26

49

26

41

45

52

26

28

40

31

28

29

26

63

30

40

35

24

51

15

38

11

25

34

30

58

32

34

27

60

34

18

32

23

41

21

16

8

28

25

22

17

14

21

30

12

27

11

15

37

11

15

19

20

28

18

38

40

42

54

31

29

34

34

25

60

39

41

36

21

37

39

60

40

17

33

27

49

40

18

29

8

12

9

78

47

16

22

14

46

42

23

15

29

44

40

17

10

16

50

56

21

32

22

28

51

30

41

15

18

18

16

24

11

10

31

23

18

27

26

21

19

34

19

25

24

32

20

28

43

22

24

31

59

56

78

33

36

51

29

67

120

24

42

47

49

59

72

60

72

26

58

48

59

46

57

52

35

32

57

20

23

34

22

84

13

32

21

13

25

18

37

16

36

33

23

54

31

12

13

22

59

26

20

133

TABLE 1.6
MAST CELL COUNT — BONE MARROW

-CA DIET

Rat
ID

Section

20

2

24

45

8

35

0

9

44

21

20

0

10

42

15

20

0

21

21

52

68

0

11

14

12

40

44

32

43

30

25

43

48

56

17

47

42

47

53

17

32

34

50

51

21

47

48

61

45

34

2

29

13

42

46

4

27

18

17

W

35

40

18

17

20

29

20

23

22

22

17

27

27

16

28

31

41

42

15

43

25

71

44

27

32

51

57

52

35

31

23

85

75

15

35

42

56

32

36

41

23

22

11

20

20

15

17

21

16

29

27

13

9

33

20

26

25

21

18

14

21

9

22

17

15

28

96

24

63

38

64

45

77

78

33

28

26

21

24

17

27

32

18

23

44

34

27

27

25

26

21

31

14

26

17

33

20

16

26

25

24

13

26

29

36

12

12

20

27

21

14

9

32

7

9

9

20

33

16

11

8

7

20

8

24

Table 1.6 continued

134

Rat
ID

Section

1 1

2

1 3 1

4

5

la

17

16

12

24

26

21

33

21

25

12

31

41

26

23

21

20

32

32

17

24

18

26

37

25

26

2a

37

13

33

14

12

23

17

22

17

53

31

30

18

15

19

20

30

28

15

15

20

28

22

12

14

3a

25

8

3

2

15

13

5

4

3

2

15

3

0

1

14

11

1

10

5

1

8

1

9

4

7

4a

16

16

19

30

30

21

14

5

18

29

1

36

17

9

20

13

28

24

19

17

19

25

24

15

20

9

36

22

33

32

30

30

23

30

36

40

24

39

53

35

23

33

27

52

27

55

17

24

26

39

47

10

29

37

40

21

15

24

11

39

16

12

26

25

41

19

19

27

12

32

13

18

17

22

16

16

18

11

10

4

17

6

8

12

6

9

4

4

5

6

9

8

3

10

3

13

9

2

5

0

6

8

2

12

18

16

29

26

10

9

17

12

21

14

7

7

19

12

44

22

17

29

27

15

22

28

14

27

31

Table 1.6 continued

135

Rat
ID

Section

13
14

15

16

5a

6a

7a

8a

49

26

18

27

37

41

28

60

10

25

25

64

30

6

13

55

27

33

51

30

21

22

20

28

16

12

33

18

20

10

1

28

12 .

23

12

4

13

9

5

16

3

1

5

18

10

2

6

17

24

19

7

13

26

0

16

9

16

2

15

0

8

10

8

12

5

15

8

9

8

11

11

19

12

16

6

14

7

18

32

7

18

0

12

24

5

16

4

11

12

2

11

13

15

48

0

11

9

16

17

4

0

20

18

21

15

16

20

17

18

8

6

16

12

21

7

24

26

12

38

8

30

20

20

46

17

21

14

26

18

21

19

13

42

47

19

20

15

20

31

36

13

18

34

20

33

27

20

26

18

25

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Table 1.6 continued

136

Rat
ID

Section

1 1

2

1 3 1

4

5

17

52

33

50

29

21

41

34

44

31

42

18

32

50

46

64

31

24

25

41

61

29

29

23

54

31

18

60

34

25

54

37

19

52

43

26

32

15

34

21

21

28

42

40

26

18

22

48

26

23

22

28

19

46

40

45

43

23

21

32

44

25

16

39

63

33

46

16

50

46

27

27

32

27

43

57

44

28

20

27

51

38

61

49

18

54

41

88

42

37

40

51

31

31

39

53

43

36

39

24

66

37

33

36

21

22

71

63

17

20

20

51

20

12

61

23

32

18

18

23

21

29

19

24

27

25

21

22

36

21

20

23

29

33

22

38

41

11

26

36

21

22

16

17

17

20

19

23

22

17

33

24

16

12

23

21

25

24

17

38

21

19

19

9

11

15

12

39

7

8

7

5

61

9

12

16

9

17

6

10

15

8

13

Table 1.6 continued

137

Rat
ID

Section

1 1

2

1 3 1

4

5

25

51

30

27

26

33

20

50

26

14

22

25

24

30

16

8

15

8

16

33

35

25

21

27

15

18

26

62

54

68

70

71

57

106

87

61

40

43

52

48

40

41

53

54

51

53

49

45

71

55

29

49

27

12

8

29

37

58

10

4

12

21

32

8

15

16

31

20

20

10

26

12

54

21

14

24

15

1 1

28

58

19

25

41

27

49

18

25

25

18

40

17

19

25

40

31

23

22

29

18

26

31

33

24

38

29

25

9

21

22

9

9

12

17

19

7

17

10

11

17

11

13

35

17

21

10

15

19

10

6

11

30

31

39

23

21

14

5

20

25

32

30

37

31

31

33

42

30

28

21

48

35

39

30

36

26

57

31

26

34

16

60

41

39

30

28

49

30

35

36

26

23

27

33

25

17

29

18

49

25

31

33

35

32

43

53

26

28

34

38

44

23

22

19

24

34

49

61

28

20

37

49

26

21

25

35

30

22

64

138

TABLE 1.7
MAST CELL COUNT — BONE MARROW

-D DIET

Rat
ID

Section |

1 1

2

1 3 1

4 1

5 1

1

0

36

16

22

38

0

37

44

20

90

4

21

18

23

34

75

60

65

15

24

77

32

29

30

38

2

16

23

17

31

47

6

38

45

22

32

12

26

35

18

13

14

23

39

11

58

34

44

21

23

38

3

23

63

41

51

19

37

58

42

72

24

46

21

46

57

15

67

38

19

80

22

27

52

16

14

44

4

19

17
4

5

52

26

24

24

34

31

31

29

39

20

26

44

51

46

28

20

70

47

39

51

20

66

64

27

59

6

15

34

8

31

5

15

20

19

32

26

18

13

48

32

18

28

14

13

27

17

18

7

35

26

19

7

8

2

10

15

56

10

3

8

26

15

40

8

6

29

6

10

33

12

21

10

13

24

16

17

7

26

Table 1.7 continued

139

Rat
ID

Section

1 1

2

1 3 1

4

5

9

65

16

52

56

32

84

29

56

24

80

31

32

47

86

54

54

60

46

68

37

41

44

42

44

58

10

133

95

55

93

74

63

88

83

76

85

92

86

57

113

94

52

42

112

101

104

101

69

90

70

116

11

36

37

20

37

42

39

18

24

14

37

16

35

18

16

21

18

35

32

16

15

12

58

30

39

26

21

39

52

16

32

38

39

26

42

42

48

13

36

70

22

62

44

46

17

16

22

27

43

56

22

53

48

63

86

30

76

32

38

56

36

30

14

50
99
79
79
58

83
72
67
72
90

15

6

20

5

31

31

36

20

13

28

33

24

14

30

26

26

20

7

24

19

12

17

2

5

10

22

16

Table 1.7 continued

140

Rat
ID

Section

11

2

1 3 1

4 1

5

17

45

0

36

12

5

18

0

42

0

34

8

3

29

7

17

32

2

28

29

36

12

26

0

0

22

18

19

44

35

13

46

24

36

36

28

45

35

35

25

39

38

30

21

10

50

36

38

35

45

31

23

55

20

5

4

18

37

21

8

2

20

11

29

5

5

^■\

14

23

9

18

5

27

28

4

24

4

21

27

21

26

73

45

91

11

26

45

22

64

41

25

26

21

59

33

40

45

43

37

46

23

32

19

34

37

22

9

20

7

22

21

21

15

19

19

13

7

16

21

8

4

4

8

9

9

12

3

10

16

15

18

23

48

63

112

38

28

58

31

70

29

63

60

60

89

75

59

56

30

50

97

43

39

35

47

85

37

24

21

17

21

54

24

34

37

19

33

12

29

18

38

14

38

72

20

55

14

37

82

19

25

17

13

Table 1.7 continued

141

Rat
ID

Section

1 1

2

1 3 1

4

5

25

30

8

2

2

11

28

2

7

5

1

9

1

9

11

3

1

26

21

28

20

32

104

18

47

9

56

59

24

57

8

24

30

50

46

8

46

50

68

14

14

49

18

27

15

19

16

15

29

29

33

44

31

15

30

31

27

11

47

8

13

36

11

32

30

43

36

24

9

28

9

2

7

12

11

18

5

13

29

16

5

21

0

14

10

15

18

15

10

2

5

29

40

12

15

45

26

17

47

24

14

33

9

9

43

22

9

30

4

16

30

10

16

16

30

3
34
10
21
28

40
18
44
54
24

31

2

9

10

14

6

1

14

26

1

19

2

7

10

14

2

0

16

4

2

3

2

9

3

3

5

32

36

24

55

20

36

21

26

48

22

17

11

26

42

23

37

9

45

58

36

46

18

63

30

26

58

142

TABLE 1.8
MAST CELL COUNT — BONE MARROW

-CA, -D DIET

Rat
ID

Section

1 1

2

I 3 1

4

5

1

30

57

42

28

15

27

39

51

12

26

85

33

43

32

16

70

39

56

43

47

69

50

57

23

38

2

19

34

9

33

11

11

6

31

18

23

35

17

43

21

20

13

25

13

20

25

35

25

45

19

3

25

65

37

53

23

22

55

24

59

18

15

24

20

23

14

33

56

19

19

21

19

18

11

45

17

4

23

18

33

60

16

18

33

31

51

49

23

32

49

50

64

41

26

38

21

52

39

34

20

14

63

5

28

39

39

25

21

69

45

17

17

38

12

24

37

37

27

32

60

31

41

33

22

62

37

29

42

6

31

35

15

46

31

23

38

22

50

25

21

38

37

39

31

33

43

16

30

15

34

28

17

42

48

7

17

35

14

14

13

4

15

16

12

12

14

22

3

13

28

8

10

7

22

20

42

57

4

15

13

8

21

15

20

23

50

10

14

25

28

36

15

28

5

22

43

40

23

34

15

37

30

23

31

22

38

Table 1.8 continued

143

Rat
ID

Section

1 1

2

1 3 1

4

5

9

28

10

17

39

25

34

19

17

37

9

25

25

8

25

29

23

20

9

9

7

13

17

13

11

12

10

26

46

35

47

35

14

15

32

18

48

32

16

40

34

27

17

36

19

21

33

20

23

43

24

37

11

29

19

35

34

24

13

20

23

25

31

34

43

51

40

26

44

37

40

20

33

38

31

32

30

25

12

15

12

17

10

14

21

14

10

15

13

17

15

14

12

14

24

21

17

15

14

25

37

22

19

21

13

23
23
23
33
35

14

11

7

58

14

55

7

10

15

37

15

9

7

10

16

17

13

16

37

19

7

16

26

9

34

23

15

24

17

12

20

20

21

20

27

20

23

16

27

14

14

14

15

27

16

20

22

24

25

16

27

20

16

13

21

17

52

30

27

26

20

60

8

29

26

11

34

7

26

15

25

26

8

30

8

10

5

18

Table 1.8 continued

144

Rat
ID

Section

1 1

2

1 3 1

4

5

17

40

28

14

28

15

14

11

23

16

22

37

34

46

24

17

67

24

20

14

16

32

38

35

11

14

18

49

54

27

10

19

32

19

11

15

10

20

15

11

14

16

19

14

8

9

16

18

15

12

24

11

19

29

38

23

36

31

50

17

53

39

43

27

28

23

22

49

23

27

17

33

43

51

16

45

2

30

20

20

78

31

14

40

37

30

48

18

23

40

28

23

32

25

41

63

26

28

31

35

64

23

28

52

21

16

79

12

16

7

110

39

23

20

21

35

57

26

17

50

51

52

53

18

26

33

67

23

24

22

22

23

6

14

20

92

8

11

35

9

71

14

11

30

33

^^

6

10

15

15

17

5

69

23

19

11

24

20

18

26

11

15

9

19

30

11

66

20

17

16

18

29

29

18

13

12

27

41

25

24

18

19

24

21

42

28

29

51

27

51

24

46

25

18

39

33

36

24

54

47

19

19

27

27

26

Table 1.8 continued

145

Rat
ID

Section

1 1

2

1 3 1

4

1 5

25

22

27

53

22

22

26

37

57

35

39

43

45

60

75

36

19

23

57

26

27

23

43

40

37

20

26

27

5

70

16

8

42

32

15

16

50

20

49

41

18

28

23

28

28

27

67

10

22

11

8

20

22

32

29

10

12

100

26

23

13

20

20

66

25

33

19

29

153

123

74

92

110

84

59

59

72

78

103

50

61

143

62

80

78

78

75

98

30

38

12

28

37

26

60

13

35

61

69

39

32

45

50

25

46

26

9

33

34

32

43

13

36

21

31

111

68

70

75

65

48

92

58

59

43

38

58

76

89

80

56

50

50

68

44

62

97

58

47

110

32

66

17

21

63

17

17

52

33

35

56

27

14

14

44

25

31

13

36

45

48

57

21

25

34

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BIOGRAPHICAL SKETCH

Rogene E. Kresak Tesar was born on June 5, 1938, on a
farm near Western, Nebraska. After attending and graduating
from Milligan High School, Milligan, Nebraska, as valedic-
torian of her class in 1956, she attended the University of
Nebraska at Lincoln for two years. In 1957 she married
Delbert Tesar; two daughters were born to this union. Upon
moving to Manhattan, Kansas, she continued her education and
obtained a Bachelor of Science in Home Economics degree in
1962.

That same year, Rogene and her family moved to Atlanta,
Georgia. She taught in the DeKalb County Public School
System for two years.

The next eight years included living for a year in
Vienna, Austria, moving to Gainesville, Florida, having two
additional children, and living for a year in Cheshire,
England.

In 1973, she enrolled at the University of Florida and
completed another Bachelor of Science degree in 1977; the
major was food science. Immediately, she began graduate
studies and graduated with a Master of Agriculture degree in
food science and human nutrition at the University of Flo-
rida in 1979. At this time, she also became a Registered
Dietitian after training at North Florida Regional Hospital.

168

169

Continuing graduate work in the Department of Animal
Science specializing in nutrition, she obtained a graduate
research assistantship at the University of Florida and
became involved in research and professional counseling,
principally concerning bone loss and osteoporosis, at the
Center for Climacteric Studies. In December, 1980, she
became a candidate for the Ph.D. After completing the
required research and dissertation, she received the Doctor
of Philosophy degree in May, 1982.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

P 'A

,f/: (

J. P. Feaster^ Chairman
Professor of Animal Science

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree o^ Doctor of Philosophy.

\\

U

mi

Morris Notelovitz, Cochairman
Associate Professor of Obstetrics
and Gynecology

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

Clarence B. Ammerman
Professor of Animal Science

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.

/James S. Dinning
Research Scientist
Food Science & Human Nutrition

This dissertation was submitted to the Graduate Faculty of
the College of Agriculture and to the Graduate Council, and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.

May 1982

Dean, College of Agriculture

Dean for Graduate Studies and
Research

UNIVERSITY OF FLORIDA

3 1262 08553 6067