THE EFFECTS OF FEEDING
B-GUANIDINGPRGPRIONIC ACID 0N
CREATINE METABOLISM AND SKELETAL
MUSCLE FUNCTION AND STRUCTURE
OF RATS

Thesis for the Degree of Ph. D.
MICHIGAN STATE UNIVERSITY
ROBERT PIERCE SHIELDS
1972

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thesis entitled

"The Effects of Feeding B-guanidinoproprionic
Acid on Creatine Metabolism and Skeletal Muscle
Function and Structure of Rats"

presented by
Robert P. Shields

has been accepted towards fulfillment
of the requirements for

Ph . D . . P
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ABSTRACT
THE EFFECTS OF FEEDING B-GUANIDINOPROPRIONIC ACID ON
CREATINE METABOLISM AND SKELETAL MUSCLE FUNCTION

AND STRUCTURE OF RATS

By

Robert Pierce Shields

This research was conducted to test the hypothesis that high intra-
cellular concentrations of creatine are essential to the integrity of
vertebrate skeletal muscle; and to provide new information on the rela-
tionship between abnormal creatine metabolism and muscle disease. To
accomplish these objectives, the structure and function of muscles were
evaluated after abnormal creatine metabolism was induced by feeding rats
a creatine analog, B-guanidin0pr0prionic acid (B-GPA).

The abnormal metabolism of creatine that occurred when rats were
fed diets containing 12 B-GPA was characterized by decreases in muscle
and brain levels of creatine, muscle N-phosphorylcreatine, urinary
excretion of creatinine, muscle-activity of creatine phosphokinase
(CPR) and by an increase in urinary excretion of creatine. The white-
type fibers from gastrocnemius muscles of normal rats contained more
creatine than the red-type fibers from the same muscles; however, the
creatine levels of both were proportionally lower than normal in fibers
from rats fed B-GPA. While pregnant rats consumed less food when their

diets contained B-GPA, there was no significant effect on food intake

Robert Pierce Shields
of young male rats when they were pair-fed rations with or without B-GPA.
In an in vitro experiment, B-GPA inhibited the reaction of creatine with
rat muscle CPK. Beta-guanidinOprOprionic acid apparently induces
abnormal creatine metabolism in rats by inhibiting the mediated entry
of creatine into tissues from plasma--and possibly by competing with
creatine for the active site of the CPR enzyme.

When muscle function was evaluated by running, the group of rats
fed B-GPA did nOt perform as well as did those that had not received
B-GPA. There were also structural changes in muscle fibers of exercised
rats fed B-GPA. The white fibers from the gastrocnemius of rats fed
B-GPA.were significantly smaller than those from rats not fed the test
compound; nevertheless, normal histochemical profiles were maintained
in these fibers. Gross pathological changes were not observed in'
experimental rats. Thus, when the normal creatine complement of skeletal
muscle is depleted (75%) by feeding B-GPA, the normal structure and
function of muscle is not.maintained.

This is the first report that tissue levels of creatine can be
reduced by altering creatine metabolism directly at a point near its
functional site. The ability to deplete skeletal muscle of its normally
high intracellular concentrations of creatine and N-phosphorylcreatine
will be of value in clarifying the relationship between abnormal creatine
metabolism and neuromuscular disease. In addition, the possibility that
the CPR enzyme acts as a translocating molecule during its reaction with
creatine should enhance our understanding of trans-membrane transport

of small molecular weight compounds.

THE EFFECTS OF FEEDING B-GUANIDINOPROPRIONIC ACID ON
CREATINE METABOLISM AND SKELETAL MUSCLE FUNCTION

AND STRUCTURE OF RATS

By

Robert Pierce Shields

A THESIS

Submitted to _
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Pathology

1972

Dedicated to my wife and family

11

ACKNOWLEDGEMENTS

I would like to exPress my appreciation to Dr. C. K. Whitehair,
my major professor, for his assistance during the course of this
study. I am also indebted to Dr. R. E. Carrow, Mrs. Barbara A.
Wheaton, and to Dr. W. W. Heusner and his staff, because without
their help the studies on muscle function and structure would not
have been possible.

A special note of thanks is due to the faculty and staff of the
Department of Pathology, who assisted in the preparation and the
photography of tissues and who so willingly and patiently provided
invaluable training in pathology.

This work was made possible by a Special Postdoctoral Fellowship

granted to the author by the National Institutes of Health.

111

TABLE OF CONTENTS

Page
INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
REVIEW OF LITERATURE. . . . . . . . . . . . . . . . . . . . . . . . 3
Historical . . . . . . . . . . . . . . . . . . . . . . . . . 3
Normal Creatine Metabolism . . . . . . . . . . . . . . . . . 4

Creatine. . . . . . . . . . . . . . . . . . . . . . .

N-phOSphOrylcreatine o o o o o o o o o o o o o o o o o
Creatine o o o o o o o o o o o o o o o o o o ‘o o o o o

\IO‘JL‘

Abnormal Creatine Metabolism and Neuromuscular Disease . . . 8
OBJECTIVES. . . . . . . . . . . . . .'. . . . . . . . . . . . . . . 11
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . 12.

General Plan . . . . . . . . . . . . . . . . . . . . . . . . 12

Source and Maintenance of Animals. . . . . . . . . . . . . . 12

Chemical Analyses. . . . . . . . . . . . . . . . . . . . . . 13

Source of B-guanidinoproprionic Acid (B-GPA) . . . . . . . . 14

Running Performance. . . . . . . . . . . . . . . . . . . . . l4

Histologic Techniques. . . . . . . . . . . . . . . . . . . . 15

Statistical Analyses . . . . . . . . . . . . . . . . . . . . 18
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l9

Feeding Trials . . . . . . . . . . . . . . . . . . . . . . . l9

Pregnant Females. . . . . . . . . . . . . . . . . . . 19
Young mle rats 0 O O O O O O O O O O O O O O O O O O 19

Chemical Analyses O O O O O O O O O O O O O O O O O O O O O O 21
Urine Excretion of Creatine and Creatinine. . . . . . 21
Muscle Creatine . . . . . . . . . . . . . . . . . . . 24
Brain Creatine O O O O O O O O O O O O O O O O O I O O 26

iv

Running Performance. . . . . . .

Histologic Results . . . . .
Paraffin-embedded Tissue.
Frozen Tissue . . . . . .
Fiber Measurements. . .

Creatine Phosphokinase Results

DISCUSSION. 0 O O 0 O O O O O O O O O O

The Effects of Feeding B-GPA on Creatine

Of the Rat O O O O O O O O O O O

The Effects of Abnormal Creatine
Muscle Structure and Function. .

SWY . C O O O O O O O O O O O O O 0
LITERATURE CITED. . . . . . . . . . .
“PENDICES O O O O I O O O O O O O C O O

VITA O O O O O O 0 O O O O O O O O O O O

Metabolism on

Metabolism

Skeletal

Page
28
31
31
31
35
39

43

43

47
51
53
59

63

Table

9A

. 10A

LIST OF TABLES

Effects of feeding B-GPA upon weight gain, number and

weight of offspring and creatine metabolism of pregnant

rats 0 I O O O O O O O O O O O O O O O O O O O O O I 0

Effects of feeding B—GPA upon body weight and food
intake of young male rats. . . . . . . . . . . . . . .

Effect of feeding B-GPA on indices of creatine metabolism

of young male rats . . . . . . . . . . . . . . . . . .

Effects of feeding B-GPA on muscle creatine concentra-
tion in predominantly red and white fiber areas of the
rat gastrocnemius. . . . . . . . . . . . . . . . . . .

Effects of feeding B-GPA to rats on the concentration
of creatine in the brain . . . . . . . . . . . . . . .

Effect of exercise and B-GPA on the histochemical
reaction in predominantly red and white fiber areas of
the gas tromemius O O O O I O O I O O U C O O O O O O

The effects of feeding B—GPA on muscle fiber size of
exercised rats . . . . . . . . . . . . . . . . . . . .

CPK activity of rats fed B-GPA and effects of B-GPA
on rat CPK activity in vitro . . . . . . . . . . . . .

Standard 8-week, short-duration, high intensity endurance
training program for postpubertal and adult male rats in

contrOIIed-running “7118618. 0 o o o o o o o o o o o o a

Summary of body weight data from rats used in running
exPeriments. . . . . . . . . . . . . . . . . . . . . .

vi

Page

20

22

23

25

27

34

38

42

59

61

LIST OF FIGURES
Figure Page

1 Diagrammatic cross section of muscle unit from the rear
leg of a rat. Specific regions studied are designated
by Roman numerals. Zone I contained predominantly red
(Type I) fibers, Zone II contained predominantly white
(Type II) fibers, and Zone III contained fibers classi-
fied as intermediate (from Edgerton et al., 1969). . . . . . 18

2 The effect of feeding B-GPA on the running performance
of rats expressed as the percent of expected revolutions.
The number of rats is given in parentheses . . . . . . . . . 29

3 The effect of feeding B-GPA on the running performance
as expressed as the percent of shock free time. Number
of animals is given in parentheses . . . . . . . .'. . . . . 3O

4 Transverse section of muscle unit from the rear leg of
a normal rat stained for LDH activity. Section includes
parts of Zone I (a), Zone III (b) and an area containing
many pale Zone II type fibers (c). . . . . . . . . . . . . 33

5 Transverse section of muscle unit from the rear leg of
a normal rat stained for myofibrillar ATPase activity.
Areas identified by letters are given in the legend of
Figure 4 . . . . . . . . . . . . . . . . . . . . . . . . . . 33

6 Transverse section of muscle unit from the rear leg of
a normal rat stained for PPL activity. Areas marked by
letters are given in the legend of Figure 4. . . . . . . . . 33

7 Cross section of muscle fibers in Zone II of the:gastnoc-
nemius of exercised rats stained for LDH activity. Figure
7a was made from muscle of a control rat and Figure 7b was
prepared from a rat fed 1% B-GPA for 75 days. White fibers
stain lighter than other fiber types . . . . . . . . . . . . 37

8 Cross section of muscle fibers in Zone II of the gastroe-
nemius of exercised rats stained for PPL activity. Figure
8a, control; Figure 8b, test. White fibers stain darker
than other fiber types . . . . . . . . . . . . . . . . . . . 37

9 Cross section of muscle fibers in Zone II of the gastroe-
nemius of exercised rats stained for myofibrillar ATPase
activity. Figure 9a, control; Figure 9b, test. White
fibers stain lighter than other fiber types. . . . . . . . . 37

vii

Figure

10

ll

12

Page

Cross section of muscle fibers in Zone II of the gastroe-

nemius of exercised rats stained with PAS. White fibers

stain darker than other fiber types and the intensity of
staining in the test rat (10b) is greater than in the

fibers from the control rat (10a). . . . . . . . . . . . . . 37

Cross section of muscle fibers in Zone I of the gastroe—

nemius of exercised rats. Cross-sectional areas are

similar in control animal #2 (11a) and animal fed 1%

B—GPA, #8 (11b). . . . . . . . . . . . . . . . . . . . . . . 41

Cross section of muscle fibers in Zone II of the gastroe—

nemius of exercised rats. The cross-sectional area of the
fibers from control rat #2 (12a) is greater than that of

test rat #8 (12b). . . . . . . . . . . . . . . . . . . . . . 41

viii

INTRODUCTION

The loss of muscle creatine, excessive creatinuria, and a reduced
excretion of creatinine in the urine are diagnostic signs of abnormal
creatine metabolism in all vertebrates. For more than 50 years these
changes have been directly associated with diseases of skeletal muscles;
nevertheless, the metabolic defect(s) responsible has not been clearly
determined. In addition, knowledge of the role of normal creatine
metabolism in the function and structure of normal muscle is incomplete.

These relationships have not been studied critically partly
because of an inability to directly alter creatine metabolism at or near
its functional site in muscle. Abnormal creatine metabolism associated
with primary muscular dystrOphy or myOpathy experimentally induced by
surgery, drugs, or diet may or may not result from a primary effect
on creatine metabolism.

Creatine or, more specifically, its functional metabolite,
N—phosphorylcreatine, is of a class of organic compounds which function
in the animal kingdom as phosphagens. A brief summary of the biological
significance of phosphagens is included, therefore, as an introduction
to a review of normal and abnormal creatine metabolism.

Ennor and Morrison (1958) defined phosphagens as:

"naturally occurring phosphorylated guanidines
which function as stores of phosphate-bond
energy from which phosphoryl groups may be trans-

ferred to adenosinediphosphate to form adenosine-
triphosphate as a result of enzymatic catalysis."

2
Although several different phosphagens such as phosphorylarginine,
phosphoryltaurocyanamine, phosphorylglycocyanime, and phosphoryl-
lombricine'have been isolated from non-vertebrates, the only phosphagen
found in vertebrates is phosphorylcreatine. In each case a specific
phosphoryl transferase enzyme has been reported. Thus, phosphagens
and their phosphoryltransferase enzymes are found throughout the animal
kingdom and may be evidence of biochemical evolution. There is no
direct support that phosphorylarginine is ontogenetically older than
phosphorylcreatine; nevertheless, since arginine is a constituent of
all proteins, it may be, and the basic phosphagen system may have

survived evolutionary selection in vertebrates as phosphorylcreatine.

REVIEW OF LITERATURE

This review focuses on the normal and the abnormal metabolism of
creatine in vertebrates. Special emphasis is placed on the relation—

ship of creatine metabolism to the function and structure of skeletal

muscle.

Historical

 

Creatine or N—methylguanidinoacetate acid was first isolated by
Chevreal in 1832 from an extract of muscle (cited by Hunter, 1928;
Wang, 1939; Milhorat, 1953). Within the next few years creatine and
its anhydride creatinine were isolated from the muscle and urine of
many vertebrate species. During the latter half of the 19th century
investigators were primarily concerned with the chemical nature of
these 2 compounds and fundamental studies such as those by Leibig (1847)
were instrumental in determining their structure. Quantitative estimates
of creatine or experimental studies of creatine metabolism were not
practical, however, until Folin (1905) develOped a colorimetric assay.
The Folin method was based on the reaction of creatinine with picric
acid.that had been previously described by Jaffe. The Folin method,
vflnile not specific for creatine, permitted reasonably accurate estimates
of muscle levels of creatine and creatinine as early as 1914. Another
'metabolic derivative of creatine, N-phosphorylcreatine, was isolated
in 1927 (Eggleton and Eggleton, 1927; Fiske and Subbarow, 1927). Thus,

by 19 30 all the presently known metabolic derivatives of creatine had

4
been isolated and an analytical method was available for experimental

consideration of creatine metabolism.

Normal Creatine Metabolism

 

The origin, biological significance and fate of creatine and its
metabolic derivatives N-phosphorylcreatine and creatinine will be

considered separately.

Creatine. Although creatine is absorbed from the gastrointestinal
tract of mammals, it has not been found to be an essential dietary
constituent (Block and Schoenheimer, 1941). Significant quantities of.
creatine are synthesized endogenously from glycine, arginine, and
methionine (Borsook and Dubnoff, 1940, 1941). The first step in the
synthesis is the formation of glycocyanamine from glycine and arginine
which occurs primarily in the kidney. Glycocyamine is subsequently
methylated to form creatine. These data were confirmed by Block at
al. (1941) and DuVigneaud et a2. (1941) using N15 labelled compounds.
As a result of their studies, Borsook and Dubnoff and Block et a1.
(1941) proposed a mechanism (below) accommodating all findings whereby
the total body requirement of creatine might be endogenously synthesized.
(aminidinotransferase)

(1) arginine + glycine guanidinoacetic acid
+ ornithine

 

(methyltransferase)
(2) guanidinoacetic acid + methionine creatine
+ homocysteine

 

Van Pilsum et al. (1963) detected L-arginine-glycine aminidino-
transferase (E.C.2.6.2.1.) activity in most body tissues but the greatest
activity per unit weight of tissue was found in the kidney and pancreas.

Studies by Cantoni et al. (1954, 1957) characterized the last reaction

5
as one which requires S-adenosylmethionine as the methyl donor.
S-adenosylmethyltransferase (E.C.2.1.1.2.) activity has been detected
in the liver (Cantoni and Vignos, 1954), pancreas (Walker, 1959) and
testicle (Salvatore and ScHlenk, 1962). The tissue distribution of
the enzymes responsible for creatine synthesis are in accord with in
vitno studies by Azzone and Carafoli (1956), who failed to detect
creatine synthesis in muscle. Earlier Baker and Miller (1940) had
failed to detect creatine synthesis in any tissues except liver and
kidney. Some synthesis of creatine in rat brain (Defalco and Davies,
1961) and testicle (Alekseeva and Arkhangel'skaya, 1964) have been
reported, but there has been no quantitative evaluation with respect
to total body creatine.

While most creatine synthesis seems to occur in the liver, kidney,
and pancreas, it has been estimated that over 95% of the total creatine
in the body is in skeletal muscle (Block and Schoenheimer, 1941).
Various types of muscle contain between 100 and 600 mg of creatine
per 100 g wet weight with the concentration arranged in descending order
of skeletal muscle > diaphragm > cardiac muscle > smooth muscle (Folin
and Buckman, 1914; Baker and Miller, 1939; Gross and Sandberg, 1942;
Eggleton et aZ., 1943; Borsook and Dubnoff, 1947). The remaining 2 to
5% of the body's creatine has been reported to be widely distributed
with the most significant concentrations in nerve and testicular tissue
(Wang, 1939). The brain contains approximately 132 mg per 100 g and
the testicles approximately 267 mg per 100 g (Hunter, 1928; Eggleton
et aZ., 1943).

Since creatine synthesis has not been detected in skeletal muscle,
it has been proposed that plasma creatine is the source of creatine

that accumulates in muscle. Values of 2.0 ml/lOO m1 of blood have

6
been reported in rats (Fitch and Payne, 1965) and rabbits (Tanzer and
Gilvarg, 1959). Blood levels of creatine are maintained by endogenous
synthesis (mentioned above), by ingestion from dietary sources (Block
and Schoenheimer, 1941), and by reabsorption in the renal tubules
(Pitts, 1943; Gayer and Krentz, 1962). In vitro studies have been
used to demonstrate a mediated system of creatine entry into muscles
from plasma (Fitch and Shields, 1966). This system was highly specific
for creatine (Fitch et aZ., 1968b) and could account for the normal
accumulation of creatine in muscles from plasma despite a significant
concentration gradient.

The major excretory route for creatine is as urine creatinine
(Borsook and Dubnoff, 1947). Small amounts of creatine have been
detected in bile, but this is not considered a major excretory route
(Jannakopulu and Impicciatore, 1961). Creatine excretion in feces has
also been reported (Fitch and Maben, 1964). Molecular degradation of
creatine may occur in the gastrointestinal tract, possibly by micro-
organisms (Twort and Mellenby, 1912), but no molecular decomposition

of creatine has been detected within mammalian tissues.

N-phosphorylcreatine. Soon after the characterization of N—phosphoryl-
creatine (Fiske and Subbarow, 1929), Meyerhof and Lohmann (cited by
Ruby and Moltman, 1962) reported the synthesis of this compound in
muscle extracts and the simultaneous hydrolysis of adenosinetriphosphate
(ATP). The enzyme adeniosinetriphosphate : creatine transphosphotrans-
ferase (CPK) [E.C.2.7.3.2.] which was responsible for this synthesis was
isolated and crystallized by Ruby et al. (1954).

The physiological function of N-phosphorylcreatine as an energy

source for skeletal muscle was pr0posed soon after its characterization

7
as a "high-energy" compound (Fiske and Subbarow, 1929). Nevertheless,
it was not until 1962 that Cain and Davies (1962) were able to detect
experimentally the depletion of muscle ATP during a single muscle
twitch. This was done by inhibiting the CPR enzyme which prevented
the rapid resynthesis of ATP from N-phosphorylcreatine. It is now
recognized that the phosphorylated form of creatine serves as an energy
store to rapidly regenerate ATP during muscle metabolism and/or the
contraction processes (West at aZ., 1966).

The tissue distribution of N-phosphorylcreatine is identical to
its precursor creatine but many of the reported tissue levels are open
to criticism. Details of these criticisms are beyond the sc0pe of this
review; however, of primary importance is the extremely labile nature
of N-phosphorylcreatine (Ennor and Morrison, 1958). Reported tissue
values are between 60 and 80% of the total creatine of the analyzed
tissue (Ennor and Rosenberg, 1952). Values of 2.4 mg of N-phosphoryl-
creatine per g of rat muscle have been reported by Fawz et al. (1962).
It has also been suggested (Beatty et aZ., 1970) that the white type
fibers of skeletal muscle contain more N-phosphorylcreatine than do the
red type fibers. N-phosphorylcreatine has not been detected in urine
or plasma (Ennor and Morrison, 1958). The metabolic fate of N-phosphoryl-
creatine is assumed to involve its hydrolysis to creatine or to

creatinine and subsequent urinary excretion.

Creatinine. In vitro, a rapid conversion of creatine to creatinine has
been observed (Borsook and Dubnoff, 1947). No enzyme has been detected
to mediate this conversion and it has been estimated that the loss of

body creatinevat the rate of 2% per day could occur by a non-enzymatic

conversion of creatine to its anhydride creatinine under body conditions

8

(Block and Schoenheimer, 1939, 1941; Borsook and Dubnoff, 1947). These
studies have also established that urinary creatinine is derived from
body creatine and that most urine creatinine comes from muscle creatine.

Tissue levels of creatinine usually range from 1 to 5 mg per 100 g
of tissue (Folin, 1914; Borsook and Dubnoff, 1947). Its concentration
in human plasma has been reported to be between 1 and 8 mg per 100 m1.
Creatinine present in the blood is filtered by the kidney and excreted
unchanged in the urine. The rate of creatinine excretion is relatively
constant and is proportional to the body muscle mass (Melvin et aZ.,

1958; Kleiner and Orten, 1962; Desp0poulous and Kolber, 1964).

Abnormal Creatine Metabolism and Neuromuscular Disease

 

Abnormalities in the metabolism of creatine were the first bio-
chemical lesion detected in patients with muscular dystrophy (Adams et
aZ., 1967). Between 1870 and 1953 it was noted that patients with a
wide variety of neuromuscular diseases exhibited one or more of the
following signs of abnormal creatine metabolism: a decreased urinary
excretion of creatine, an increase in urinary excretion of creatine, or
a decrease in muscle levels of creatine (Nevin, 1934; Milhorat, 1953).
These changes were observed in humans as well as in experimental animals
with generalized myOpathy, regardless of the etiologic agent. Thus,
in 1953 Milhorat (1953) concluded that the relationship between abnormal
creatine metabolism and neuromuscular disease lacks etiological speci-
ficity. The abnormal patterns of creatine excretion and loss of muscle
creatine were therefore assumed to be secondary to muscle cell injury
and not related to the cause of such maladies. Since 1953 newer knowledge
of the dynamics of body creatine metabolism, of muscle function and

structure, and improvements in analytical procedures suggest that

9
etiologic specificity exists and that probably the relationship is more
complex.

Using isotOpic creatine, Fitch and Sinton (1964) provided evidence
that although the clinical signs of abnormal creatine metabolism may be
the same regardless of the etiology of the muscle injury, the mechanism
by which these signs develop may differ. They were able to measure
the turnover (T 1/2-time) of muscle creatine in patients with abnormal
creatine metabolism due to various neuromuscular diseases. The T 1/2—
time of muscle creatine of normal patients and of patients with poly—
myositis was approximately 38 days. Patients with neurogenic (amyo-
trophic lateral sclerosis) or primary muscular dystrophy (Duchenne's
type) had significantly increased and decreased T 1/2-times, respect-
ively. These data were interpreted in relation to the ability of skele—
tal muscle to maintain its normal complement of creatine. In one case
(neurogenic) it was proposed that plasma creatine could not enter muscle
at the normal rate and was excreted in the urine. In the case of pri-
mary muscular dystrophy, it was porposed that muscle could not success-
fully trap or bind creatine that had entered and it returned to the
blood and was excreted. With polymyositis creatine entry or binding
within the muscle was normal but muscle cells were being destroyed by
the inflammatory process and their creatine was lost more rapidly than
normal. In each case there was creatinuria and lower-than-normal levels
of muscle creatine, but the mechanism by which these signs occurred
seemed to differ. These data (Fitch et aZ., 1964, 1968a) were the first
evidence relating abnormal creatine metabolism to the etiology of neuro-
muscular disease. subsequent studies of the entry of creatine from
plasma (Fitch et aZ., 1966, 1968b) indicated that (at least in rats)

creatine enters muscle by a specific mediated process.

10

To more clearly understand the relationship between creatine
metabolism and skeletal muscle function and structure one must also con—
sider certain adaptive responses of muscle. In the muscle of the new-
born, CPK activity is lower than in adults (Perry, 1970). Within the
first 10 days after birth when walking begins, there is a dramatic
increase in muscle CPK activity. There are also changes in CPR isoenzyme
patterns from the BB type in fetal muscle to the MM type which is pre—
dominant in adults (Goto et aZ., 1969). As the animal matures certain
muscle fibers develop a different biochemical profile (white type
fibers) that also seems to be dependent upon muscle activity (Dubowitz,
1970). These adaptive changes also suggest that there may be a direct
relationship between creatine metabolism and normal skeletal muscle
function.

In addition to the above data, indirect evidence, such as the role
of N-phosphorylcreatine in maintaining tissue ATP levels, the ability
of muscle to store large quantities of creatine, and the preservation
of the phosphagen system through evolutionary selection, indicate that
additional knowledge of creatine metabolism could provide a better
understanding of vertebrate muscle function. It would also clarify the
relationship between abnormal creatine metabolism and neuromuscular

disease.

OBJECTIVES

The purpose of this research was to provide a better understanding
of the role of creatine in muscle integrity and to clarify the relation-
ship between abnormal creatine metabolism and pathological changes in
skeletal muscle. Previous research by the author had demonstrated that
an analog of creatine, beta-guanidinOprOprionic acid (B—GPA), would
inhibit creatine entry into muscle in vitro. The initial phase of
these studies was to determine the in viva effect on creatine metabolism
when B—GPA was fed to rats. The second phase included an evaluation
of the performance and structure of muscles after they had been deprived
of their normal complement of creatine by feeding B-GPA.

The specific objectives were: (1) to determine the effects of
feeding B-GPA on creatine metabolism of rats; (2) to determine the gross
and microscopic tissue changes that occur when rats are fed B-GPA;

(3) to evaluate muscle performance or rats that have been fed B-GPA;
and (4) to correlate the biochemical and morphologic data from the
above experiments to permit a better understanding of the role of
creatine in the function, structure and disease processes of skeletal

muscle.

11

MATERIALS AND METHODS

General Plan

 

Creatine metabolism of rats fed B—GPA was evaluated by determin—
ing urine, muscle, and brain creatine; urine creatinine, muscle B-GPA;
and muscle CPK activity. Possible effects on muscle function and
structure were studied by gross and microscopic examination of selected
muscles and by evaluating individual animal performance when subjected
to controlled muscular stress in a running wheel. General or whole
body effects were studied by determining body weight gain, food intake,

and routine cage performance.

Source and Maintenance of Animals

Rats of the Sprague-Dawley strain purchased from commercial
sources* were used for all experiments. Except during nursing periods,
they were housed in individual wire-bottomed cages. Stainless steel
metabolism cages were used for urine collection and for obtaining food-
intake data. Water was given free choice at all times. Diets consisted
of either commercial laboratory chow** or a diet prepared from purified
ingredients (Fitch et aZ., 1960). The ingredient analysis as well as
subsequent chemical analysis during the course of these studies indi-

cated that the commercial diet contained creatine. The purified diet

 

*
Spartan Research Animals, Inc., Haslett, Michigan, or Hormone
Assay Laboratories, Chicago, Illinois.

**
Ralston Purina Co., St. Louis, Missouri.

12

13
was therefore used in one study to test the possible influence of
dietary creatine on the effect of B-GPA. The test compound, B-GPA,
was incorporated into both of these diets by batch mixing in a commercial
paddle-type table mixer. In one trial pregnant rats were fed 0.5, 1.0
or 2% B-GPA. In additional feeding trials young male rats were pair-

fed diets containing 1 or 2% B-GPA.

Source of B-guanidinoprgprionic Acid
The B—GPA used in these experiments was obtained by special synthe-
sis from the Cyclo Chemical Company, Los Angeles, California. Purity

was determined by gas and thin layer chromatography.

Chemical Analyses

 

Urinary creatine and creatinine were determined on 24-hour urine
samples collected under toluene by the alkaline picrate method described
by Folin (1914). Muscle creatine was estimated on water homogenates
by the alkaline picrate method and by the enzymatic analysis of Tanzer
and Gilvarg (1959) as modified by Bernt et a1. (1963) and by Marymont
et a2. (1968). Muscle B-GPA was determined on water homogenates by
Sakaguchi reaction using 8-hydroxyquinoline as prOposed by Gilboe and
Williams (1955) and by paper chromatography (Block et aZ., 1958). The
enzymatic assay of Tanzer and Gilvarg (1959) was also used to determine
CPK activity but modifications were necessary. Immediately after decapi-
tation, the left gastrocnemius, soleus, and plantaris were removed as
a unit and frozen by submersing in iSOpentane that had been precooled
in liquid nitrogen. The frozen muscles were stored in individual sealed
containers at -20 C. Muscle for analysis was sliced from the posterior
aspect of the gastrocnemius (Zone I, see Figure 1) without thawing the

sample. Creatine phosphokinase was extracted according to Oliver (1955)

14
except that the muscle was homogenized in 20-volumes of ice-cold 0.1M
KCl instead of 6-volumes. After homogenization in an all-glass homogenizer
the sample was centrifuged at 15,000 x g for 20 minutes while at 0 to
-4 C. The resultant supernate was diluted 1:200 in 1.0M glycine buffer
and assayed for CPK activity. Protein of the diluted extract was
determined by the Lowry (1951) method. The final assay mixture was
that recommended by Marymont et a1. (1968) except that 0.01M mercapto-
ethanol was included (Kar and Pearson, 1965) and the amount of creatine

and B-GPA varied as described for each individual experiment.

RunningrPerformance

 

Muscle performance was tested by running rats in the Controlled-
Running Wheel of Wells and Heusner (1971). Rats to be tested were run
according to the 4—week, short duration, high intensity endurance program
developed by these investigators (Appendix A). This was a progressively
increasing program in which rats eventually ran at speeds of 4 feet-per-
second for 4 bouts of 10 seconds each. Individual performance was moni-
tored by calculating the percent-of-expected revolutions (PER) and the per-
cent-shock—free-time (PSF). This program was designed for at least 75%
of the rats to perform at better than a 75% rate in both parameters. Body
weights were recorded both before and after running and each animal was
necropsied after the program was completed. Multiple tissues were taken
for histopathologic examination and muscles from the rear limbs (gastroe-
nemius, soleus and plantaris) were frozen for chemical and histochemical.
analyses.

Histologig_Techniques

All experimental animals were killed by decapitation and exsanguina-

tion and examined grossly for lesions. Multiple tissues were taken at

time of necropsy for histologic examination and placed either in Zenker's

15
fixative or in 10% neutral buffered formalin. ’After 24 to 48 hours
tissues were retrimmed, embedded in paraffin, sectioned, and stained
with hematoxylin and eosin, Giemsa, Gomori's trichrome and the periodic
acid-Schiff reaction (PAS). All procedures were those recommended in,
the Manual of’HistoZogic Staining Methods of the Armed Forces Institute
of Pathology (1968).

Histochemical procedures were performed on muscles removed and
frozen immediately after decapitation. The rear legs were skinned and
the superficial posterior crural muscles were exposed by reflecting
overlying tissue. The right gastrocnemius, soleus and plantaris were
removed as one unit, rolled in talcum powder and lowered with forceps
into isopentane for approximately 60 seconds. The iSOpentane was pre-
cooled to -l40 C. to -185 C. by liquid nitrogen. The frozen muscle
was stored in individual sealed containers at -20 C. At a later, more
convenient time small sandwich blocks (10 mm thick) were cut from the
midsection of each muscle unit with a precooled microtome knife. These
smaller blocks were immediately mounted on chucks with 5% gum tragacanth.
Serial cross sections 10 microns thick were then prepared using a
rotary microtome-cryostat.* The sections were mounted on glass cover-
slips and air dried.

The following histochemical procedures were applied to sections
from each muscle unit: phosphorylase (PPL), by the method of Takeuchi
(1958); succinate dehydrogenase (SDH), by the method of Barks and
Anderson (1963); intermyofibrillar adenosine triphosphatase (ATPase),
by the method of Wachstein and Meisel (1957) as described by Thompson

and Hunt (1966); cytochrome oxidase (CYO), by the method of Burstone

 

*
International—Harris Microtome-Cryostate, Model CTI Inter-
national (IEC) Equipment Co., Needham Heights, Massachusetts.

16

(1959) as modified by Pearse (1960); and lactic dehydrogenase (LDH),
by the method of Hess et al. (1958) as described by Pearse (1960).
Sections were also stained with Sudan black B (Lillie, 1954), Gomori's
trichrome (Engle and Cunningham, 1963), and with Giemsa, PAS and hema-
toxylin (Harris alum) and eosin as described in the Manual of’Histologic
Staining.Methodb of the Armed Forces Institute of Pathology (1968).

Tissue morphology and staining reactions were evaluated by light
microscopy. In addition, specific areas of these muscle units were
selected for more critical study. The areas selected corresponded to
foci where the fibers were predominantly red (Zone-I), white (Zone-II)
or were of intermediate (Zone-III) fiber type (Figure 1). In these
zones the intensity of staining was determined by the percent light
transmitted through each of 30 fibers in the zone. To be sure of
identifying the same fibers in each stain, the hematoxylin and eosin
stained sections were projected with a microprojector* on a white sheet
of paper (x200) and at least 30 cross sections of adjacent individual
fibers were sketched. These drawings were then used to identify indi-
vidual and groups of fibers. The percent of transmitted light was then
determined by projecting each muscle fiber in an area over a photocube
coupled to a digital readout (Wells et aZ., 1972). The fiber sketches
were also used to determine mean fiber area after tracing with a compen-
sating polar planimeter.** Fiber area was read in square centimeters
but, because of the initial magnification, divided by 40,000 to indicate

actual fiber diameter in square centimeters.

 

*
Prado Universal, Ernst Leitz GMBH, Wetzlar, Germany.

**
Keuffel and Esser Co., New York.

17
Statistical Analyses
Data were analyzed statistically by methods described by Lewis

(1966).

18

®

GASTROCNEMIUS
PLAN- ®
TARIS ‘ LATERAL
\\ HEAD

 

MEDIAL
HEAD

 

 

 

Figure 1. Diagrammatic cross section of muscle
unit from the rear leg of a rat. Specific regions studied
are designated by Roman numerals. Zone I contained pre-
dominantly red (Type I) fibers, Zone 11 contained pre—
dominantly white (Type II) fibers, and Zone III contained
fibers classified as intermediate (from Edgerton et aZ.,
1969).

RESULTS

Feedinngrials

 

Pregnant Females. Eight pregnant rats (first litter) were fed various
levels of B-GPA during the last 6 to 8 days of pregnancy and during
lactation, as outlined in Table 1. Three others that received no

B-GPA served as controls. The females fed 1 or 2% B-GPA in a commercial
laboratory chow failed to consume adequate amounts of the diet to
maintain normal weight gains. Therefore, 4 days after parturition,

2 of the rats that had received 1% B-GPA were changed to a 0.5% level
for the remainder of the test period. Only 1 female that had received
1 or 2% B-GPA weaned any pups. The other pups were either eaten by

the mother or died--apparently from starvation. The effects of feeding
B-GPA to pregnant female rats is summarized in Table 1. Pregnant rats
receiving B-GPA gained less weight during the last 7 days of pregnancy,
gave birth to fewer pups and weaned fewer pups than did the control
animals. In addition, there was a marked effect on creatine metabolism,
as indicated by the drop in levels of muscle creatine and the increase

in the urinary creatine : creatinine ratio.

Young_Male Rats. Since pregnant rats ate the diet containing 1 or 2%
B-GPA reluctantly, 2 paired-feeding trials were conducted to determine
the effect of B-GPA of food intake in younger rats. In the first, 10
28-day—old rats were divided into 3 groups. Group 1, containing 4 rats,'
received 1% B-GPA in a commercial ration for 8 days followed by 2%

19

20

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a

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.aoauausuuma umumm m%mm e «noun Nm.o Ou comcmnua

.waficmosvmuomon vaAMHHUMm madmaom I haao mean:
a

«a

a»

 

 

 

 

 

 

 

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om.m Ashen mmv ao.a mace Awm.mv «H OH +
«more No.N
Amm.o um meme NNHINH meme OHV om.s saexmm.m~v a Awa.ov m as +
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mm.m Amman «no No.3 one: Amo.nv as me +
ms.m Asses «me OH.H mace Awa.mv a mm +
«anus No.s
Amsme mmav m~.~ Ams.mmv a Amo.AV ca He +
Ashes muse mm.H Amm.-v a Awm.aV oa so +
«molm Nm.o
Amame mNHV ~s.m AmN.Hev NH Amm.mv ma AHH+
sam.o “meme smv so.s «*Awm.sev s Awm.sv as NHH+
mam.o Amsme «no mm.s ««Amm.aev a Ame.ev es N~H+
been Hopscoo
0H m mawawummuo on u umou so mmmu .oa mam A.u3 ummuo>mv A.u3 ammuo>mv housewoum mo
« .u m unaummuo H D Am\m8v mafiummuo saunas mam modems mam penned a« mhmv n ummH
mesa umnasz mean umnasz afimw .uz

 

 

amfiaonmume mcfiummuo mam

.mumu unmawwua mo
mcflunmmwo mo unwfims mam yonaaa .Gamw usmwoa some <mclm savoum mo muuommm

.H magma

21
B-GPA for 6 days. Group II, which also contained 4 animals, were pair-
fed the same diet but without B-GPA. The remaining 2 rats (Group III)
were fed the same diet without B—GPA but in ad Zibitum quantities.
There was no significant difference in the average daily body weight
gain or in the average daily food intake of the groups of rats in this
study (Table 2, Part A).

The second paired-feeding trial included 17 21—day-old male rats
that were divided into 3 groups. Beta-guanidinOproprionic acid was.mixed
into a purified ration (Fitch et aZ., 1960) at the 1% level and was
fed for 21 days. As in the first study, Group I (6 rats) received
diet containing B-GPA, Group II (6 rats) was pair-fed with Group I but
received no B-GPA, and Group III (5 rats) served as ad Zibitum controls.
Data from this study are given in Table 2, Part B. The rats fed B-GPA
did not have a significantly different mean daily food intake or weight

gain than rats in the control groups.

Chemical Analyses

Urine Excretion of Creatine and Creatinine. Urine excretion of creatine
and creatinine was determined at selected intervals during all feeding
trials as well as on samples from animals used in the running experi-
ments. The pregnant females were removed from their cages and placed

in metabolism cages for 24 hours for urine collection.‘ Feeding trials
in younger rats were conducted in metabolism cages and 24-hour urine
collections were made without moving them. In every case, feeding
B-GPA significantly increased urinary levels of creatine, decreased the
excretion of creatinine, and therefore increased the urinary creatine :

creatinine ratio. These data are summarized in Table 3.

22

Table 2, Effects of feeding B—GPA upon body weight and food intake
of young male rats

 

 

Average daily Average daily
Group weight gain* food intake*

 

Part A: Commercial Laboratory Diet

Data for 14-day feeding trial:

I (4)** 6.52 i_ .50 18.0 i 1.20
II (4) 7.04 :_ .29 17.8 i_ .87
III (2) 7.57 19.1

Data for 8 days - fed 1% B-GPA:
I 7.84 -_I_-_ .81 17.6 3; 2.10
II 8.12 i. .82 16.9 i 1.20
III 9.18 18.6

Data for 6 days - fed 2% B-GPA:
I 4.75 i .58 18.7 i .39
II 5.58 :_l.10 18.5 i. .51
III 5.45 19.8

Part B: Purified Ingredient Diet

Data for 20 days - fed 1% B—GPA:
I (6) 5.76 i .69 12.6 i-l.1
II (6) 4.87 i .67*** 10.7 i 1.3
III (5) 5.53 i .35 12.0 i .8

 

*
Mean values :_S.D. of mean invg per day.
**
Number in parentheses indicates no. of rats in group.

***
Probability = .3>P>.2; between this value and mean from Group
I of this trial.

23

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.uofip uaofivoumefi vmfimauae mw>fioomu macaw

flies

.vofiuma wawmuaa mawusv «menu Nm.o pom mama
««

.msme o “om NN sum meme m now «moum NH emu mama uses
an

.mommnucoumm aw am>fiw ma vmummu mum» mo

 

 

 

 

 

 

 

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vamaausm.s Aevo~.mam.s *AstoaHNo.m «mule Am\mav
amuum means:
Asvaoauam.s Amvsm.uem.s moveaaumm.m Hoseaoo Am\mav
Amvmo.uwo.a onaoauaw.o onaoxmwG.H «scum masseuse «Hams:
Assoc Hmm.o Amvmoawma.o AmHVGOaHNG.o Aevaoauom.o Houuaoo oasesummuo
Amvwaaumm.~ vaweauwN.e *AoHvAmaHne.s Asemarmsm.m «muIA oaumu masummuo
levoo.H~m.o Amveoaume.o Amavsoauom.o leveOaHmA.o Houueou Aaa\mav
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flavmoausm.o AmvHOAHoH.o Assesoauem.o AevaaHoe.o Houuaoo Asa\wav
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Teams some *e«ams zoom Ame sues sue nun been mamsamaa

 

umMH no mNmm wcficmmsumom

 

 

mums mama waaom mo Bmwaoemuma maaumouo mo muofivafi do <molm weavmmm mo uommmm .m manms

+

24
Muscle Creatine. Muscle creatine was determined on the triceps brachii
of male rats that had received B-GPA for 14, 50 and 75 days postweaning.
In each case, the muscles from rats that had been fed B-GPA contained
less creatine than did the muscles from rats that were fed diets cone
taining no B-GPA (Table 3). Muscle creatine levels decreased to approxi-
mately 1.0 mg/g of muscle in rats fed B-GPA for 50 days and, in spite
of continued feeding, the values did not decrease further.

The values for muscle creatine given in Table 3 were estimated by
the alkaline-picrate method. Muscles from rats fed B-GPA for 75 days
were also analyzed for creatine by the enzymatic assay of Tanzer and
Gilvarg (1959). The mean values for the alkaline-picrate method were
4.87 mg/g muscle for control rats and 1.08 mg/g muscle for the rats
fed B-GPA. Mean values using the enzymatic assay on normal and test
rats were 4.54 mg/g muscle and 0.73 mg/g muscle, respectively. Thus,
there was close agreement between the 2 methods.

Muscle creatine was also determined on small samples of muscles
taken from areas of the gastrocnemius that contained either predominantly
red (Zone I) or predominantly white (Zone II) type fibers. The samples
were taken from rats that had received 1% B-GPA in a purified ration
for 50 days. The results, summarized in Table 4, indicate that the
white fibers normally contain more creatine than the red type and that
feeding B-GPA resulted in proportionally lower levels of creatine in
both fiber types.

Exercise did not seem to affect the amount of creatine in the.
muscles of rats. Muscle creatine levels of rats that were used in the
running experiments were compared to those of rats of the same age that
had not been forcibly run. Four exercised control rats had a mean

muscle creatine level of 4.87 :_.02 S.D. mg/g muscle compared to that

25

 

 

 

 

 

Table 4. Effects of feeding B—GPA on muscle creatine concentra-
tion in predominantly red and white fiber areas of the rat
gastrocnemius
Muscle creatine(mg£g~wet wt.)
Rat No. Diet Red fiber area White fiber area
1 1% B-GPA* 0.920 1.051
2 1% B—GPA 0.980 .935
3 1% B-GPA 0.540 .669
4 1% B-GPA 0.478 .717
5 1% B-GPA 0.544 .560
6 1% B—GPA 0.462 .707
Mean - 0.654 -_I-_ .23** 0.773 1 .18
13 Control 5.647 7.159
14 Control 4.389 5.526
15 Control 5.066 6.095
16. Control 4.374 5.711
17 Control 4.553 5.838
18 Control 5.235 5.329
Mean - 4.877 1 .52 5.943 : .65
Summary:
(3) Control: white fiber areas a 5.943 i_.65 [P < 05]
red fiber areas = 4.877 + .52 '

(b)

Effect of B—GPA:

white fiber areas
red fiber areas

87% decrease in creatine
87% decrease in creatine

 

*

*
Animals fed 1% B-GPA in a purified ingredient diet for 50 days.

*
Mean : S.D. of mean.

26
of 4.93 for 2 sedimentary controls. 0f the groups that had received
B-GPA, the 8 exercised rats had a mean muscle creatine of 1.08 i_.05 S.D.

and 5 non-exercised rats had a mean of 0.99 i_.63 S.D. mg/g muscle.

Brain Creatine. Although most of the body's creatine is in skeletal
muscle, significant quantities are present in nerve tissue (Wang, 1939).
It was of interest, therefore, to determine if feeding B—GPA to rats
would also lessen the concentration of creatine within brain tissue.
Analyses were done by the enzymatic assay on frozen brains from control
rats as well as rats that had been fed B-GPA for 50 and for 75 days.
These data (Table 5) indicate that feeding B-GPA will also effectively

lower brain creatine concentration.

Muscle B—GPA. Muscle levels of B-GPA were determined on the triceps

brachii of rats fed the experimental rations for 14, 50 and 75 days.

The results, which are included in Table 3, indicate that significant
quantities of B-GPA would accumulate in muscles when it was included

in the diet at the 1% level.

The Sakaguchi reaction used for these analyses is not specific for
B—GPA but only for a monosubstituted guanidine (Gilboe and Williams,
1955). Thus arginine and other monosubstituted guanidines present in
the water extracts of muscle might be expected to react positively.
Because the concentration of free arginine in muscle is relatively low
(approximately 0.25 uM/g, Rogers and Harper [1968]), it was possible
to dilute the muscle extracts and obtain virtually a zero Optical density
reading on control samples while still maintaining an adequate reading
from the samples of muscle taken from animals fed B-GPA. To confirm

that the reacting compound was B-GPA, aliquots of extracts were compared

27

Table 5, Effects of feeding B-GPA to rats on the concentration of
Creatine in the brain

 

 

Brain Creatine

 

 

 

Animal (mg/g wet weight)
No. Diet Days fed Diet :_S.D. of mean
2/2 1% B-GPA 75 0.975
3/2 1% B-GPA 75 1.040
3/4 1% B—GPA 75 0.970

8 1% B-GPA 75 1.003

11 1% B-GPA 75 0.830

13 1% B-GPA 75 0.910

15 1% B—GPA 75 1.050
(Mean - 0.968 _-t .08)

2 1% B-GPA 50 1.310

4 1% B-GPA 50 0.810

6 1% B-GPA 50 0.974
(Mean = 1.031 i .26)

Mean for all rats fed B-GPA 8 0.987 :_.l4*

2 Control 75 1.250

5 Control 75 1.485

(Mean = 1.368)

14 Control 50 1.340

16 Control 50 1.400

18 Control 50 1.418

(Mean = 1.386)

 

Mean for all rats fed control diets - 1.379 i;.09*

 

A
P < .001.

28
to standard solutions of B-GPA, creatine, creatinine and arginine by

paper chromatography using 2 different solvent systems.

Running Performance

 

Sixteen 68-day-old male rats were used in these experiments. Ten
were raised by females that had received 0.5% B—GPA during pregnancy
and lactation. These 10 were then continued from weaning on 1% B-GPA.
Six rats of the same age but which had received no B—GPA served as
controls. All rats were exercised in the controlled running wheel of
Wells and Heusner (1971). After a 4-day training period, 2 rats from
each group were removed on the basis of their performance and the
remaining 12 rats (8-test and 4-controls) were run according to the
4dweek running program given in Appendix A. Data pertaining to body
weight gain during these experiments are given in Appendix B. The final
mean body weight and the average daily weight gain for the rats that
were fed B-GPA were slightly less than for control animals; nevertheless
there was overlap in these data.

The running performance of the rats in this study was evaluated
in terms of the percent of expected revolutions (PER) and by percent
of shock free time (PSF). These results are expressed graphically in
Figures 2 and 3, respectively. The expected daily PER of 75% was
accomplished by 3 of the 4 rats in the control group and therefore the
overall group performance was within the expected range of 75%. Four
of the animals fed B-GPA (50%) performed below 75% PER on more than 5
(75%) days, indicating that the group performance was less than the
expected 75% PER. A critical comparison of these groups is not possible
because of the small numbers of rats tested. Individual rats in the

test group, however, performed as well or better than individuals in

29

1

I60

I40-

I20

IOO

80

 

o—-—o . RATS FED I‘lo B-GPAIB)
o---o s RATS FED CONTROL DIETI4)

1 0

4O

 

PERCENT OF EXPECTED REVOLUTIONS (PER)

 

Figure 2. The effect of feeding B-GPA on the running
performance of rats expressed as the percent of expected
revolutions. The number of rats is given in parentheses.

' ‘——‘—. *

Mc?‘ . - "‘

 

3O

IZCIr

IOO

 

I:
m
o.
m
E
l-
m 80
m
c:
IL
x
E; €ND-
I o——o = RATS FED I95 B-GPAIB)
co o---o . RATs FED CONTROL DIETI4)
.—
z 40-
In
0
a:
3.:
2C)-

 

DAYS

“ .__._
V

Figure 3. The effect of feeding B—GPA on the running
performance as expressed as the percent of shock free
time. Number of animals is given in parentheses.

31
the control group in spite of the fact that their muscles contained

less than the normal amount of creatine.

Histologic Results
Histologic studies consisted of (1) the examination of multiple
paraffin-embedded tissues taken at time of euthanasia; (2) the histo-
chemical analysis of frozen muscle sections; and (3) the measurement

of individual muscle fiber cross-sectional area.

Paraffin-embedded Tissue. Multiple tissues were taken from all animals
and embedded in paraffin. Sections were prepared from the following
organs: liver, kidney, heart, lungs, testicle, thyroid, intestine,
thymus, spleen, and the triceps brachii, psoas major, and tibialis
anterialis muscles. Paraffin sections were also prepared from the
gastrocnemius from all animals except those in the performance studies.
At the time of necropsy the brains of experimental animals were removed
whole and either frozen or fixed in formalin.

No significant histological changes were observed in sections
from paraffin-embedded tissues when stained with hematoxylin and eosin,

Giemsa, Gomori's trichrome, or PASA

Frozen Tissue. Muscles (gastmcnemius, soleus and plantaris) were

 

removed as a unit and rapidly frozen. Subsequently, serial sections
prepared from these tissues were stained with a series of histochemical
and conventional stains. Some of the differential staining reactions
of the 3 fiber types studied are presented in Figures 4, 5 and 6.

The staining reactions of red (Zone I) and white (Zone II) fibers
were compared by determining the percent of light transmitted through

30 adjacent fibers in each zone. These data are summarized in Table 6.

32

Figure 4. Transverse section of muscle unit from the rear leg of
a normal rat stained for LDH activity. Section includes parts of Zone
I (a), Zone III (b) and an area containing many pale Zone II type
fibers (c). x 75.

Figure 5. Transverse section of muscle unit from the rear leg
of a normal rat stained for myofibrillar ATPase activity.‘ Areas
identified by letters are given in the legend of Figure 4. x 75.

Figure 6. Transverse section of muscle unit from the rear leg of
a normal rat stained for PPL activity. Areas marked by letters are
given in the legend of Figure 4. x 75.

33

\

 

 

Figure 6

34

Table 6. Effect of exercise and B-GPA on the histochemical reaction
in predominantly red and white fiber areas of the

 

 

 

 

gastrocnemius
Muscle % light transmitted**
Stain Zone* 1% B-GPA(5) Control(4)
PAS I 43:10 . 6 55:12 .9
II 4l:_5.8 60: 6.2
PPL I 26: 8.1 20t_6.8
II 6: 2.6 11:_4.8
LDH I 40: 0.9 42: 3.2
II 68: 511 7Qt_6.6
ATPase I 50i_2.7 53: 6.5
II 66: 4.4 681 6.2
SDH I 35: 7.8 34: 3.0
II 54: 3.3 52: 2.4
cy—o I 75: 2.4 80i_4.7
II 76: 4.0 8P: 0.7
Sudan I 801- 2.4 79: 5.0
II 82:_4.1 85: 2.8

 

*
Zone I-predominantly red fibers, Zone II=predominantly white
fibers. See Materials and Methods.

**

% light transmitted through 30 individual fibers in each area
and from each animal. Values given are means :_S.D.' Numbers in
parentheses indicate number of animals tested.

35

The red fibers of control rats stained more intensely (lower % trans-
mission) with stains for SDH, LDH, and ATPase activity and possibly
with the Sudan stain. White type fibers stained more intensely with
the reaction for PPL. The staining of red and white fibers from rats
fed B-GPA was, with one exception, the same as was observed in the
control animals. The only exception was the PAS reaction which, in
Zone II, was slightly darker in the rats fed B-GPA. Thus, in these
studies both the red and white type fibers stained normally (Engle,
1970) and no significant changes were detected in animals fed B-GPA.
Typical staining reactions of fibers from Zone II of test and control

rats are illustrated by Figures 7 through 10.

Fiber Measurements. The cross-sectional area of 30 adjacent muscles
was determined in each of the 3 different zones of the gastrocnemius
that are shown in Figure 1. The mean cross-sectional area of fibers
from each zone is given in Table 7. Fibers in Zone I (predominantly
red) were approximately the same size in test and control rats and
were the smallest of the fibers measured. The fibers in Zone 11
(predominantly white) were the largest and the white fibers from the
control rats were larger than those from rats receiving B-GPA. Zone
III fibers were intermediate in mean fiber area and also somewhat
larger in the control animals.

Since the mean body weight of the rats that received B-GPA was
slightly less than the mean body weight of control rats, a ratio was
calculated which consisted of the mean fiber area divided by the indi-
vidual rat body weight. These data are given in Table 7 and support
the conclusion that fibers in Zone II of test rats were proportionally

smaller than those in Zone II of the animals that did not receive B-GPA.

36

* ~ <
Figure 7. Cross section of muscle fibers in Zone II of the.
gastrocnemius of exercised rats stained for LDH activity. Figure 7a

was made from muscle of a control rat and Figure 7b was prepared from
a rat fed 1% B-GPA for 75 days.

White fibers stain lighter than other
fiber types. x 189.

Figure 8.* Cross section of muscle fibers in Zone II of the
gastrocnemius of exercised rats stained for PPL activity. Figure 8a,
control; Figure 8b, test. White fibers stain darker than other fiber
types. x 189.

* .
Figure 9. Cross section of muscle fibers in Zone II of the

gastrocnemius of exercised rats stained for myofibrillar ATPase
activity. Figure 9a, control; Figure 9b, test. White fibers stain
lighter than other fiber types. x 189.

Figure 10. Cross section of muscle fibers in Zone 11 of the
gastrocnemius of exercised rats stained with PAS. White fibers stain
darker than other fiber types and the intensity of staining in the
test rat (10b) is greater than in the fibers from the control rat
(10a). x 189.

I

 

These sections are serial sections taken from control rat #2 and
test rat #8 (see Appendix).

37

 

__4J
Figure 8

 

Figure 9

 

Figure 10

38

Table 7. The effects of feeding B-GPA on muscle fiber size of
exercised ratsTT

 

 

Group Treatment Zone I Zone II Zone III

 

A. Mean Fiber Area*

 

 

Control (4H 1.18:0.3a 0.861.009}, 0.561.004
Fed 17. B-GPA (5) 0.61:0.1‘3l 0.64:.004b 0.491.004
B. Mean Fiber Area/Total Body WeightTTT
Control (4) . 3.96:.27‘: 2.87:.14 1.90
Fed 17. B-GPA (5) 2.27:.11c 2.46:.90 1.85
a? < .001.
bP < .005.
C? < .001.

* _
Area expressed in sq.cm. x 2.5 x 10 5 and are mean values

tNumber in parentheses = number of animals tested.

+1.From dams that received 0.5% B-GPA during nursing and fed 1%
B-GPA for 75 days. Exercised for 20 days in running wheel.

+++Figures given are mean values i_S.D. in sq.cm. x 2.5 x 10-8.

39
The small difference in mean area of fibers in Zone III was not evident
when divided by body weight. Thus, the only real difference in fiber
size between test and control animals would appear to be in Zone 11.

This difference in fiber size is visible in Figure 12.

Creatine Phosphokinase Results

It had been previously reported (Fitch et aZ., 1968) that B-GPA
would not serve as substrate for rabbit muscle CPK in the in vitro assay
of Tanzer and Gilvarg (1959). This fact was confirmed during the course
of these studies. In addition, it was found that B-GPA would not inter-
fere with the reaction of creatine under the assay conditions. These
data, however, may not apply to rat muscle CPK.~ Under the assay condi-
tions the enzyme is not saturated with substrate which would limit one's
ability to detect interference between B-GPA and creatine for the active
site of the enzyme. Because the mediated entry of creatine into rat
muscle from plasma could involve the CPK enzyme, studies were initiated
to determine the effect of feeding B-GPA on the activity of rat muscle
creatine phosphokinase and to study the in vitro reaction of creatine
and B-GPA with this enzyme. The results given in Table 8 indicate that
rats fed B-GPA have lower muscle CPK activity and that B-GPA effectively

inhibits the reaction of creatine with the enzyme.

40

Figure 11. Cross section of muscle fibers in Zone I of the
gastrocnemius of exercised rats. Cross—sectional areas are similar
in control animal #2 (11a) and animal fed 1% B—GPA, #8 (11b).. H &'E
stain. x 189.

Figure 12. Cross section of muscle fibers in Zone II of the
gastrocnemius of exercised rats. The cross-sectional area of the
fibers from control rat #2 (12a) is greater than that of test rat
#8 (12b). x 198.

41

     

Figure 11

 

42

Table 8. CPK activity of rats fed B—GPA and effects of B-GPA on rat
CPK activity in vitro

*
PART A: Muscle CPK activity of normal rats and rats fed 1% B—GPA

 

 

 

 

**
mM Creatine Activityi _
in Assay + B-GPA Control
42 6.96 8.18
6.31 . 7.77
9.91
Mean 6.64 8.62
25 3.08 5.31
3.00 3.60

 

PART B: Effect of B-GPA (52.5 mM) on rat CPK activity in vitro

 

 

 

 

mM Creatine Activigy
in Assay Without B-GPA With B-GPA
25.20 3.60 3.39
18.90 3.17 ___
12.60 2.94 2.14
7.65 2.13 1.48
6.30 2.00 1.44
2.52 1.09 .75_

 

6

*
Fed 1% B—GPA for 75 postweaning days.

**
Activity expressed as umoles creatine phosphorylated/minute/mg
protein in extract.

DISCUSSION

The ultimate objective of these studies was to provide a better
understanding of the relationship between creatine metabolism and
skeletal muscle function and structure. To this end, muscle function
and structure were evaluated after abnormal creatine metabolism was
induced by feeding B-GPA. There were, therefore, 2 separate but inter-
related phases of this research. One served to monitor creatine
metabolism and in the other muscle function and structure were evalu-
ated. To facilitate an analysis of the data obtained, this discussion
has therefore been divided into 2-parts. In the first part, the data
pertaining to the effects of feeding BécPA on creatine metabolismuwill
be considered. The second part of this discussion will deal more spe-
cifically with the effects of abnormal creatine metabolism on skeletal

muscle function and structure.

The Effects of Feedinng-GPA on Creatine Metabolism of the Rat

 

To accurately evaluate the effects of any dietary supplement it
must be ascertained if the addition of the test compound to the diet
will alter or otherwise modify the intake of other essential dietary
ingredients. During the studies using pregnant rats, a marked difference
in food intake was observed when 1 or 2% B-GPA was included in the diet.
With the possible exception of a significant effect on creatine metabol-
ism, it is believed that most of the effects observed when pregnant rats

were fed B-GPA were manifestations of a reduced food intake.

43

44

In contrast to the studies in mature female rats, the inclusion of
l or 2% B-GPA did not alter the food intake in young male rats. There
was no significant difference in mean daily body weight gain or in mean
daily food intake when young rats were pair-fed diets containing B-GPA
for periods of up to 21 days. Food intake was not measured over longer
periods; nevertheless, the body weights of rats fed 1% B-GPA for 75
days were slightly less than those of rats that did not receive B—GPA
(Appendix B). The difference in these 2 groups might represent a
moderate, long-term effect on food intake; or, in light of the smaller
size of the Type II fibers in the rats fed B-GPA, the difference in.
body weight might be a reflection of the failure of these white fibers
to hypertrophy. Since there are no estimates of the size of the total-
body white fiber population, this latter explanation would be difficult
to evaluate further. With this one possible exception, it would appear
that feeding B-GPA to young male rats had no significant effects on
food intake. It would, therefore, be reasonable to conclude that any
effects of B-GPA such as those on creatine metabolism occur directly
because of the presence of B-GPA in the diet and are probably not due
to a secondary effect on food intake.

All tests employed in this research to monitor the effects of
B—GPA indicated that there were marked changes in creatine metabolism.
The alterations were manifested by a decrease in muscle and brain levels
of creatine, an excessive creatinuria and a diminished excretion of
creatinine in the urine.

The mechanism(s) by which B-GPA alters creatine metabolism is
probably complex; however, based on these studies and previous data the
most plausible explanation would be that it inhibits creatine entry

from plasma into muscle. The in vitro studies of Fitch and Shields

45

(1966) and by Fitch et al. (1968b) indicate that creatine enters rat
skeletal muscle by a mediated system and that B-GPA will competitively
inhibit this entry. When rats that have been fed B-GPA are injected
with a small dose of creatine-l-14C, less radioactive creatine
accumulates in their muscles and a greater portion is excreted in the
urine. These results were attributed to the ability of B-GPA to inhibit
the entry of the radioactive creatine into muscle from plasma in vitro
(Shields and Whitehair, 1972). In these present studies, brain levels
of creatine as well as creatine levels in muscle were significantly
lowered when rats-were fed B-GPA. This suggests that the same entry
system may also account for a portion of the normal brain complement
of creatine. Moreover, it is tempting to regard a mediated system of
creatine entry from plasma as of practical significance for many body
tissues.

It has been estimated that between 60 and 80% of the creatine in,
muscle is in the form of N-phosphorylcreatine. When muscle creatine
is lowered to only 25% of its normal concentration as in these present
studies, the muscle concentration of N-phosphorylcreatine must also be
significantly lower than normal. This conclusion has been recently
substantiated by Jellinek and Fitch (1972). In addition, they found
that after feeding rats 1% B-GPA for 26 days, muscle ATP, ADP and
phosphoenolpyruvate (PEP) levels were also significantly lower than
normal.

The inability to lower tissue levels of creatine appreciably below
1.0 mg/g tends to support the theory that more than one entry system is
involved in the entry of creatine into muscle (Fitch and Shields, 1966).
Based on in vitro data a small increment of creatine enters muscle by

simple diffusion. At physiologic plasma levels of creatine (ca. 0.1 mM),

46

however, this mechanism alone might not be capable of maintaining an
intracellular concentration of approximately 7.6 mM. Entry by diffusion
plus intracellular binding, as has been prOposed (Fitch and Sinton,
1964), might be a better explanation.

The excessive excretion of creatine in the urine that developed
in all animals fed B-GPA could have resulted from (a) a blockage of
renal reabsorption, (b) an excessive loss of creatine from tissues,
(c) an increase in the synthesis of creatine and/or (d) a blockage of
creatine entry into tissue. There is no evidence to implicate any of
these mechanisms except that already presented regarding the blockage
of creatine entry into muscle. The reabsorption of creatine by the
kidney has been reported (Gayer and Kienitz, 1962), but the specificity
of this mechanism has not been studied in detail. Since most mechanisms
at renal reabsorption, however, are subject to some analog competition
(West at aZ., 1966), it would seem possible that B—GPA could inhibit
creatine reabsorption and result in creatinuria. 0f the mechanisms
proposed, only an increase in the synthesis of creatine might have been
capable of maintaining normal tissue creatine levels. In this research,
however, normal muscle creatine values were not maintained regardless
of the status of creatine synthesis.

The excretion of creatinine in the urine of all rats fed B—GPA
was decreased. Since urine creatinine is derived primarily from muscle
creatine (Block and Schoenheimer, 1939, 1941; Borsook and Dubnoff, 1947),
a depletion of muscle creatine concentration should result in a decreased
creatinine excretion in the urine. Patients with myopathy and concomitant
low levels of muscle creatine also exhibit this same change (Milhorat,

1953).

47

Although creatine entry into skeletal muscle by a mediated system
has been proposed (Fitch and Shields, 1966), there has been no indica-
tion of the specific mechanism involved. The CPK studies of this.
research suggest that this enzyme may be at least a part of the creatine
entry mechanism. The phosphorylation of creatine during transport
across a cellular membrane would possibly remove the then intracellular
phosphorylated creatine from a gradient against entry. The conforma-
tional changes that reportedly occur during the reaction of this enzyme
with its substrates (James and Morrison, 1966), as well as the fact
that most CPK is bound to cellular membranes (Baskin and Deamer, 1970;
Jacobus, 1972) would tend to add support to the participation of CPK
in the transport of creatine. In addition, it has been recently pro-
posed that B-GPA is phosphorylated when it accumulates in muscle after
feeding (Jellinek and Fitch, 1972). Future studies of the kinetics
of the reaction of creatine and B-GPA.with rat muscle CPK may clarify
this hypothesis.

The Effects of Abnormal Creatine Metabolism on
Skeletal Muscle Structure and Function

 

No marked differences were observed in the histochemical staining
reactions of white (Type II) or red (Type I) fibers when abnormal creatine
metabolism was produced by feeding B-GPA to rats. The small difference
in the intensity of PAS staining in Type II fibers of rats fed B—GPA
may have been a result of the smaller size of these white fibers. Thus
muscle fibers seemed to maintain most of their characteristic histo-
chemical patterns in spite of the low levels of creatine.

Of particular significance is the fact that the white fibers of
rats fed B-GPA were not as large as those of control rats after both

groups had been exercised. This difference in fiber size was not due

48
to a difference in total body weight as was indicated by the body
weight : fiber size ratio (Table 7). This is even apparent when fibers
from animals of similar body weights are visually compared (Figure 12).
The specific cause of the difference in fiber size is not known.
Also, it is not known if this change represents atrOphy or a failure
of the normal processes of hypertrophy. It has been demonstrated that
the Type II white fibers differentiate soon after birth and hypertrOphy
is concomitant with increasing muscular fiber activity (Ashmore et aZ.,
1972). During this same period the metabolic profile of these fibers
also changes. There is an increase in CPK activity and N-phosphoryl-
creatine levels (Perry, 1970) and, since muscle creatine levels and
CPK activity were below normal in rats fed B-GPA, the normal processes
of differentiation may have been at least partially limited. Not all
normal differential processes were impaired, however, because the Type
II fibers retained most of their normal histochemical characteristics.
In a review, Engle (1970) has summarized experimental and clinical
findings of Type 11 fiber atrophy. Atrophy of Type II fibers is one
of the most common myopathies. It has been observed experimentally in
cats and guinea pigs after nerve resection and clinically in cases of
cashexia, disuse, chronic corticosteroid intoxication, myasthenia
gravis and untreated collagen vascular diseases such as panencephalitis.
It has been also proposed by Engle that preferential atrOphy of Type
II fibers occurs because they are more susceptible to a lack of trephic
influence than are the Type I fibers. In the case of the rats in this
present study, their running performance would make it difficult to
argue that there was a marked difference in the nerve impulses received

by the white fibers of control and test animals. A more plausible

49
explanation might be that the fibers were unable to respond to the
impulses in a normal manner to produce fiber enlargement.

During the course of these studies the rats fed B-GPA were able

to perform routine cage movements without noticeable impairment, in
spite of lower levels of muscle creatine. A more critical test of
muscle performance was conducted in the running wheel.- Rats fed B-GPA
did not run quite as well as did normal rats in this test;.however,
some of the animals that had low muscle creatine levels performed as
well as the controls. It is believed, therefore, that the difference
in running ability, as measured in_these tests, is minimal.(Heusner,
1972). It is tempting to propose that high levels of muscle creatine
are not necessary for muscle function but any such hypothesis must be
qualified by a consideration of the type of performance test used.
The stress required by the running studies in this work may not have
been sufficient to stress the performance of white fibers (Faulkner,
1970). Alternative tests such as jumping might be required before a
significant impairment of performance is observed.

In these studies high intracellular levels of creatine did not
appear essential for survival of muscle cells. Thus, it can be reasoned
that either (a) compensatory mecahnisms were adequate for metabolic
needs under the experimental conditions or (b) the high levels of intra-
cellular creatine are in excess of metabolic needs. While it is possible
that the compensatory reactions may have permitted relatively normal
performance, they did not permit the Type II fibers to attain their
normal size, nor were they dramatic enough to be detected by histo-
chemistry. Furthermore, the fact that levels of N-phosphorylcreatine,
ATP, ADP, and PEP are much lower than normal when rats are fed B-GPA

would argue against the presence of effective compensating mechanisms,

50
especially those functioning as phosphagens. Since the CPK enzyme and
N-phosphorylcreatine are detectable in most body tissues, it is possible
that part of the normal creatine concentration present in skeletal muscle
is acting only as a reservoir to maintain constant blood levels. In.
this hypothetical case, body tissues would be served with a constant
blood supply of creatine and could utilize the N-phosphorylcreatine
system to regenerate ATP without the necessity of storing large quanti-

ties of creatine.

SUMMARY

This research was conducted to test the hypothesis that high intra—
cellular concentrations of creatine are essential to the integrity of
vertebrate skeletal muscle; and to provide new information on the
relationship between abnormal creatine metabolism and muscle disease.
To accomplish these objectives, the structure and function of muscles
were evaluated after abnormal creatine metabolism was induced by feed-
ing rats a creatine analog, B-guanidinOprOPrionic acid (B-GPA).

The abnormal metabolism of creatine that occurred when rats were
fed diets containing 1% B—GPA was characterized by decreases in muscle
and brain levels of creatine, muscle N-phosphorylcreatine, urinary
excretion of creatinine, muscle activity of creatine phosphokinase
(CPK) and by an increase in urinary excretion of creatine. The white-
type fibers from gastrocnemius muscles of normal rats contained more
creatine than the red-type fibers from the same muscles; however, the
creatine levels of both were proportionally lower than normal in
fibers from rats fed B-GPA. While pregnant rats consumed less food
when their diets contained B—GPA, there was no significant effect on
food intake of young male rats when they were pair-fed rations with or
without B-GPA. In an in vitro experiment, B-GPA inhibited the reaction
of creatine with rat muscle CPK. Beta-guanidinOproprionic acid.
apparently induces abnormal creatine metabolism in rats by inhibiting
the mediated entry of creatine into tissues from p1asma--and possibly
by competing with creatine for the active site of the CPK enzyme.

51

52

When muscle function was evaluated by running, the group of rats
fed B-GPA did not perform as well as did those that had not received
B-GPA. There were also structural changes in muscle fibers of exercised
rats fed B-GPA. The white fibers from the gastrocnemius of rats fed
B-GPA were significantly smaller than.those from rats not fed the test
compound; nevertheless, normal histochemical profiles were maintained
in these fibers. Gross pathological changes were not observed in,
experimental rats. Thus, when the normal creatine complement of
skeletal muscle is depleted (75%) by feeding B-GPA, the normal structure
and function of muscle is not maintained.

This is the first report that tissue levels of creatine can be
reduced by altering creating metabolism directly at a point near its
functional site. The ability to deplete skeletal muscle of its normally
high intracellular concentrations of creatine and N-phosphorylcreatine
will be of value in clarifying the relationship between abnormal creatine
metabolism and neuromuscular disease. In addition, the possibility
that the CPR enzyme acts as a translocating molecule during its reac—
tion with creatine should enhance our understanding of trans—membrane

transport of small molecular weight compounds.

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APPENDICES

APPENDIX A
(Table 9A)

59

 

 

 

ONO mom oeumm m.m ~.H O.m e mm ON OHuOO m.H mH mum

ONO mow oeumm m.m ~.H O.m H mm ON OH“OO m.H «H are

ONO mom oeumm n.m ~.H O.m e mm ON OHuOO m.H mH um

ONO mom Oqumm m.m ~.H O.m q mm ON OH“OO m.H NH Hum

OOOH OHO ooumm O.m N.H O.m e um mH OHuOO m.H HH zuH m
OOOH OHO OOAOm O.m N.H O.m e RN mH OHHOO m.H OH mum

OOOH OHm OOumm O.m N.H O.m e um mH OHuOO m.H m Hue

OOOH OHO oonmm O.m N.H O.m e um mH OHuOO m.H m 3um

OOOH OHO oouam O.m ~.H O.m e um mH OHAOO m.H n Hum

ONHH OON oeuHm m.~ N.H O.m e ON OH OHuOO m.H O ZuH N
OONH OOO Omume O.~ N.H O.m m oo OH OHAOO O.~ m mum

OONH OOO Omuae O.~ ~.H O.m m OH OH OHuOO m.~ e Hue

OONH qu Omumq m.H N.H O.n m oe OH OHuOO O.m m 3am

OONH Ome Omnme m.H N.H O.m m Oq OH OHAOO O.m N Ham

OONH one Omnme m.H ~.H O.m m OH OH OHHOO O.m H an H
III III OOHOe m.H 0.0 O.m H H OH OO0O¢ O.m HI mum

III III oouoq m.H 0.0 O.m H ,H OH OOAOq O.m NI Hue O

.HMH .mmm Aoom Aoom\umv Amav AaHav mason unom use Aoomv HummusHav Aoomv .uH .33 .33
Aoomv mnoHu usHaO pooem xoonm munom mo maoHuHu maHB oaHH uaHH mo mo
oaHH InHo>om .moum cam soosuom .oz Ioaom umom xuoz GOHumuo hon hon
xuos .exm mo oaHs oaHH IHouo<
HouoH Hmuoa HmuoH

«mHoona wananuIOoHHouusoo aH mums oHna uHanm

Ono Hmuuoonnumoa you Emumoua msHaHnuu oosmuapno huHmnounHIans .aoHumuaOquonm .xoosIm Ounvsmum .4m oHan

6O

.amuwoua onu ou hHunouommHO

Osoamou ou mouoomxo on OHnoo mHnaHnm mo come no msHmuum nonuo .moaHm> omega nH newsman oommmm ou mom: on coo

mason consume oaHu no .munon mo Hanan: .unoo Hon mHOHuHumaou .mBHu umou use aH msoHumuouH< .m3o03 N HmsHm

onu wnHunp poann no me no mouoom 33m use mmm o>ms vHaonm mHmaHsm zoom HHm mo Nnn umnu om posmHHnoumm onus

anemone men mo muHmsousH pom soHumunO any .amuwoua can mo msHsanon any um own mo when OmH new ON newsman
mama mHmaHsm HH< .aHmuum moHsmnIoawmuam mnu mo many ona maHm: postmoO mp3 amuwoum Oumvsmum mH3H

.HNOH .uoamnom pom mHHo3
x

 

 

OOO OOm OOAOO O o O H O m e ON mN OHAOO m.H ON mum

OOO OOO OOAOO O.q O.H O.m 3 ON mN OHAOO m.H OH Hue

OOO OOO OOHOO O.e O.H O.m q ON mN OHHOO m.H OH 3um

OOO OOO OOHOO O.q O.H O.m q ON mN OHAOO m.H mH HuN

ONO mow Oqumm m.m N.H O.m q mN ON OHHOO n.H OH zuH q
.HmH .mmm Aoom Aoom\uwv Amav AnHav muaom uaom Hoe Aoomv AommuaHav Aoomv .uH .33 .33

Aoomv mGOHu uaHaV Omoem 3oonm muaom mo mGOHuHu maHH oaHH oaHH mo «0
oaHH InHo>om .woum =33 soosuom .oz Ioaom umom 3uo3 aoHuouo man man
3uo3 .Oxm mo oaHH oaHH IHmoo<

Hoooe Hoooa Hoooa

A.v.uooov <m oHomH

APPENDIX B
(Table 10A)

61

.mhmu «N was we amm3umn mmmv om now cam
**

.maHanmB vacuum vmoHMHHUMm mmHmbm I hHao mmHmz
an

 

 

 

 

 

Nam NoN «Na NHH mom ma «muum NH

omN own mNN «HH «H _<mUIm NH

Nmm New mHN mNH mH <muum NH

zmu\mma.mumwo~ aha HNN zmu\mqm.q NNN hmv\mwo.q NNH mmm NH m «mwum NH

New «on «Hm NNH HH «main NH

emu ¢wm mmm NNH m dmulm NH

owN ¢N~ mum mNH wwu m N <munm NH

mma onN NHN mHH N <muum NH

Non mam NqN MMH m Houuaou

mm m . um com own mhm umw mmm % w . HMH m e Houuaou

n\ oq m Now mwm cum A vhmw\wwmmw HNN mv\ 00 q NNH * Hq N H Houuaoo

mom Ham qu wNH H Houuaou

unmaHummxm hmvlmn wdHuav m%mv mu mhmv NN mhmv me mmmv mH .u3 .02 .oz .uMHQ
aHmw hHHmv mwmum>m van waHdmmz umm awn

msouw mo .uB hvon.nmmz ammuo><
mquBHummxm wcHaasu nH.wmm: mum“ aoum aunt usmHma mvon mo humaasm .<OH anma

VITA

VITA

Robert Pierce Shields was born in Nashville, Tennessee, on June
11, 1932. He was educated in the public school system and was graduated
from Hillsboro High School, Nashville, Tennessee, in June 1950.

In September 1950, he enrolled in the pre-veterinary curriculum
at the University of Tennessee in Knoxville, Tennessee. In September
1952, he was admitted to the School of Veterinary Medicine of Auburn-
University at Auburn, Alabama. The degree of Doctor of Veterinary
Medicine was awarded to the author in June 1956.

From September 1956 to September 1959 he served as an instructor
in the Department of Pathology and Parasitology at the School of
Veterinary Medicine of Auburn University. He was awarded a Master of
Science degree in August of 1959. In September 1959 he accepted a
position as Veterinary Pathologist for the Ralston Purina Company of
St. Louis, Missouri.

In 1962 he was awarded a National Institutes of Health Fellowship
at the University of Arkansas Medical Center in Little Rock, Arkansas.
During the next four years he served as a resident in the Department of
Pathology and in 1966 he was awarded a Master of Science degree in.
Biochemistry from the University of Arkansas.

From 1962 to 1970, the author served as an Associate Professor at
Auburn University in the Department of Pathology and Parasitology in
the School of Veterinary Medicine and the Department of Animal Science
in the School of Agriculture.

62

63
In July 1970, he was awarded a Special Postdoctoral Fellowship by
the National Institutes of Health for a study of the relationship of
phosphogens to neuromuscular disease and the completion of the Doctor
of PhilosoPhy degree in Pathology.
The author is married to the former Shirley Clayton Salter of
Thomaston, Georgia, and has two children, Carolyn Carey, age eight,

and Robert Bradley, age six.

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