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The influence of thyroxine and desiccated thyroid preparation on the metabolic and heart rates of the rat

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The influence of thyroxine and desiccated thyroid preparation on the metabolic and heart rates of the rat

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Content THE INFLUENCE OF THYROXINE AND DESICCATED THYROID PREPARATION
ON THE METABOLIC AND HEART RATES OF THE RAT
A Thesis
Presented to
the Faculty of the Department of Biochemistry
The University of Southern California
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
by
George Dalton Kryder
June 1949
UMI Number: EP41294
All rights reserved
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a note will indicate the deletion.
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UMI EP41294
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This thesis, written by
, G - E Q E G - S : . . J D4LTm.MXDEE...........
under the guidance of Faculty Committee,
and approved by all its members, has been
presented to and accepted by the Council on
Graduate Study and Research in partial fu lfill
ment of the requirements for the degree of
MASTER OP SCIENCE
Date June 1949
FaculJiLjCommittee
j y y Chtnrmatt ^
/z&fcxf:.
...
TABLE OF CONTENTS
CHAPTER PAGE
I. STATEMENT OF THE PROBLEM . 1
II. HISTORY AND REVIEW OF THE LITERATURE ... 2
Discovery of the Colloid Substance ... 2
Elucidation of the Thyroid as an
Endocrine Gland • .................. 3
Isolation of Thyroxine ................ 3
Effects of Administration of Thyroid
Preparations ........................ 4
Summary of Literature Survey......... 10
III. METHODS............................... 11
Determination of the Heart Rate .... 11
Determination of the Metabolic Rate . • 13
Training the Animal............... . 18
Preparation of Thyroxine for Injection . 19
Preparation of Desiccated Thyroid
for Injection ................. 20
IV. PLAN OF THE EXPERIMENT......... 22
V. RESULTS ......... 23
VI. DISCUSSION ............. 30
VII. SUMMARY . . . ........... 38
VIII. BIBLIOGRAPHY ..................... 39
LIST OP TABLES
TABLE PAGE
I. Constants for the Calculation of Metabolic
Hate .......... 17
II. Experimental Results with their Standard Error .. 29
LIST OF FIGURES
FIGURE PAGE
1. A Typical Electrocardiogram Made by the Method
Described in the Text................... . 13
2. Schematic Representation of the Apparatus Used
for the Determination of Oxygen Consumption ....... 14
3. The Heart Rates of Two Different Animals Plotted
Against Time .................................... 20
4. Metabolic Rate Plotted Against the Various Dosages .. 24
5. Heart Rate Plotted Against the Various Dosages .... 25
6. The Heart Rate Plotted Against the Metabolic Rate ... 26
7. The Heart Rate Plotted Against the Metabolic Rate
with the Points Taken from the Average...... 27
CH&JPTER I
STATEMENT OP THE PROBLEM
It has long been known that heart rate and metabolism
is stimulated by thyroxine or other thyroid preparations.
There has been, however, some controversy as to whether
thyroxine is the only factor elaborated by the thyroid which
affects these processes. In particular, it has been
suggested that desiccated thyroid contains a factor other
than thyroxine which stimulates the heart rate.
This work is primarily concerned with an examination
of the question of the presence or absence of such an
independent cardiac stimulating factor in desiccated thyroid.
CHAPTER II
HISTORY AND REVIEW OP THE LITERATURE
Years before the thyroid was known to be an endocrine
gland, It was the subject of study by many investigators,
Cytological work done in 1836 by King showed the presence of
a colloid substance in the "cells” of the gland. This
material was described by him as being a viscoid fluid sub
ject to coagulation by both heat and alcohol. These
assertions gave the first hint as to the protein nature of
the colloid substance which is now called thyroglobulin.
Further work in the field of thyroid was stimulated greatly
some forty years later by the discovery that the thyroid
gland secreted a hormone. That the effects of thyroidectomy
could be offset by thyroid administration was shown by
Kocher in 1883. In a matter of years it was demonstrated
without a doubt that the gland was endocrine in nature and
the consequences of insufficient or excess secretion of the
thyroid hormone appeared to be an explanation of many
previously misunderstood symptoms.
The discovery of the endocrine nature of the gland
attracted a number of chemists who had previously been
interested in the colloid substance found in the thyroid
follicles. An important contribution in thyroid chemistry
was made by Baumann in 1896, when he discovered the high
concentration of iodine in thyroid tissue and noted its high
affinity for the colloid matter. Oswald, in 1899, extracted
this colloid from the thyroid and studied its chemical
properties. In the extract he found a protein, globulin in
nature, which was physiologically active in relieving the
symptoms of hypothyroidism. Following this discovery many
attempts were made to obtain more potent preparations of
thyroid substance from the colloid or whole gland. Acid
hydrolysis led to the purification of a substance called
iodothyrin by Baumann. Further hydrolysis by Oswald in 1911
enabled his isolation of a pure substance, the biologically
inactive amino acid diiodotyrosine. Alkaline hydrolysis,
though early abandoned because of its detrimental effect on
the activity of the hydrolysates, was begun again in 1915 by
Kendall. He eventually worked out a procedure which resulted .
in the isolation of a crystalline substance containing
sixty per cent iodine and possessing full physiologic
activity. It was Kendall who named this substance ’ 'thyroxine1 ' .
It remained for Harington, eleven years later,' to work out
the structure of the newly discovered substance. The-struc
ture postulated by this worker in 1926 was confirmed a year
later by synthesis, the synthetic material showing activity
equal to that of the natural form.
The discovery of thyroxine stimulated the study of the
effects of administration of thyroid gland and thyroxine.
From a biochemical viewpoint these have been manifold and
include a stimulation of glucose absorption and utilization,
an increase in gluconeogenesis, respiratory quotient, and
nitrogen excretion as well as effects on salt metabolism and
water balance• Clinically the effects noted are on growth
rates, maturation, heat production, the nervous system and
circulatory system. Numerous reports are to be found in the
literature in connection with these phenomena. However, the
primary concern of this paper is with the effect of thyroid
preparations on two of the aforementioned phenomena, heart
rate and metabolic rate.
The change of metabolic rate in thyroid disturbances
was first postulated by Friedrich Muller in 1893 on the
basis that there must be increased food oxidation when
hyperthyroid patients lost weight in spite of greatly
increased food intake. That this inference was correct was
shown two years later by Magnus-Levy, whose measurement of
the gas exchange led to widespread use of the method as a
diagnostic measure in people suspected of thyroid disorders.
The same principles which were laid down by these workers
are still being used today, although the methods have under
gone great modification.
With the advent of a convenient measuring stick such
as the basal metabolic rate, there arose the first dis
crepancy noted between the action of preparations of the
5
thyroid gland and that of thyroxine, which at that time was
generally considered to he the thyroid hormone. One of the
first indications of this nuance was described in work with
thyroxine polypeptide and dried whole thyroid gland. ' The
polypeptide, a product of proteolytic digestion of whole
thyroid gland, was shown by Salter et al. (1933) to have a
calorigenic activity equal to that of thyroxine when given
parenterally to human subjects. With this in mind, Means,
Salter and Lerman (1933) began experiments to show that the
calorigenic action of desiccated thyroid was to be deter
mined by its content of thyroxine iodine. The expected
result was not obtained. The dried thyroid, containing an
amount of chemically determined thyroxine, exerted a
strikingly greater calorigenic effect when fed to the human
test subjects, than did an equal amount of crystalline
thyroxine. Although Thompson (1934) has claimed that the
diiodotyrosine found in the thyroid has little if any calori
genic activity, Means et^ al.« at this time, came to the
conclusion that the calorigenic activity of whole thyroid is
related to its total organic.iodine, not its thyroxine iodine.
A volume of experimental evidence, while not neces
sarily confirming the conclusions reached by the above
workers, has nevertheless verified their results. Barnes
(1934), using thyroglobulin split with pepsin, showed that
the activities of the acid soluble and acid insoluble
portions of the hydrolyzed gland in increasing the metabolic
rate of rabbits,,were not equal, although the iodine content
of the two was approximately the same. Palmer and Leland
(1935), using the metabolic rate of guinea pigs as a gauge
of activity, came to the conclusion that the action of whole
thyroid gland- is determined by its thyroxine iodine, not its
total organic iodine content, if it is assumed that the
naturally occurring 1-isomer possesses twice the activity of
the d-isomer. Poster (1936) came to the same conclusions
when testing the metabolic rate and weight loss of guinea
pigs given thyroxine and dried whole gland. In the same
paper, evidence was offered to show that the 1-isomer of
thyroxine had an activity twice that of its enantiomorph.
Thompson (1935) gave evidence contradictory to the
above by showing that the activity of thyroid preparations
was greatly lowered by alkaline hydrolysis while the iodine
content remained the same. Meyer and Danow (1940), using
thyroidectomized rats whose basal metabolic rates were
raised to normal with the thyroid preparations, reached the
conclusion that the iodine content of thyroid was an
approximate guide for its calorigenic activity. A recent
paper by Prieden and Winzler (1948) compares the goiter-'
preventing activity of thyroxine, natural and synthetic
thyroproteins in thiouracil fed rats. Again it was noted
that the activity of the natural thyroproteins could not be
compared with, thyroxine on the basis of thyroxine iodine
content or spacial isomerism.
Correlation of the activity of thyroid materials with
respect to other symptoms noted in thyroid disease are to be
found mainly in connection with heart rate. Lewis and
McCachern (1931) isolated hearts from normal and hyperthy
roid rabbits. Comparison of rates showed that the hearts of
the hyperthyroid rabbits, whether treated with thyroxine or
desiccated thyroid, showed a relatively higher rate in every
case. Markowitz and Yater (1932) isolated hearts from chick
embryos and noted an increased pulsation rate when thyroxine
was added to the culture media.
Comparative influence of thyroxine and desiccated
thyroid preparations on the heart rate and metabolic rate
were done by Meyer and Yost (1939). Using thyroidectomized
rats, they administered dosages of their test materials
which would bring the metabolic level to normal. Comparison
of the heart rates at this common metabolic level gave
indication as to the effect of each material on the heart.
Thyroxine and complete thyroglobulin were found to have only
a slight heart effect, the rate being only half way to
normal when the metabolic rate was at that level. The
hydrolyzed products of the thyroglobulin, on the other hand,
were shown to have a greatly increased heart action; a
hyperthyroid-like tachycardia occurring when metabolic rates
were still below the normal range. The acid-insoluble
portion of the hydrolysate was much the stronger in this ef
fect. Dried whole thyroid was much the same in this respect
and seemed to have a very decided action on the heart.
A paper by Meyer and Danow (1940), using the same criteria
as mentioned previously, made a study of the thyroid
hydrolysates and thyroxine and their activity in coincidence
with iodine content. They assumed that the iodine content
of the thyroid preparations was an approximate guide for the
estimation of the metabolic effect, although no such corre
lation was found between iodine content and heart
stimulation. Thyroxine, according to them, has little
demonstrable proportionality on either physiologic effect.
In two different publications, Meyer and Thompson (1940) and
Meyer and Ferguson (1942) noted that blood extracts from
untreated toxic goiter contained a substance specific for
heart stimulation. A similar heart rate effect was shown by
feeding thyroids removed from human patients suffering from
toxic goiter which had been treated with iodine. Both of
the above experiments were done on thyroidectomized rats
whose basal metabolism was raised to normal with the partic
ular preparation used. Meyer concluded from these
experiments that the feeding of iodine caused an accumu
lation in the thyroid of a substance which accelerates the
heart beat in rats.
Evidence against such a hypothesis was offered by
LeBlond and Hoff (1944). They found that graded doses of
thyroxine increase the metabolic rate and heart rate in a
parallel fashion in thyroidectomized rats. The feeding of
dinitrophenol, while causing a considerable rise in oxygen
consumption, had little noticeable stimulating effect on the
heart. From this they concluded that the increase in heart
rate found in hyperthyroidism is not a result of general
metabolic stimulation, but can be attributed directly to
thyroxine.
Salter (1945) has stated that one effect of thyroxine
is to block the vagal inhibition of the heart. Hoffman
et al. (1947), working with rats, cats and rabbits, also
postulated a decrease in the sensitivity of the heart to
vagus stimulation. They pointed out that perfusates from
isolated hearts of hyperthyroid animals contained large
amounts of an epinephrine-like substance when small amounts
of acetyl choline were added to the perfusing solution.
Hearts of thyroidectomized-animals, on ..the other hand,
showed an intense depression with the same drug.
Work mentioned previously, done by Markowitz and
Yater (1932) with thyroxine addition to isolated chick heart
media, was interpreted by them to indicate direct muscle
stimulation by the material, since the forty-eight hour em
bryo has no cardiac innervation.
10
Summary of Literature Survey. The amount of work done
in connection with the action of thyroxine and thyroid
preparations has yielded conflicting results. Some authors
t
have stated that the activity of desiccated whole thyroid
or thyroglobulin is.proportional to the organic iodine con
tent, while others have shown that the best criteria is the
thyroxine iodine content. The relatively greater biological
activity of the natural 1-isomer of thyroxine has been sug
gested by some as being the logical basis for determining
activity.
The comparative action of thyroid or thyroxine on
heart and metabolic rates has been the subject of contro
versy as well. Postulation of a second thyroid hormone
specific for heart acceleration has been made. Thyroxine
has been demonstrated by others as the sole agent responsible
for tachycardia in hyperthyroidism. Methods of action of
this effect of thyroxine have been given as a result of a
decrease in vagan sensitivity while experiments on isolated
embryonic heart with no innervation have indicated that the
response to thyroxine is a direct action upon the cardiac
muscle cell.
CHAPTER III
METHODS
Determination of Heart Rate. The determination of
the heart rate in the rat has been accomplished by many
methods. One of the most satisfactory procedures and
probably the most consistent is by use of the electrocardio
graph. The most common electrode which has been used with
the rat is a needle placed under the skin. However, this
type of electrode did not seem advisable in the present work
for several reasons. It did not seem probable that a basal
heart rate could be obtained with such attachments. The use
of local anesthesia was contraindicated because of its pos
sible effects on the metabolism or heart rate. It therefore
remained to devise some sort of electrode which would be
sensitive enough to pick up the cardiac impulses without it
being too cumbersome or uncomfortable for the rat. The
electrodes and leads decided upon were made of small pieces
of tin about half an inch square, on which was soldered fine
insulated copper wire. These contacts were placed upon each
side of the animal immediately behind the forelegs on an
area which had been depiliated and rubbed with electrode
paste. Adhesive tape was then wrapped once about the body
and over the metal squares to hold them in place and to
insulate them from the surrounding metal of the cage.
12
The normal trained animal reacted favorably to this method
and its heart rate, after a reasonable waiting period, was
at levels corresponding to the lowest averages found in the
literature.
The accommodation of eight animals by the metabolic
apparatus and the desire for simultaneous measurement of both
heart and metabolic rates, necessitated the development of a
means of drawing the leads from each of the eight metabolic
chambers. This was accomplished by running the wires through
the air outlet tube of each bottle. The tubes were closed
by means of clamps and the openings at either end sealed
with wax to insure against possible leakage. All of the
lead wires were sent to a control box containing an eight
way switch. Attachment of the leads from the electrocardio
graph to this control center made possible the selection of
any one of the eight cages at random. By this method, it
was possible to determine the heart rate of the animal while
measurements of the oxygen consumption were in progress.
Figure 1, below, is an example of the electrocardio
graphic determinations of heart rate which were made by the
method described above.
Figure 1. A typical electrocardiogram
made by the method described in the text*
Rate is 322 beats per minute*
Determination of the Metabolic Rate. A method
devised by Mason and Winzler was used in the determination
of the oxygen consumption of the test animals. The apparatus
is shown in Figure 2. The principal involved is a measure
ment of the rate of fall of pressure using a fluid manometer
in a closed system to give a figure which can be used in
*
calculating the basal oxygen consumption of the animal.
In practice, the following procedure is carried out:
The first step is to place the rat in a wire cage just large
enough to fit into the mouth of the bottle which is used as
the metabolic chamber. This cage rests on a low plastic
stand, under which is placed 25 ml. of thirty-five per cent
sodium hydroxide. With the cage in place, the lid of the
9
49
16
59^84399
5954
52
93
19359
V'ft* T o e l e c t r o c a r d i o g r a p h
W ater.
M a n o m e t e r
O utlet
Figure 2. Schematic representation of the apparatus
used for the determination of oxygen consumption.
jar is screwed on tightly, the seal being made secure with
vacuum grease. The bottle and the stand upon which it rests
are then lowered into a water bath maintained at a constant
temperature of twenty-eight degrees centigrade. The water
around the chamber is kept in.-circulation' by a stream of air
under the surface. Oxygen is blown into the system under
sufficient pressure to raise the level of the water
manometer to the 65 cm. mark. Turning the stopcock to the
appropriate position cuts off the supply of oxygen and leaves
only the metabolic chamber and the manometer connected. The
carbon dioxide expired by the animal is rapidly absorbed by
the sodium hydroxide and any change noted in the level of
the manometer can be attributed solely to the consumption of
oxygen in the system.
Calibration of the apparatus as done by Mason and
Winzler gave a series of constants which facilitated the
calculation of the metabolic rate for any animal of any
weight. Fuhrman et al. (1946) devised a similar apparatus
whose constants were calculated in the same manner. The
metabolic rate is obtained by substitution in the following
formula:
Metabolic Rate = (Vg - W) KR
W
in which
Vg is the gas volume of the chamber containing CO2
absorbent and platform
16
W is the weight of the animal in grams, assuming a
specific gravity of 1.0
27S
K = f c where P is the normal pressure of the
*o
manometric fluid in centimeters of water
* v ■
*
R is the change in manometric reading in millimeters
per five minutes.
The combination of the above figures into the con
venient form of a constant enabled the derivation of a more
simple formula:
o Metabolic Rate - (const.)(change in manometer in mm. / 5 min.)
weight of the animal
The resulting figure represents the cubic centimeters
of oxygen consumed per one hundred grams per hour.
A list of the constants used for the determination of
metabolic rate is given in Table I.
To obtain the figure for the rate of fall of the manom
eter, it was found that a minimum of six five-minute readings
were necessary for accurate metabolic rates. The average of
the two lowest readings during that period could then be,
considered as the basal rate of oxygen consumption to be sub
stituted into the formula. Correction of this figure due to
changes in barometric pressure or water temperature during
the determinations was. made possible by the incorporation
TABLE I
17
Animal Weight Constant* Constant*
(grams) (Odd Numbered Cages) (Even Numbered Cages)
100 186.3 188.4
105 185.8 187.9
110 185.2 187.5
115 184.7 186.9
120 184.2 186.4
125 183.6 185.8
130 183.1 185.4
135 182.5 184.9
140 182.0 184.4
145 181.4 184.0
150 180.9 183.4
155 180.4 183.1
160 179.9 182.4
165 179.3 182.0
170 178.8 181.4
175 178.3 180.9
180 177.8 180.3
185 177.4 179.8
190 176.7 179.3
195 176.2 178.8
200 175.7 178.3
205 175.2 177.8
210 174.6 177.3
215 174.1 176.6
220 173.6 176.2
225 173.1 175.7
230 172.6 175.2
235
172.0 174.6
240 171.5 174.1
245 171.0 173.6
250
170.5 173.1
255
169.9
172.6
260
169.4
172.0
265
168.9
171.5
270
168.4 171.0
275 167.9
170.5
280 167.3
169.9
285 166.8
169.4
290 166.3
168.8
295
165.8
168.4
300 165.3
167.9
305 164.8
167.4
310
164.3
166.9
315
163.8
166.4
320 163.3
165.9
*• Constants for odd and even numbered cages differ due to
the extra volume caused by longer hose attachment to the
oxygen supply.
18
into the system of a thermobarometer. Readings of this
gauge were taken every five minutes, along with the other
readings and the necessary corrections made.
Training the Animal. The necessity of training
animals in preparation for measurements such as were contem
plated is quite well known. However, the degree of this
t
preliminary period again varies in literature reports. For
this reason, it was decided to determine the minimum
training necessary for the strain of rats used and for the
particular conditions under which they would be studied.
Exact duplication of the experimental procedure was
carried out on several test animals until they showed what
appeared to be basal levels of both heart rate and metabolic
rate on two consecutive days. A minimum of three one-hour
experimental runs on three consecutive days was found to
thoroughly train the rats. A rat, trained in this manner,
could be expected to give basal levels for both determina
tions, if used for experimentation within a week. Ideally,
the three days prior to the actual determinations are
preferable.
The frequent handling during the injection period,
plus the indoctrination training, accustomed the animals to
the routine sufficiently well so that only half an hour was
required in the metabolic chamber before minimum rates were
obtained. The necessity for this waiting period is well
19
shown in Figure 3, which represents the heart rate at five-
minute intervals for two normal, trained animals. Similar
results as to time and metabolic rate were obtained.
Preparation of Material for Injection.
1. Thyroxine
Roche synthetic DL-thyroxine was weighed to the
nearest tenth of a milligram and placed in a volumetric
flask containing hot distilled water made slightly acid with
dilute hydrochloric acid. The resulting suspension was made
alkaline with sodium hydroxide to dissolve the material, and
was then brought back to the isoelectric point where a fine
precipitate was observed. The thyroxine was finally dis
solved by adding sodium hydroxide to make a final pH of
about 7.4 as shown by nitrazine indicator paper. The
solution was then made to the mark and preserved in a brown
bottle kept refrigerated when not in use.
Twenty milligrams of the synthetic compound In one
hundred milliliters of water gave a solution containing two
hundred micrograms per milliliter.
2. Desiccated Thyroid
The desiccated thyroid powder was found to be highly
insoluble in solutions which would be compatible to intra-
peritoneal injection. However, it was found that a satis
factory injection mixture could be obtained by suspending
' the material in a finely divided state.
20
N o r m a l T r a i n e d A n im a l s
3 7 5
/O
x o
Figure 3. The heart rates of two. different animals
are shown plotted against time. This graph indicates the
importance of the preliminary waiting period before actual
rate determinations are begun.
21
This was done by placing the carefully weighed powder
in a mortar and grinding while adding small amounts of one
per cent sodium bicarbonate until a total of ten milliliters
had been added. The mixture was then transferred quantita
tively to a volumetric flask and made to the mark with
0.5 per cent chlorobutanol. The desiccated thyroid powder
was known to contain 0.26 per cent thyroxine by chemical
determination. One gram of the material was suspended in
one hundred milliliters of the solution to make an injection
solution containing twenty-six micrograms per milliliter.
CHAPTER IV
PLAN OF THE EXPERIMENT
Seven'groups of eight male rats of the University ofi
Southern California strain were used in the experiments.
Of these seven groups, one remained untreated to he U3ed as
a control. The rest of the animals were injected intraperi-
toneally with thyroxine or desiccated thyroid in three
predetermined dosage levels designed to increase the
metabolic rate 10, 90 and 175 per cent above the normal.
For the desiccated thyroid the level was found to be 3.75,
7.5 and 20 micrograms of thyroxine per one hundred grams.
Amounts of 30, 60 and 150 micrograms per one hundred grams
of dl-thyroxine were given. The thyroxine given as desic
cated thyroid was determined chemically from the iodine
content of the dried substance.
Each of the rats was injected intraperitoneally for
ten days, the training period beginning on the fifth day and
continuing an hour each day until the actual experiment was
begun.. The determinations of heart and metabolic rates
were done on the eighth, ninth and tenth day3, to give a
total of three separate readings on each animal.
CHAPTER V
RESULTS
The data accumulated during the course of the exper
iments can best be presented in graphic form. The figures
which are used in the graphs are given in Table II.
Figures 4 and 6 represent the metabolic and heart
rates, plotted against the dosage of thyroxine administered,
as such, or as chemically determined in the thyroid powder.
These graphs verify the results of many previous investiga
tors who have noted that the action of desiccated thyroid
gland is greater than can be accounted for by its content of
thyroxine. It can also be noted that the effect of the thy
roid material is of the same magnitude on both heart rate and
oxygen consumption. This can better be seen in Figures 6 and
7, in which the two effects are plotted against one another.
Figure & represents the plot of each single determination
made and does not take into consideration the thyroxine or
desiccated thyroid dosage given to the animals. The large
number of points in this graph have been consolidated in
' . '
Figure 7 as averages for all determinations at a given
injection level of the thyroid and thyroxine. It can be
seen from this graph that the points fall approximately in a
single straight line. In interpreting the data, this be
comes very significant since it would appear that, regardless
M etabolic R ate f e e Ot / / o o ^ . J h r .)
24
2oo
200
/go
160
IS O
^ = D e s ic c a t e d Th y r o id
0 = Th y r o x in e
120
n o
to o
Figure 4. Graphic representation of the data presented
in Table II, showing metabolic rate plotted against the
various dosages.
H ear? Rate (Beats /minute)
25
420
4/0
400
340
380
370
360
D E S IC C A T E D THYRO ID
o= T h y r o x / n e
JO
Figure 5. Graphic representation of the data presented
in Table II, showing the heart rate plotted against the
various dosages.
26
*20
210
^200
s
r 190
o
"^1/80
O
so^/ro
Ui
& 160
$
!J /s o
©
i / «
0
K
Ui A
130
b O G
720
O Q
$© o T
I/O
O© A
O
A
O
A
AA © 0
* A g o
o
A
©
* * -A ¥ @°
A
A
*> ° g- A G A
A O Q
© °A A °
300 3/0 3» 330 340 350 3Ao 3T0 38o 3H e a r t Ra t e ( bea tsfm H v re )
+ - v , 4 . Represents the heart rate plotted against
the metabolic rate.^ Dosage levels were neglected. Circles
designate dl~thyroxine; triangles represent thyroxine as
desiccated thyroid.
METABOLIC Rate ( c c Okj lO O C jm j h r)
27
n o
too
A
2/0
ZOO
t?o
/80 - O
/T O
160
IS O
140
130
A= O e s /c c A T E O Th y Ro io
! * ° \ ■ m O s T h y r o x in e
A
s C o n t r o l a n im a l s
300 3/0 3X0 330 340 3SO 360 370 380 390 400 4io 4X0 430 440 4S0
H e a r t R a t e ( b e a t s J m in u te)
Figure 7. Heart rate plotted against metabolic rate
Each point represents the average rates for a given dosage
level. *
28
of the type of thyroid preparation administered, the heart
rate and metabolic rate are increased in a parallel fashion.
Referring again to Figure 6, it can be seen that a
rather large spread of single points was obtained by
plotting the data in this manner. This variation noted in'
the effect of the preparations on metabolic and heart rates
suggested that the observed rates could not be correlated
with one.another. The large number of determinations, how
ever, made possible a statistical treatment, and by this
means it was shown that the correlation coefficient was of a
magnitude that rendered the data statistically significant.
29
TABLE II
No. of Heart Rate Metabolic Rate
Dosage Deter*- (beats per (cc. Og per
(gamma) initiations minute) 100 gm. per h r. )
Controls
— — “ 310 ± 21.2* 117
+
13.2
30 23 329 d t 22.5 134 ± 13.8
dl-thyroxine 60
18 352 ± 22.7 153 d b 22.1
150 16 395 ±
23.9 179 ± 22.9
Thyroxine as 3.25 14 349 ± 22.9 136 ± 14.5
Desiccated 7.50 19 385 ± 43.6 170 ±
18.1
Thyroid 20.0
13 405 d b 38.2 207
+
25.6
Standard Errors S.E. = d2
m(m-l)
CHAPTER VI
DISCUSSION
The most striking result that can.he seen from the
data is the great difference in quantitative response between
crystalline thyroxine and thyroxine as desiccated thyroid
gland. That this phenomena would occur was indicated by pre
liminary work done to determine the dosage levels of the test
substances. For example, thirty micrograms of dl-thyroxine
gave approximately the same metabolic and heart rate
increases as did 3.25 micrograms of thyroxine in the form of
desiccated thyroid. The ratio of one dosage to the other,
nine to one at these levels, appears to increase with the
dosage in the manner indicated in the graphs. The same dif
ferences, although not as pronounced, were observed in a
large number of cases by many workers. Recent quantitative
data has been presented by Frieden and Winzler (1948) in
experiments with the goiter prevention method in rats*
t
These workers observed a factor of two in comparing desic
cated thyroid and thyroxine as the active 1-form. Hamilton,
Albert and' Power (1948) obtained the same factor when com
paring the calorigenic effects of these two materials upon
tadpoles. The present work shows an even greater differe’ nce'
at the higher test levels used.
31
The explanations proposed for this apparent discrep
ancy of action between the two thyroid materials are
numerous•
As previously mentioned, Means et al. (1933) attempted
a correlation between total iodine content and biological
activity. This possibility has not been borne out by later
work. A much nearer approximation for thyroid activity has
been based on the assumption that the thyroxine of the thy
roid occurs only as the 1-isomer. This was first postulated
by Palmer and Leland (1935) in work with the oxygen consump
tion of albino rats. They assumed that the dosage of
crystalline thyroxine in the racemic form would be only one
half as effective as a similar quantity of the 1-form.
While this undoubtedly contributes to the activity of thyroid
which is in excess of its calculated thyroxine content, it
still does not offer a full explanation.
At a first approximation, it may seem possible, as
has been suggested in connection with heart rate, that the
thyroid contains another hormone whose action is identical
with that of thyroxine. A closer examination, however, would
make this seem unlikely. Harrington (1939) and Frieden and
Winzler (1948) have postulated a skeleton structure which
illustrates the maximal deviation possible from the thyroxine •
structure while the thyroxine-like activity was retained.
This structure retains the diphenyl ether linkage, halogens
at the 3, 5 positions, a functional group in the para posi
tion and a hydroxyl group at either ortho prime or para
prime. With this in mind, it seems unlikely that there is
such a compound or one if its variations in the thyroid
which has not been isolated and shown to have activity of the
same magnitude as thyroxine. It is still more difficult to
imagine a compound having physiological action identical to
that of thyroxine while having a structure completely
different.
A second possibility which might explain the discrep
ancy in action of thyroid gland in regard to thyroxine
content may be the conversion of inactive compounds present
in dried thyroid into physiologically active materials in
the test animal. The conversion of diiodotyrosine, known to
be present in thyroid, has been observed in vitro and
in vivo. Harington (1926) and Harington and Barger (1927)
have shown that the synthesis of thyroxine from diiodotyro
sine is possible by an oxidation process which removes the
alanine side chain from one of the molecules and causes a
coupling to form thyroxine. Thyronine, the uniodinated form
of thyroxine, as well as 5,5-diiodothyronine and other pos
sible precursors of thyroxine are administered as an integral
part of desiccated thyroid. The conversion of any of these
to thyroxine, or at least to an active thyroxine-like com
pound seems possible. However, it has beeri shown that the
33
administration of these various possible precursors does not
give rise to a significant metabolic or heart stimulation
except in rather large doses, amounts of which are not found
in the thyroid, LeBlond and Hoff (1944) measured the meta
bolic and heart stimulation of rats by diiodotyrosine,
thyronine, inorganic iodide and thyroxine. Their data showed
that only thyroxine had a marked effect on the heart and
metabolic rate.
Rapport and Canzanelli (1931) compared the calori-
genic activity of these same compounds on guinea pigs.
Their interpretation of the data led them to the same con
clusions reached by LeBlond at al.
The possibility that for activity thyroxine requires
its incorporation into a peptide has been suggested by
Harington (1937)., Means and Salter (1935) obtained such a
thyroxine peptide by subjecting thyroid to proteolytic
digestion. Their observations on human patients led them to
the conclusion that on an iodine basis, thyroxine is fully
as active as was the polypeptide. While the possibility
remains that the circulating hormone may be a peptide, there
is no evidence to indicate that this form of linkage
increases the activity of the compound. Kendall (1935) has
suggested that thyroxine in peptide linkage prevents its
escaping from the body.
34
The incorporation of thyroxine into a large protein
molecule, as is the case in thyroglobulin, could possibly be
an explanation for its observed greater activity in that
form as opposed to crystalline thyroxine. That the circu
lating hormone could not be thyroglobulin, however, was
proved rather conclusively by Lerman (1940). He produced
an antiserum to thyroglobulin in rabbits. By testing the
serum of normal and hyperthyroid human patients with the rab
bit antiserum, he was able to show that thyroglobulin was
not present to any extent in the circulation of these
people.
Tarug and Chaikoff (1948) have recently completed
work which indicated the "association” of thyroxine with the
plasma proteins and that this was the manner in which the
compound was transported in the blood stream. They made this
postulation on the basis of studies with radioactive iodine
in which they found that the iodine of normal rat plasma is
almost completely extractable with N-butyl alcohol at room
temperature. Addition of crystalline thyroxine to the plasma
of these rats and repeated crystallization of the added
thyroxine did not lower the radioactivity of the crystallized
material. Prom this data, it can be intimated that thyroxine,
as the thyroid hormone, is released from the protein at the
site of its action.
On this assumption that thyroxine is the thyroid
hormone, another explanation for the apparent excessive action
of whole thyroid has been offered. It is known that crystal
line thyroxine, due to its high insolubility, is absorbed
erratically from the intestine. In the intestine and in
vitro thyroxine which is incorporated with protein is ab
sorbed at a more rapid rate. It would appear from this that
the thyroxine of the desiccated thyroid preparations, which
is assumed to occur in some peptide or other protein combi
nation, is absorbed more easily into the circulation. While
this hypothesis has never been proved, there is some evidence
that this would account for the unexplained potency of thy
roid gland in stimulating metabolic rate.
The data presented in this paper shows that the heart
stimulating effect of desiccated thyroid is approximately,
the same as on the metabolic rate. On the assumption that
thyroxine per se is the thyroid hormone, the same arguments
can be used here that were mentioned as possible explanations
for the excessive metabolic stimulation.
However, in this respect, there have been a number of
other theories advanced to explain the observed phenomena.
The bulk of the work done in correlating the metabolic and
heart rates in hyperthyroidism has been published by Meyer
et al. (1940, 1941, 1939, 1942). In his reports he has attri-
4
buted the cardiac stimulation to a second thyroid factor.
36
While his conclusions can be understood in view of the data
m
obtained, the main consideration has been the evaluation of
the methods used in accumulating the results, A very large
possible error was introduced into his work by separating
determinations of heart, rates and oxygen consumption. It is
quite well known that the heart rate of the rat varies
greatly within short periods. The rats used by this group
were manually held while the heart rate was being determined
by palpitation. Experience during preliminary work for this
paper indicated that the handling of the test animals during
heart rate studies caused unpredictable increases in rate.
It might be argued that the heart rates are based on relative
values, but there are a great number of outside factors which
might influence the heart rate and it seems unlikely that the
heart acceleration caused by picking up the rat could be
considered constant.
Work mentioned previously has resulted in direct
opposition to the conclusions reached by Meyer. Some of
this experimental evidence has been interpreted freely to
mean that thyroxine is responsible for the cardiac stimula
tion, in hyperthyroidism. For example, Fishburn and
Cunningham (1938) claimed that thyroxine, as the active prin
ciple of the thyroid gland, was responsible for heart rate
regulation in the rat, since they were able to maintain nor
mal heart rates in thyroidectomized animals by the
37
administration of thyroxine. There has been, however, more
conclusive data offered which would verify this opinion. In
their experiments with thyroid and dinitrophenol, LeBlond and
Hoff (1944) gave evidence which was discussed previously and
which would indicate this to he true.
The continued higher pulsation rates of'explanted
cardiac tissue from animals which had been treated with
either thyroxine or thyroid would again indicate that thyrox
ine is the agent responsible for cardiac stimulation in
hyperthyroidism.
In the present work, while it was found that thyroid
had a greater action than thyroxine on a common basis, it
can be noted that at a similar metabolic level the corres-
c *
ponding heart rate was approximately the same, regardless of
the type of material administered. The presence of a second
material in the thyroid gland other than thyroxine with
specific action on the heart would have resulted in an obvious
deviation from the data obtained. Conversely, if thyroxine
had been more active in exciting heart rate than in increasing
metabolic rate, it would likewise have been reflected in the
data.
It can be stated, therefore, that the data presented
in this paper indicates that the heart effects noted in
hyperthyroidism are due solely to the action of thyroxine.
SUMMARY
The problem about which this paper is concerned is
the relationship between thyroxine and desiccated thyroid in
stimulating the heart rate of animals made hyperthyroid with
these substances.
A review of the literature shows many conflicting
viewpoints as to the cause of the observed tachycardia.
Some claim that thyroxine is the causative agent, while
others maintain the presence of an auxiliary thyroid substance
specific for heart stimulation. This work was undertaken to
show the presence or absence of such a secondary thyroid hor
mone •
Methods were developed which enabled the determination
of heart rate and metabolic rate simultaneously in rats
which had been injected with various doses of thyroxine and
desiccated thyroid.
The data obtained during the course of the experiment
would seem to show that the effects of the two materials
were parallel in their action of the heart when compared at
a common metabolic level. Prom this it was concluded that
it is thyroxine which is responsible for the tachycardia
noted in hyperthyroidism.
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oi S au th am California UMlI

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Creator Kryder, George Dalton (author)

Core Title The influence of thyroxine and desiccated thyroid preparation on the metabolic and heart rates of the rat

Degree Master of Science

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