A STUDY O F THYROID ACTIVITY IN DAIRY GOATS R ELA TI NG TO AGE,
LACTATION, P R E G N A N C Y AND SEASON O F THE YEAR

E U G E N E E. FLAMBOE

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies of
Michi ga n State University of Agriculture and
A p p l i e d Science in partial fulfillment of
the requirements for the degree of
DOCTOR OF P HILOSOPHY
Department of Physiology and Pharm ac olo gy

Year
Appio ve d

1958

Eugene E. Flamboe
1

ABS T R A C T

The technique

for measuring individual thyroid secretion

rates using 1-131 and 1-thyroxine injections
419,

(J. Animal Sci.,

14:

1955) was applied to dairy goats for the purpose of determining

thyroid activity in relation to age,
season.

lactation,

preg na ncy and

The comparatively rapid output rate of this species

(tVz = 7.3 + 0.35 days) without thiouracil treatment permits rather
pre cis e determination of the amount of 1-thyroxine needed daily to
prevent

1-131 output.

secretion rate.

This endpoint was used to measure thyroid

The mean thyroid secretion of pregnant goats,

cluding 2, aged and 3 two-year-old animals,

in­

during February was

0.262 _+ 0.026 mg. per 100 pounds body weight daily.

A similar

group of non-pregnant goats h ad a mean secretion rate value of
0.277 _¥ 0.05 mg.

The mean thyroid secretion rate of the young

animals in this experiment was 0.325 _+ 0.019 mg.

This is s i g n i f i ­

cantly higher than the mean value 0.187 _+ 0.016 mg.
aged group.

found in the

The mean thyroid secretion rates of a group of open

non-lactating goats including a group of 5 aged and a group of 4
two-year-old animals were 0.199 _+ 0.024 mg., and 0.278
respectively for the month of May.

In July the mean values for

this same group of animals were 0.099 _+ 0.02 mg.
mg.

respectively.

and 0.177 j- 0.013

In August these values were 0.197 _+ 0.033 mg.

and 0.213 _+ 0.017 mg.
_+ 0.03 mg.

+_ 0.036 mg.

respectively for the same groups,

and 0.336 _+ 0.018 mg.

of goats in October.

and 0.243

respectively for a similar group

In comparing these data the difference b e ­

tween the 2 age groups was sta tistically significant during February,
July and October.

The values in July were also significantly lower

Eugene E. Flamboe
2

ABS TR ACT

for both age groups than at any other time of the year.

The mean

thyroid secretion rate of a group of 4 aged and a group of 7 you ng
lactating goats during May were respectively 0.188 _+ 0.053 mg.
0.263 _f 0.030 mg.

During July the mean thyroid secretion rates of

the aged and young groups were 0.05 +_ 0.016 mg.
mg.

respectively.

were 0.121

and

In August these values

+■ 0.040 mg,

nificant difference was

for aged and young goats

and 0.177 _t_ 0.040 mg.

respectively.

found between the 2 age groups

of the three experiments.
groupss was significant.

and 0.07 _+ 0.0074

However,

the seasonal

No s i g ­

in either

decline

in both

In July the mean secretion rate of the

no n-lactating animal was significantly higher than that of lactating
goats.
Although thiourecil
iodine output rate,

treatment increased the thyroidal

the mean thyroid secretion rate was net s i g n i ­

ficantly different in thiouracilized than in normal

goats.

Although no correlation with thyroid secretion rate
existed,

except for the month of August,

there was a definitely

increased thyroidal uptake of iodine during the summer months.

The

thyroids of non-lac ta tin g animals also took up more iodine than
lactators.

No correlation was found between thyroid secretion

rate as determined by the substitution me thod and output half-time
except during the month of May.

A STU DY OF THYROID ACT I V I T Y IN DAIRY GOATS REL A T I N G TO AGE,
LACTATION,

PRE GNA NC Y AND SEASON OF THE YEAR

by
EU GENE E. FLAMBOE

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michi ga n State University of Agriculture and
Applied Science in partial fulfillment of
the re qu irements for the degree of

DO C T O R OF PHILOSOPHY
Department of Physiology and Pharmacology

1958

ProQuest Number: 10008614

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Dedicated

to

my wife, Louise

ACKNOWLEDGMENTS

The author wishes to express his sincere gratitude to
Doctor E. P. Reineke

for his wise council and guidance in conducting

this investigation and for his helpful suggestions in the p r e p a r a ­
tion of this manuscript.
Special thanks are due to Doctor B. V. Alfredson

for the

use of the facilities and equipment of the Department of Physiology
and Pharmacology and to the Michigan State University Agricultural
Experiment Station for financial help in this project.
The author is also indebted to his fellow graduate students
for their technical assistance and helpful
this study and to Mr.

criticisms throughout

Howard Hardy for the building and maintenance

of much of the equipment used in this work.
Grateful appreciation is extended to Mr. Arthur B u t t e r ­
field for his cooperation in the management and care of the e x p e r i ­
mental animals.

TABLE OF CONTENTS
Page
I NTR ODUCTION

........................................................

1

SURVEY OF LITER ATU RE
. . ...........................................
3
A. Ea r l y Investigations
.............................
3
B. Formation of Thyroid Hormone
...............................
4
5
1. Iodide Accumulation by the Thyroid Gland ...............
2. Org an ification of Iodine
..............................
5
3. Proteolysis of Thyroglobulin
.........................
10
C.
Hormonal Transport and Utilization
...................... . 11
1. Transport
................................................. 11
13
2. U t i l i z a t i o n ................................
D. Factors Influencing Thyroid Function
. ...................
15
1. P ituitary Thyrotropic Hormone
. . . . .
..............
15
2. T e m p e r a t u r e ................................................. 16
3. Season and Light
................................... 17
4. Thyroid-Gonad Relationship
............................
18
5. Thyroid-Adrenal Relationship
. . . .
18
6. A ntithyroid Compounds
............................... 20
E. Economic Aspects of the Thyroid
............................. 20
1. Effect of Thyroid on Growth
............................ 20
2. Effect of Thyroid on R e p r o d u c t i o n ........................ 21
3. Effect of Thyroid on Milk Production
.
22
F. Methods of Determining Thyroid Activity
.................
25
1. Oxygen Consumption and B . M . R ................................ 25
2. Body Weight and Replacement Theory
..................... 25
3. Goitrogenic Method
.....................................
26
4. PBI Method
................................................. 27
5. Thyroid Studies with Radioactive Iodine
..............
28
MATERIALS A ND M E T H O D S ..................... . .
................... 33
A. Experimental Animals
.................................... 33
....................................................... 33
B. Apparatus
C. Thyroxine
....................................................... 36
D.
Radioactive I o d i n e ....................................... . . 36
E. T h i o u r a c i l ....................................................... 37
F. M et ho d
......................................................... 37
E XPE RIMENTAL P R O CED URE AND RESULTS
Experiment 1
. . .
Experiment 2
Expe rim en t 3
Experiment 4
Experiment 5
G E N E R A L DISCUSSION

.....................

S U MMA RY AND C ONCLUSIONS
BIBLIOGRAPHY
APPENDIX

..............................

............................................

43
43
44
50
51
53
72
87
91
104

LIST OF TABLES

TAB LE
I,

II.

III.

IV.

V.

VI.

VII.

VIII.

PAGE
The values obtained for thyroid secretion rates,
output half-time, 48 hour percent uptake and c o r r e l a ­
tion coefficients (r) for individual pregnant goats
. . .

45

The values obtained for thyroid secretion rates, o u t ­
put half-time, 48 hour percent uptake and correlation
coefficients (r) for individual non-pregnant goats
. . .

46

The values obtained for thyroid secretion rates, o u t ­
put half-time, 48 hour percent uptake and correlation
..............
coefficients (r) for individual aged goats

47

The values obtained for thyroid secretion rates, o u t ­
put half-time, 48 hour percent uptake and correlation
coefficients (r) for individual young goats
...............

48

A s ummary of the mean values obtained for output halftimes, 48 hour percent uptake, peak percent uptake and
thyroid secretion rates for all groups at various times
of the year
............................................

66-67

The t values and degrees of freedom obtained when mean
secretion rates of various groups of animals are c o m ­
pared with regard to season and a g e ............
68-69
The t values and degrees of freedom obtained when mean
secretion rates of various groups of animals are
com­
pared with regard to age, season and lactation
.. .

70

The values obtained for coefficients of correlation (r)
when 48 hour percent uptake and output half-time is
compared with thyroid secretion rate
................

71

APPENDIX TABLE
A.

B.

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation
coefficients (r) for aged lactating g o a t s ..............
The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation
coefficients (r) for individual young lactating goats

107

.

108

APPENDIX TABLE
C.

D.

E.

F.

G.

H.

J.

K

L

M.

N.

0.

PAGE

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual aged no n-lactating goats .

1G9

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual young no n-lactating goats

110

The values obtained for thyroid secretion r a t e s , output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual aged lactating goats
. .

Ill

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual young lactating goats
. .

112

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual aged n on -lactating goats .

113

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual young non-lactating goats.

114

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual aged lactating goats . . .

115

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual young lactating goats
. .

116

The values obtained for thyroid secretion rates, output
half-time, 48 hour
percent uptake and correlation c o ­
efficients
(r) for individual aged non-lactating goats

.

117

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual young non-lactating goats.

118

The values obtained for thyroid secretion rates, output
half-time
48 hour percent uptake and correlation c o ­
efficients
(r) for individual immature goats ............

119

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients
(r) for individuc.l aged goats
. . . . . . .

120

APPENDIX TABLE
P

Q.

R.

PAGE

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual young goats

121

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual young thiouracilized goats

122

The values obtained for thyroid secretion rates, output
half-time, 48 hour percent uptake and correlation c o ­
efficients (r) for individual immature goats
. . .

123

LIST OF FIGURES

FIG UR E
1.

2.

3.

4.

5.

6.

Page
Apparatus and radiation counting equipment used in
determining thyroidal radioactivity in the goat

. . . .

35

A graphic illustration of thyroidal 1-131 uptake and
output, and the .influence of thyroxine injections in
graded doses on the output portion of the curve . . . .

39

Graphic illustration of the dose response relationship
between thyroidal 1-131 output and thyroxine injections,
and the extrapolation of the data in order to obtain
the estimated thyroid secretion rate
....................

42

Graphic illustration of thyroidal 1-131 uptake and o u t ­
put for both normal and thiouracilized animals and the
effect of thyroxine injections in graded doses on the
........................
output portion of these curves

56

A graphic summary comparing the mean thyroid secretion
rate data obtained from all five experiments for the
various groups of non-lactating animals at various
times of the year

59

A graphic summary comparing the mean thyroid secretion
rate data obtained at various times of the year for
different age groups of lactating and non-lactating
animals

61

1

INTRODUCTION

Recent advances

in the field of e nd ocrinology have made it

increasingly evident that an interplay of many hormones
in various physiological processes.
hormone

is involved

The notion that one particular

is concerned with the adjustment of one particular process

is no longer compatible with present

findings.

The thyroid gland

presents a good example of this since it appears to play a role in
conjunction with other hormones in the physiological processes of
growth,
turn,

lactation,

reproduction and various other functions.

In

the activity of the thyroid gland is profoundly affected by

other endocrine substances as well as external and internal changes
of environment.
In the p a s t , investigators in the field of endocrinology have
had their efforts hampered by the fact that necessary hormone p r e p a r ­
ations were expensive,

the supply was limited and moreover they were

not very uniformly standardized.

However,

in the case of the t h y ­

roid gland it is now possible to produce varying degrees of g l a n d u ­
lar activity.

By administering certain iodinated proteins or

synthetic thyroxine,
duced;

varying degrees of h ype rthyroidism can be p r o ­

a hypothyroid state can be readily brought about simply by

feeding with various goitrogens.

The impetus of such a control

over the thyroid has produc ed great strides in thyroid physiology,
especially in the field of animal production.
the voluminous amount of material

This is evident by

c oncerning the subject found in

2

such publications as the Journal of Dairy Science,
Animal Science and the various agricultural
letins.

However,

the Journal

of

experiment station b u l ­

it requires only a casual perusal

of this l i t e r a ­

ture to reveal many contradictory results obtained by a number of
investigators.

It has been pointed out by Maqsood and others that

much of this antithetical information is due to a lack of knowledge
of the normal

thyroxine secretion rate during the time of a d m i n i s ­

tration of thyroidal materials.

As stated by Maqsood:

In the p a s t , factors such as breed and strain d i f f e r ­
ences, sex and age of the animal, stage of sexual d e v elo p­
ment, dose, mode and duration of the hormone administered,
seasonal variations in the length of daylight, and e n v i r o n ­
mental temperature, all of which appear to affect the normal
thyroxine secretion rate, have not been taken into c o n s i d e r ­
ation while studying the role of the thyroid status on
maturity and fertility.
This could be better understood if
expressed quantitatively in terms of the amount of thyroxine
secreted per day by the a n i m a l .
Until recently the techniques employed to determine thyroid
secretion rates have been difficult to apply to larger animeils b e ­
cause they necessitated slaughtering large numbers.

However,

a

meth od has now been developed which employs radioactive iodine and
allows a quantitative measurement of thyroid activity in the intact
animal.
In the present study,

the method introduced b y H e n n e m a n ,

et a l . (1955) was adapted to obtain information concerning the
normal thyroid secretion rate of the dairy goat.

The relative ease

of handling plus the short output half-time of 1-131 without the
use of thiouracil make this animal

ideal

for such studies.

3

SURVEY OF L IT ERA TUR E

A,

Early Investigations

Since ancient times the superficial anatomical

location of

the thyroid gland has made it possible to associate certain diseases
with gross changes in this gland.

During the time of Hippocrates it

was known that certain substances such as burnt sponge and seaweed
when added to the diet resulted in the relief of enlarged thyroids.
As early as 1820 iodine was knowingly administered for the relief of
goiter and physiological

studies made it apparent that the thyroid

elaborated a substance which was necessary for normal body function.
Murray

(1891) reported that a glycerine extract of sheep's thyroid

when admin ist er ed to human patients satisfactorily alleviated h y p o ­
thyroidism.

Magnus-Levy

(1895),

in his calorimetric studies,

found

that patients suffering from Gull's disease were markedly below normal
in heat production.

It was also shown by Magnus-Levy that the a d ­

mi nistration of thyroid tissue raised the level of m e t a b o l i s m in such
patients as well as in normal persons.
stimulate much biochemical
Baumann

(1896)

findings served to

interest in the thyroid and, soon after,

succeeded in demonstrating the presence of icdirie in

thyroid material.

Kendall

hormone in crystalline
ton

These

(1914) was the first to isolate the thyroid

form and gave it the name thyroxine.

Haring-

(1926), using improved extraction, techniques, was able to isolate

a sufficient quantity of thyroxine to determine the empirical

4

and structural

formula.

A year later Harington and Barger

(1927)

s ucc eeded in s ynt hesizing thyroxine.

B.

Formation of Thyroid Hormone

Although it was reco gn ize d early that the thyroid contained
iodine an d that this element was necessary for the normal
of the gland,

the accumulation of iodine by the thyroid was not

directly demonstrated until Marine and Rogoff
within

function

five minutes

(1916) reported that

following the intravenous injection of 50 mg.

of po tas si um iodide the iodine content of the
be increased several hundred percent.

thyroid of a dog may

Since this time in vitro e x ­

per ime nts involving the use of radioactive iodine and thiouracil have
reveale d the presence of two distinct and independent mechanisms
the handli ng of iodide by the thyroid gland.
responsible

for

One mechanism is

for concentrating iodide and another for organic binding.

When thyroid tissue slices are incubated in a me di um c o n ­
taining 1-131 the labeled iodine is found in the tissue both as iodide
ions and as iodine in organic combination

(Chaikoff and Taurog,

1949).

The addition of thiouracil to the m e d i u m results in iodide continuing
to be accumulated,

but organic b i n d in g fails to occur.

p e r i men ts by A s t wo od

Iri vivo e x ­

(1944-45) revealed that thiouracilized rats d e ­

p le ted of iodine are still able to concentrate rapidly considerable
quantities of icdine when injected with po ta ssi um iodide.
and Sharp

McGinty

(1946) noted that increased amounts of dietary iodine raise

the thyroid iodine content above the usual low levels in rats c h r o n ­
ically treated with thiouracil.

When the protein of the gland is then

5

p r e c i p i t a t e d with Somogyi's reagent this increased thyroid iodine is
soluble in the supernatant

fluid.

In studying the effects of thio-

cyanate in rats Vanderlaan and Vanderlaan

(1947)

found that thio-

cyanate inhibited the preferential concentration of iodide by thyroid
tissue and also caused the discharge of iodide from the thyroid.

1.

Iodide Accumulation by the Thyroid Gland
Histological studies have shown the uptake of iodide by the

thyroid to be directly related to the size of the thyroid epithelial
cells.

For this reason,

it is believed that the iodide-trapping

m e c h a n i s m resides in these cells.

Wollman

(1953) observed that the

pow e r to concentrate iodide by the thyroid was dependent upon the
presence of fully formed epithelial tissue and occurred in the absence
of any visible

colloid.

It is not fully understood by what me chanism

the epithelial cells are able to concentrate iodide and maintain a
gradient against plasma but it is presumed to be one which requires
energy.

The extent and efficiency of the iodide-trapping mech ani sm

can be quantitated by simultaneous measurement of the concentration
of iodide in the thyroid and in the blood.
t hyroid-serum or T /S ratio.
found to be 25/1.
uracil

In normal rats the T/S ratio has been

When the animals were pretreated with p r o p y l t h i o ­

the ratio of thyroid iodide to serum iodide rose to a value

of 250/1

2.

This is expressed as a

(Vanderlaan and Vanderlaan,

1947).

O rganification of Iodine
After the inorganic iodide has been trapped more than 90

per ce nt of it is readily converted to organically bound forms.

Gross

6

a nd L e b lon d

(1947),

using autoradiography,

found o rganically bound

iodide in the epithel ium and colloid as early as thirty minutes after
ad ministration of radioiodine.

After an interval of 24 hours it was

found almost e xclusively in the colloid.

The organic

forms of iodine

have been quantitatively measured by chromatographic and colorimetric
procedures.

At any given time the normal thyroid has been found to

contain 25 percent of total iodine as thyroxine and triiodothyronine,
ap proximately 65 percent as diiodotyrosine and monoiodotyrosine
m ono iodohistidine and diiodothyronine are also present
Michel,

Some

(Roche and

1954).
Most of the organic iodine

in the thyroid is bound to protein

and believed to be in peptide linkage.
matic hydrolysis results

Prolonged alkaline or e n z y ­

in the liberation of iodinated amino acids.

The m e c h a n i s m of organification of iodine

is not completely understood

but at least three steps are concerned in the ultimate synthesis of
thyroxine:
1.

Oxidation of iodide to iodine or a more highly
oxid ize d form.

2.

Iodination of tyrosine radicals of p rotein to form
monoiodotyrosyl and diiodotyrosyl radicals.

3.

Oxidative coupli ng of the iodinated tyrosine
radical s to form thyroxine and triiodothyronine.

Evidence has accumulated which strongly suggests that the synthesis
of thyroxine
Chaikoff

is dependent upon oxidative enzyme systems.

(1943) noted the necessity for tissue organization in the

in vitro conversion of inorganic
sine.

Morton and

iodide to thyroxine and d i i o d o t y r o ­

This observation suggested to them the participation of an

int ra cel lul ar enzyme system.

Further studies were made on the effects

7

of anaerobiosis and of substances that are inhibitors of cytochrome
oxidase.

In both cases there were marked inhibitory effects on the

formation of thyroxine and diiodotyrosine.
agreed that other,
also involved.

and probably more important,

This is shown by the

oxidation produced by thiourea,

1949).

it is fairly well

enzyme systems are

fact that the inhibition of

thiouracil and sulfa compounds is

not mediat ed through the cytochrome
(Astwood,

However,

- cytochrome oxidase system

Evidence review ed b y Astwood suggested to him that

the most likely enzyme to be involved directly in the synthesis of
thyroid hormone

is a peroxidase or a peroxidase

type compound.

enzyme is capable of promoting the oxidation of iodine and,
tioned by Westerfield and Lowe

(1942),

This

as m e n ­

it could also carry out the

oxidative coupling of two diiodotyrosine molecules to form thyroxine.
Dempsey

(1944) pr esented histological evidence of the presence of fine

granules in the thyroid cell which give histochemical reactions t y p i ­
cal of peroxidase and has shown that thiouracil
tions.

More recently Weiss

inhibits these r e a c ­

(1953) reported the synthesis of

or gan ically bound iodine by cell

free preparations of thyroid

tissue when such homogenates were supplemented with cupric ion and
tyrosine.

Fawcett and Kirkw ood

it is possible to extract

(1953, 1954) have also shown that

from thyroid and salivary gland tissue a

soluble enzyme which catalyzes the iodination of tyrosine in the
presence of cupric ion.
that the requirement

In view of this work it was at first believed

for copper indicated that either a copper c o n ­

taining enzyme was involved

(Weiss,

1953) or the cupric

ion p a r t i c i ­

p a t e d as a non-enzymatic entity in the oxidation of iodide to iodine

8

(Fawcett and Kirkwood,
that the

1953).

Those latter workers

further suggested

function of cupric ion in this iri vitro reaction is to a c ­

cept electrons directly from iodide ion and thus replace an ’’iodide
oxidase" which causes the formation of elemental
thyroid gland.

However,

iodine in the intact

in the light of further study on the effect

of iodide concentration on the rate of iodination of tyrosine Serif
and K ir kwo od

(1956) have concluded that the iodine in this in vitro

reaction is bein g produced by an enzyme system.
this enzyme

They presume

that

is the iodide oxidase which functions in intact thyroid

and salivary gland tissue.

This enzjme is believed to have the

prope rt y of catalyzing an equilibrium between electron donors or
acceptors and the iodide-iodine system.

In this way it has the ability

to catalyze either the oxidation of iodide,
depending on the redox potential
active

or the reduction of iodine,

of the system.

With respect to the

form of iodine in the iodination process two possibilities

have been postu lat ed by Serif and Kirkwood

(1956).

In the first case,

in the presence of iodide oxidase two electrons are removed from
iodide ion to produce
ating enzyme
However,

I+ .

This m a y be passed directly to an iodin-

(tyrosine iodinase) and organification takes place.

it is pointed out by these workers that this I+ m ay also

react with substances other than tyrosine iodinase and,
it will also react with iodide to form elemental
this,

iodine.

therefore,
In view of

I + is not thought to be the active iodir.ating species of iodine.

The second pos sib il ity is that elemental iodine is the product of
iodide oxidase and that it is released to the environment where it
is taken up by tyrosine iodinase.

It is further p ostulated that

9

the rate cf reaction would be greatly augmented if the tyrosine
iodinase were to catalyze the iodination of tyrosine by causing the
elemental

iodine to dissociate

to I + as well as dissociating the

ph eno li c hydroxyl of tyrosine.
In the past it was believed that these oxidative processes
occurred within the epithelial cell and the thyroglobulin with its
iodinated residues was then secreted into the colloid of the follicle.
However,

it has been observed by Pitt-Rivers and Trotter

(1953) using

ra d ioa uto gr aph y that inorganic iodide accumulated in the colloid and
it was found that organification of iodine took place
p h e r y of the colloid near the colloid-cell
of these observations

interface.

in the p e r i ­
On the basis

it is theorized that the cells continuously

secrete an oxidizing enzyme into the colloid where iodination of the
thyroglobulin takes place.
vations of Roche.

In support of this notion are the o b s e r ­

Using the best techniques the only iodinated

pr o tei n that he was able to find in the thyroid was t h y r o g l o b u l i n ,
whereas Reineke and Turner

(1942) have shown that it is possible

to iodinate casein and other proteins.
took place within the cell we would,

If organification of iodine

therefore,

besides thyroglobulin to become iodinated.

expect other proteins

However,

in the light of

this new concept the cell, where the various proteins are available,
contains only the iodide ion.

The free iodine necessary for iodina-

tion of pr ote in is found only in the cclloid and in the presence of
only one tyrosine-containing protein and that one is thyroglobulin.
By way of summary,

the present evidence

indicates that i n o r ­

ganic iodide is p re fer entially remo ved from circulating blo od by the

10

thyroid epithelial cells and secreted into the colloid.

In the

presenc e of oxidative enzymes this iodide is converted to nascent
iodine.

Tyrcsine residues contained within the protein thyroglobulin

molecule are then iodinated to form monoiodotyrosine and d i i o d o t y r o s i n e .
Two molecules of these latter residues,
p eroxidase-like enzymes,

still

in the presence of

are oxidatively condensed to form thyroxyl

and triiodothyronine residues of thyroglobulin.
then acts as a storage

3.

This thyroglobulin

form of the thyroid hormone.

Proteolysis of Thyroglobulin
Since thyroglobulin as such has no hormone

properties it must

be p r esu med that the next step in hormone synthesis is the liberation
of the thyroid active substances.

DeRobertis

(1941) has shown that

thyroid proteolysis leading to diffusable products of low molecular
weight

takes place in the thyroid.

He has demonstrated the presence

of a proteolytic enzyme in the colloid capable of releasing tyrosine
from a protein substrate and has characterized it as a cathepsin
type compound.

The free iodinated products which have been found in

the thyroid include m ono iodotyrosine and d i i o d o t y r o s i n e , traces of
m o n o i o d o h i s t i d i n e , thyroxine,

and triiodothyronine.

The thyroxine

and triiodothyronine contained in this pool are believed to be the
source of the plasma thyroid hormone.

Gross and Leblond

found that after the injection of radioiodide

(1951)

in rats the specific

a cti vit y of the free thyroxine is at first higher in the thyroid
than in the plasma and later becomes approximately equal in both.
It is poi nte d out by these workers that the passage of thyroxine

from

11

gland to plasma is facilitated by a concentration gradient of over
100:1.

More recently Taurog,

1-131 to horses and sheep.

Wheat and Chaikoff

(1955) administered

Using chromatography they analyzed a

bloo d plasma sample obtained from the thyroid vein and found mainly
thyroxine and triiodothyronine;

only slight

were detectable.

found earlier by Gross and Leblond

This was also

traces of iodotyrosine

(1951) using autoradiographs of paper chromatograms.

These latter

workers concluded that all of the iodothyronine compounds go into
the circulation directly while the iod.otyrosines are metabolized
w ithin the gland.

The fate of the iodotyrosines then would be to

undergo dehalogenaticn and act as hormone precursors.
the thyroid handles iodine with maximum efficiency.

In this way
Roche has p o s t u ­

lated that practically all of the iodine leaves the gland as hormone
iodine but only after one or more cycles of organification with t h y r o ­
globulin.

In support of this,

Roche has shown that thyroid slices

or homogenates are able to dehalogenate diiodotyrosine to m o n o i o d o ­
tyrosine and this in turn is deiodinated to form tyrosine.

This

de halogenating m echanism depends upon an enzyme system since it
varies with pH and is stopped by heating.

It is also shown to be

inhibited by thyroxine and activated by TSH.

Furthermore,

incubation

of thyroid slices in a med ium containing iodotyrosine and thyroxine
results in the deiodination of the tyrosine but not the thyroxine.

C.

1.

Hormonal Transport and Utilization

Transport
The mode of transport and nature of circulating thyroid

hormone after its release into the blood stream has been the object

12

of much investigation.

It has been known for some time that the

hormone is r eadily bound to plasma protein.

Recen tl y the use of

paper zone electrophoresis to separate plasma proteins and radioactive
iodine as a tracer has made it possible to study the behavior of
iodine containing compounds

(Albright,

et a l ., 1956; Recant,

1956).

Therapeutic amounts of 1-131 were administered to euthyroid and h y p e r ­
thyroid subjects,

and their serum was then studied.

It was found

that initially most of the 1-131 was unbound by protein but there
was a definite localization in the albumin fraction.

After several

days there appeared in the normal subjects a gradually increasing
localization of ra dioactivity in an area just ahead of the alpha-2
globulin in addition to the albumin-bound radioactivity.

In the

hyperthyroid subjects the sequence was essentially the same; however,
the radioact ivi ty in the inter-alpha zone reached higher levels at a
more rapid rate and then declined more sharply.

Albright and his c o­

workers suggest that this more rapid turnover of 1-131 in the h y p e r ­
thyroid individual

is an indication that the circulating hormonal

iodine is carried by an alpha globulin.

Further studies involving

the use of starch zone electrophoresis and paper chromotography
revealed that the radioactive material present in the alpha zone
consisted entirely of thyroxine.

No triiodothyronine was found.

In

vitro experiments have shown both compounds do associate with alpha
globulin but thyroxine binds more firmly and can displace trii odo ­
thyronine.

This is believed to afford an explanation for the absence

of triiodothyronine in the alpha globulin in in vivo experiments and

13

m a y also account

for the greater speed of biologic activity of t r i ­

iodothyronine .
Recant

(1956) has reported an alteration in the transport

m ec h a n i s m cf thyroxine as a result of certain conditions.

She has

found that 70 to 100 percent of 1-131 labeled thyroxine incubated in
vitro with serum from patients with nephrosis was
alpha-2 globulin area.

found in the

In contrast, both normal and myxedematous

subjects bound most of the thyroxine in the albumin fraction.
abnormal nephrotic pattern was

The

found to be reversed within forty-eight

hours after steroid therapy.
Dowling

(1956) has found a marked increase of thyroxine b i n d ­

ing to serum alpha globulin in pregnant women,

beginning as early as

the twenty-first day after ovulation and persisting throughout
pregnancy.

2.

A similar change occurs after estrogen administration.

Utilization
At present one of the major problems confronting the thyroid-

ologist concerns the metabo li sm of thyroxine by the peripheral

cells

of the body.
Gross and Pitt-Rivers

(1951) have suggested that t r i i o d o ­

thyronine is derived froir. a deiodination pr oce ss of thyroxine by
peripheral

tissues.

This notion carried further led to the hypothesis

that thyroxine itself has nc metabolic activity but acts as a p r e ­
cursor for triiodothyronine which is met abolically active.

Direct

evidence in support of this concept was afforded by Albright,
(1954).

et a l .

These workers demonstrated the deiodination of thyroxine to

14

triiodothyronine _in vitro by rat kidney slices.

Albright,

et a l .

(1956) have extended these studies of thyroxine utilization to subcellular fractions of ki dne y tissue.

When 1-131 labeled thyroxine

was incubated with an enzyme system derived from mitochondria the
reaction product was rot the expected triiodothyronine but tetraiodothyroacetic acid.

When triiodothyronine was similarly incubated

the reaction yielded triiodothyroacetic acid.

The true significance

of this reaction has not as yet been ascertained but it is believed
by these workers that it may represent a degradative process.
ever,

the point was not overlooked that

How­

this transformation may

represent an intermediate compound in the formation of a specific
metabolic all y active derivative of the thyroid hormone.
P itt-Rivers

Lerman and

(1956) have reported that these iodothyroacetic acid

compounds act qualitatively like thyroxine and triiodothyronine but
are quantitatively weaker.

Tetraiodothyroacetic acid,

intravenously,

has 1/ 1 5tb the activity of 1-thyroxine in raising the B.M.R.
with myxedema.

of patients

Triiodothyroacetic acid has 1/4tb to l/6tl? the activity

of thyroxine.
Several

theories as to mechanisms of thyroid hormone action

have been postulated.

Lehninger has proposed that thyroxine acts

pri mar ily on the mitochondrial membrane.

He observed that thyroxine

p r o m o t e d water imbibition by isolated mitochondria.

Lardy has p o s ­

tulated that oxidative phosphorylation is the primary site of action
of thyroid hormone.

He bases his theory on the i£ vitro evidence

for

the u ncoupling of oxygen uptake from phosphate esterification in the
presence of thyroxine.

15

D.

Factors

Influencing Thyroid Function

A v ari et y of conditions and agents are kn ow n to influence
thyroid function.

These include the anterior pit ui tar y thyrotropic

h o r m o n e , e n v i r o n m e n t , adrenal

cortex and gonadal h o r m o n e s , i o d i n e ,

and an ti th y ro i d d r u g s . ,

1.

Pi t ui ta r y Thyrotropic Hormone
The relati on sh ip between the pituitary and thyroid has been

studied in considerable detail

in various laboratory animals.

It is

generally agreed that se cretory activity of the thyroid is r egu lated
by a balance between thyroid hormone and the anterior pituitary
thyrotropin ho rmone
45; Taurog,

(Cortell and R a w s o n , 1944; Keating,

Chaiko ff and Bennett,

Stanley and Astwood,

1949).

et a l ., 1944-

1946; L ebl on d and Gross,

1948;

It has been found by these workers that

under the st imulating influence of thyrotropic hormone,
iodide is more rapidly taken up, bound to protein,
the hormone dis ch ar ge d into the circulation.

inorganic

p ro teolyzed and

Associated with these

p ro ce s se s is a hype rt ro ph y and hyperplasia of the thyroid.

With

increased co ncentration of thyroid hormone in the blood the activity
of the anterior pi t ui t ar y is modified in such a w a y that d imi nished
amounts of thyrotropin are secreted.
of action of thyroxine

With regard to the m e c h an i sm

in su ppressing TSH secretion D'Angelo has a d ­

vanced the most recent theory.

He suggests that the excess thyroxine

so speeds up me ta bolic processes that the rate of disposal of t h y r o ­
tropin from the circulation is increased and therefore lower c o n c e n ­
trations of TSH are available to the thyroid gland.

In support of

16

this concept is the observation that TSH and thyroxine given s i m u l ­
ta ne ou sl y result in less thyroid stimulation than when TSH is given
alone.

It has also been po inted out by D*Angelo that the rate of

disappearance of TSH is slower in h y p o p h y s e c t o m i z e d , t h y r o i d e c t o m i z e d ,
or goitrous animals.

In each of these three cases the animals are

existing at lower metabolic levels due to a deficiency of thyroid
hormone.

The s par in g of TSH under these conditions may reflect an

inhibition of an inactivation process due to a lowered rate of
metabolism.
Although the activity of the thyroid is pr imarily under the
control

of the anterior pituitary it has recently been shown by

D*Angelo that the thyroid gland is able to cariy on a residual
function even in the complete absence of the hypophysis.
residual

This

function has been estimated at approxi ma te ly 10 percent of

the n o r m a l .

2.

Temperature
It is now held as a general principle that environmental

temperature modif ie s thyroid activity

(Dempsey and A s t w o o d , 1943;

Leblond,

1949; S c h a c h n e r , Gierlach and

Krebs,

et a l ., 1944;

Dempsey,

1949; Blincoe and Brody,

1944,

1955; H e n n e m a n , et a l ., 1955).

E x is t in g evidence reveals that exposure of rats to cold leads to
c onsi de ra ble increases in p ro duction of thyroid stimul at in g hormone
by the pituitary,

follicular cell activity resulting in a heightened

p ro du ct io n and release of thyroid hormone,
hormone by body tissues.

and u ti li zat io n of thyroid

The converse of this takes place when the

1?

a ni ma ls are e xp os ed to high temperatures.

The thyroid r esponse to

change of environmental temperature is generally assumed to be r e ­
lated to mo di fi ca ti ons of pi tuitary T SH secretion
However,

Wolf and Greep

(Uotila,

1939).

(1937) have shown that the thyroids of hypo-

p hy s ec to m iz e d animals do reveal minimal histologic signs of s t i m u l a ­
tion by cold.

Rand,

Riggs and Talbot

(1952) have suggested that

the m ec ha ni sm involved in response to cold is ar. increased utilization
of thyroxine by the tissues.

This results in a compensatory response

of the pit ui ta ry to a reduced concentration of thyroid hormone in
the blood.

However, B ro wn - Gr a nt points out that this may be true

for chronic cold experiments but it seems more probable that some
neural mechanism,

independent of the hormone concentration, may be

involved in the ra pi d response to acute cold exposure.
of this Uot il a

In support

(1939), Purvis and Griesbach (1946) have found that

p it u it a ry stalk transection results in a loss of thyroid response
to cold exposure.

3.

Season and Light
Seasonal variations have been found to occur in the over-all

activity of the thyroid in m an y species
Reineke and Turner,
Reineke,

1956).

1945; Henneman,

(Starr and Roskelly,

1940;

et a l ., 1955; Lodge, Lewis and

So far no definite physiological

explanation other

than that due to temperature or light change has been forthcoming.
Pu nt ri an o and Meites

(1951) have found that continuous light induced

significant reductions in thyroid weight,
uracil and thyroid uptake of radioactive

thyroid reaction to thioiodine in rats.

Continuous

18

d arkness was o bs erved to have the reverse effects.

These workers

concluded that continuous light depresses while continuous darkness
increases thyroid secretion in mice.

4.

T hy ro id - Go n ad Relationship
There is a great deal of conflicting evidence p re se n te d in

the literature concerning the relationship between the thyroid gland
and the gonads.

However,

it seems to be agreed in general

relati on sh ip is a reciprocal

one.

that the

There is some evidence to show

that castration produces a slow involution of the thyroid and a
slight re du ct io n in total met ab ol is m (K o re n ch ev s ky , 1930).
trast, K i pp en and L oeb

In c o n ­

(1936) reported thau gonadectomy in the guinea

p ig r es ul te d in thyrotropin secretion and thyroid proliferation.

The

effects of estrogen on thyroid function are apparently related to
dosage and the pe ri o d over which the hormone is ad ministered
1941).

(Lerman,

Both estrogens and androgens in physiologic amounts are

capable of stim ul at in g thyroid activity (Lewis and McCullagh,

1942).

When the dose of estrogen is increased thyroid function is definitely
depressed

5.

(Gardiner,

1949).

Thy roid-Adrenal Relat io ns hi p
Cortical extracts of the adrenal

affect thyroid function
O e h m e , 1939).

(Oehme,

gland have been shown to

1936; Hoen, M c G a v a c k , Langfe ld and

When cortical extracts are administered in large

doses they will prevent the rise of basal metabolic rate which would
o rd in ar il y follow the administration of thyrcid hormone.
et a l . (1946),

as a result of their enzyme studies,

Tipton,

suggest that this

19

a cti o n m ay be due to the cortical hormones inhibiting the mec ha ni sm s
which are re sponsible
chrome oxidase
tered,

there

for the activation of succinoxidase and c y t o ­

in hyperthyroidism.

When p hy siologic doses are a d m i n i s ­

is an augmentation of the characteristic action of t h y ­

roxine upon growth and calorigenesis
Hoffman and Talesnik,

1948).

(Vergara Soto,

1948; Hoffman,

In the ad renalectomized animal there is

an increase in heat p ro duction which is prevented by prior t h y r o i d ­
ectomy

(Marine and Baumann,

thyroidal

1922).

There is also a rapid loss of

iodine following adrenal injury.

Animals expo se d to various

situations of stress show an increase in the secretion of thyrotropin,
a dim in is hed thyroidal uptake of 1-131 and a diminution of hormone
in the plasma.

This could be interpreted as denoting an increase in

thyroid activity.

The lowered amount of circulating thyroid hormone

may reflect a heightened state of m e ta bo li sm and result in an increase
in p it uitary thyrotropin release.

Moreover,

it has been shown that

di minished storage of 1-131 is associated with the more active thyroid
in colder temperatures and a greater 1-131 storage occurs in the
summer when the thyroid is least active
Kendall and Simonsen,

1928).

However,

(Seidell and F e n g e r , 1913;
this picture is complicated by

the findings of Meites and W ol terink (1950).

They observed that rats

m a in t a i n e d on a c al orically inadequate diet had a decreased thyroidal
uptake of r adi oa ct iv e iodine due to a decrease in size of the thyroid.
The r a d i oa c ti vi t y per unit weight of the stressed animals was not
s i gn if ic an tl y different

from that of the well

fed animals.

20

6.

Antit hy ro id Compounds
The te rm antit hy ro id substance is applied to any agent which

is capable of supp re ss in g the formation of thyroid hormone by the
thyroid gland of the intact animal.

A vast amount of m aterial has

been p ub li sh ed concerning these antithyroid compounds and this review
does not attempt to cover all of it.Much of this literature
s u mm ar iz ed and the

following reviews

fication in relat io n to activity
of the nature of their action

deal with their chemical

(Anderson,

(Astwood,

1.

1951),

1949),

rev ie w of the entire subject by McGavack

E.

has

been
classi­

a differentiation

and a rather complete

(1951).

Economic Aspects of the Thyroid

Effect of Thyroid on Growth
It has been well established

induced or spontaneous,

that hypothyroidism,

depresses growth in all animals.

whether
This effect

is illustrated by the stunted growth and retarded mental and sexual
d eve lopment of cretins.

Likewise,

tadpoles immersed in water c o n ­

t ai ni ng a n ti t hy ro id drugs are delayed in their metamorphosis.

De­

p ression in growth has also been reported in th yr oi dectomized cattle
b y Br od y and Fran ke nb ach

(1942) and Spielman,

et a l . (1945).

These

authors also noted that this effect was much greater in the young
than in older animals.

B l a x t e r , et a l . (1949) suggested that some

economic value may be derived from partial suppression of thyroid
a c ti v it y prior to m ar ket in g calves or older animals.

Their logic

such treatment is that depression of basal meta bo li sm and activity

for

21

w ou ld result in more of the ingested net energy being available

for

deposition in the tissues as fat.
There is a pauc it y of literature concerning the effects of
mild hy pe rt hy r oi di s m on growth.

In severe hyp er th yroidism marked

losses of weight occur, presumably due to an elevation in metabolism.
Reine ke

(1946) stated that

"there is some evidence that a properly

r e gu la te d dosage of thyroid substance will cause an increase in
growth rate at least in some species."

In a later paper

the growth of swine the same author reports:

(1948) on

"Administration of

thyroprotein in proper dosages will cause some increase in the growth
rate and will also increase the rate at which maturation changes
occur."

Millen,

Nevens and Gardner

(1948) administered 1.3 grams

of iodinated casein per- 100 pounds body weight to two dairy calves
and noted a slight increase in growth above normal

controls.

Ho.w-

e v e r , when four grams per 100 pounds of body weight were given the
usual symptoms of severe h yp erthyroidism resulted.

2.

Effect of Thyroid on Reproduction
Complete agreement has not been reached on the role of the

thyroid in male

fertility.

There is some evidence to support

the

view that the thyroid has no direct effect on the testis but rather,
any reprod uc ti ve disturbance

in the male in h yp ot hyr oi di sm and h y p e r ­

thyroidism is due to changed metabolic status.

Petersen,

(1941) r ep or te d that thyroidectomy in the bull,

r es ulting in complete

clinical
estrual

myxedema,
female.

et a l .

results in a loss of libido and interest in the

No effect on spermatogenesis was apparent since

22

e j ac ul at es obtai ne d by ampullae ma ss aging were normal
morphology,

activity,

in sperm

longevity and fertilizing ability.

When t h y ­

roidal material was administered libido was completely restored.
Reineke
was

(1946) report ed favorable results when iodinated casein

fed to aged bulls.

He found that ten out of fourteen animals

s ho we d increased vigor and more speedy ejaculation.
In th yroidectomized rams the picture appears to be just the
reverse of that

found in bulls.

Bogart and Mayer

ma rk e d decline in sperm number and motility,
abnormal

forms in thiouraci li ze d rams.

(1946) noted a

and an increase in

It appears that sex interest

was not impaired in these experiments since semen was collected with
an artificial vagina.

The adverse effects on sperm prod uc ti on were

r e ve r se d by feeding iodinated casein.
With regard to r ep roduction in the female,

thyroidectomy in

the cow results in a complete absence of normal estrual behavior
(Brody and F r a n k e n b a c h , 1941).

However,

even in myxedematous animals

ov ulation and a normal ovarian cycle occurs and impregnation followed
b y normal p reg nancy can take place.

Petersen and his coworkers b e ­

lieve that the thyroid plays only an indirect role in reproduction
through maint en an ce of an optimum rate of metabolic processes.

3.

Effect of Thyroid on Milk Production
The use of thyroidal m a te ri al s in milk production is u n ­

d o u b t ed l y the most

important economic aspect of thyroid physiology

at the present time.

The

fact is new well established that the

feeding of thyroidal substances will increase milk and milk-fat
produc t i o n .

23

G ra ha m

(1934)

first demonstrated a definite relationship

be tween milk secretion and the thyroid.

In his thyroidectomized

animals there was not a very significant difference

in the fall of

milk secretion when compared to that accompanying control o p e r a ­
tions.

However,

there was a rapid rise in milk and milk-fat p r o ­

duction during the d ec lining phase of lactation when a small amount
of thyroid material was added to the diet of both thyroidectomized
and control animals.

In later experiments

(1934a) he noted that

there was always a mark ed rise in milk-fat production,

but there

were large variations in the increase of milk secretion.
of Graham has been confirmed by Jack and Bechdel
Graham and Turner
al.

(1938); Ralston,

et a l . (1940);

The work

(1935); Herman,
and Blaxter,

et

(1949).
This work served to stimulate speculation on the use of

iodinated protei ns to increase milk production.

Turner

(1940) r e ­

por te d an increase of milk production in goats

following the feeding

of small amounts of iodinated protein.

in 1942, Reineke e x ­

Later,

tended these preli mi na ry experiments to cattle.

He reported an

increase in meta bo li sm of twenty to thirty percent accomp an ie d by
an improvement in appearance and vigor and an increase in milk yield
and m il k- fa t percentage.

In 1943 Reineke reported on successful

field trials of feeding synthetic thyroprotein to cattle.

Since

this time an iodinated casein preparation has been developed by
Turner and Reineke and is now marketed under the name of protamone.
It has been re ported by Graham

(1948)

that the feeding of p rotamone

to commercial herds in the Los Ange le s milk shed resulted in increases

24

in m il k s ecr etion of as much as 5 pounds of milk and 0.3 pound of
butterfat

daily during p ea k production.

The effect on lactation of feeding thyroidal materials has
be en

found to be dependent upon several

factors.

The response

in

milk production, with rega rd to dosage has been studied by Reineke,
et al. (1944); Blaxter

(1945) and Reece

(1944).

The influence of

age and size cf the animal has been repor ted on by Blaxter
and Booth,

et a l . (1947).

(1946)

Bla.xter has also found that in cattle

breed has rel at iv el y little effect on response.

In their treatise

on the role of thyroidal materials in animal production Blaxter,
Reineke,

Crampton and Petersen

(1949) state that the most

important

factors i nfluencing the response of the cow to thyroprotein feeding
are her stage of lactation and producing ability.
is ba se d on the wo rk of Herman,
(1940);

and Blaxter

(1945),

This conclusion

et a l . (1938); Ralston,

et al,

Length of stimulation and nutritional

status have also been studied as factors influencing lactational
response

(Reece,

1947; Thomas and Moore,

1948; Reineke,

1943).

With regard to the effect of feeding iodinated protein on
milk composition Blaxter,

et a l , (1949) have concluded in their r e ­

v ie w that there is an increase in the lactose content and possibly
a small decrease

in the protein and nor,-protein nitrogen content.

These authors further state that "as far as milk composition is
concerned,

the m il k p r o d uc ed by cows receiving iodinated casein is

perf ec tl y safe,

and nu tr itionally adequate."

25

F.

1.

Methods of D et ermining Thyroid Ac tivity

Oxygen C on sumption and B.M.R.
The observations of Magnus-Levy

(1895),

concerning the in­

crease in oxygen consumption and carbon dioxide output following the
a dm in is tr at io n of thyroid material,

formed the basis for the most

w i de l y used met ho d of studying thyroid activity.

The method c o n ­

sists of determ in ing the heat production of a resting,
dividual

in a thermo-neutral environment.

fasting in­

This basal metabolism

rate is ex pr es se d as calories per hour per square meter surface area
and is u su al ly d et ermined indirectly by m e as uri ng the carbon dioxide
and oxygen respi rat or y exchange.

With the advent of radioactive

iodine

the met ho d has fallen into more and more disuse as a d i a g ­

nostic

tool.

for thyroxine.

It is still wid el y used,

however,

as an assay p ro cedure

The technique does not lend itself very readily

the study of large animals and most especially the ruminant.

to

It is

difficult to maint ai n the animals in a subdued state and p rac ti cal ly
impossible to obtain a fasted ruminant animal.
the rumen also interfere in this determination.

Gases p ro duc ed in
In measuring the

carbon dioxide p ro du c ed there is no way of d if ferentiating between
that produc ed by m e ta b o l i s m of the bo dy and that resulting from
bac te ri al

2.

fermentation.

Body Weight and Replacement Therapy
The loss of body weight in thyroidectomized animals and the

r est or at io n of growth

following the administration of -thyroid active

m a te ri al s has been used as a metho d of assay.

However,

this type of

26

stu dy has net found wide acceptance because it lacks the necessary
s en s iti vi ty for accurate quantitative m e a s u r e m j n t s .

3.

Goitrogenic Method
Fo ll ow in g the discovery of certain compounds such as t h io ­

urea and thiouracil which prevent the formation of thyroid hormone,
Dempsey and Astwood

(1943) described a new method of assay based on

the action of these compounds.

Mixner, Reineke and Turner

(1944)

report ed a similar type of assay using the one-day old chick as the
test animal.

In the absence of thyroid hormone there is a c o n s e ­

quent increased pr od uction of pituitary thyrotropic hormone.

As a

result there is a marked stimulation of the thyroid and this is r e ­
flected in histological

changes and increased size of the gland.

These changes vary directly with the rate of secretion of the t h y r o ­
tropic hormone and indirectly with the amount of circulating thyroid
hormone.

The end point us ed in this determination is that amount of

exogenous thyroxine needed daily to maintain a normal hormonal
balance between

the thyroid and pituitary as determined by the a b ­

sence of weight change in the thiouracilized animal.
Mixner and Turner

(1945)

found good agreement between the data o b ­

tained by this m e th od and that obtained
measurements.

Reineke,

from basal m et ab ol is m

The technique has gained widespread acceptance and

most of our present day knowledge of thyroid activity and factors
a f fe ct in g the activi ty have resulted from its use.

However,

the

a pp li ca ti on of this m e t ho d to the large domestic animal has been
limit ed by the necessity for s la ughtering large numbers.

27

4.

PBI Method
Because of the limitations of the goitrogenic technique in

larger animals ma ny investigators in this field have used p r o t e i n bound iodine m ea sur em en ts as an indication of thyroid activity.
has been confirmed by Morton,
et a l ■ (1942)

Perlman,

and Chaikoff

(1941); Morton,

that disturbances in thyroid activity are reflected

in the t h y r o x i n e -1 ike fraction of plasma iodine.
workers

It

L on g and his c o ­

(1951) measured the protein bound iodine content of serum

in dairy cattle and found significant differences between breeds.
However,

in connection with the determination of protein-bound

iodine it has been p oi nt ed out by Salter,
iodine,

when present in excess,

bination with protein,
active fractions.

et a l . (1949)

that

inorganic

is capable of forming a loose c om ­

and may be preci pi ta te d with the hormone

Values for pro te in -b ou nd iodine may therefore

become me an ingless if iodine is being taken in by the subject.
a review,

Rapport and Curtiss

In

(1950) have stated that the chief

disadvantage of the PBI method of d et ermining thyroid activity is
that iodine in any form has a demonstrable effect on protein-bound
iodine levels in the serum.

In this regard, Reece and Man (1952)

have shown the presence of a nonthyroidai
p r ot e in - b o u n d iodine of cattle.

Lewis

fraction

in the plasma

(1952) observed no effect on

PBI of cattle when the ration was supplemented with iodized salt.
However,

he further stated that the iodine in the supplemented

ratio n may not have been in excess of the animal's needs and if an
excess were present the results might have been different.
there is much evidence

Although

for and against the PBI met ho d of ascert ai ni ng

28

t hyro id ac ti vi ty many clinicians still regard it as a valuable
l abo ra to ry aid in diagnosis of thyroid function.

5.

Thyroid Studies with Radioactive

Iodine

Soon after the discovery of artificial
Joliot and Curie
of radioactive

(1934),

iodine.

by Lawrence and Cook se y
vestigators.

ra dioactivity by

Fermi report ed the successful preparation
Fol lo wi ng the development of the cyclotron
(1936) this isotope became available to i n ­

Since this time much knowledge of the thyroid has

been gained through the use of radioiodine.
Kelsey,
r adioactive
biological

Heines and Ke a ti ng (1949) have identified fourteen

isotopes of iodine.
investigations.

Four of these have been employ ed in

1-128 with a half-life of 25 minutes

was the first to be prepared.
of slow neutrons on iodine.

This was accomplished by the action
1-126 with a half-life of 13 days is

pr ep ar ed by the action of fast neutrons on iodine.

The bombardment

of metallic te ll urium with slow neutrons in the chain reacting pile
gives rise to 1-131 with a half-life of 8 days;
life of 12.6 hours also results

1-130 with a half-

froir. the bombardment of tellurium.

1-131 is used almost exclu si ve ly in biological studies at the present
time because of its convenient half-life and it can be obtained in
carrier-free

form.

Hertz and his coworkers

(1958,

to report the use of radioiodine
this element w it hi n the body.
the thyroid,

1940,

1941) were the first

in following the distribution of

The acti on of factors whi<h affect

such as thyrotropin and iodine were

by these workers.

further elucidated

29

Morton,

Chaikoff,

et a l . (1943, 1944),

using iri vitro t e c h ­

niques involving the incubation of thyroid slices in Ringer's s o l u ­
tion c on taining tracer amounts of radioactive

iodine, were able to

d etermine the fractions of iodine-containing compounds in the thyroid
gland.
Hamilton and Soley

(1938, 1939,

use of ra di oactive isotopes in humans.

1940)

first reported the

By mak in g measurements

directly over the thyroid they were able to follow the uptake of
iodine and compared the values obtained from normal persons with
those

from patients with various thyroid diseases.

and Schmidt

(1949), w or k in g with humans,

Werner, Quimby

reported the uptake of

radioiodine by the thyroid after an interval of 24 hours p o s t ­
injection.

It was deemed by these workers as the most

favorable

time for a ss ay in g thyroid function in humans because at this time
uptake has leve le d off in both the euthyroid and hyperthyroid gland.
M e as u re me nt s at the 24 hour period also provide a normal uptake
range whereby a large m aj or it y of patients with hyperthyr oi di sm and
h y po th yr oi dis m can be distinguished from those with normal

function.

At the present time the 24 hour thyroidal uptake of radioiodine is
p r o b a b l y the most w i d e l y used technique in diagnosing thyroid d i s ­
orders.
minimal
However,

It has the advantage of being simple to perform and requires
cooperation by the patient and minimal dose of radioiodine.
in the past few years some criticism of this method has been

expressed.

In view of various

the blo o d iodide pool,
iodine,

factors which m a y have an effect on

from wh ic h the thyroid draws for its hormonal

it is felt that uptake determinations do not always reflect

30

true

thyroid activity.

rates,

Such variables as differences in excretion

e nterohepatic circulation,

and exogenous iodine

from known

and unkn ow n sources would be expected to affect the blood iodide
pool.

McConahey-,

et a l . (1956) have re por te d on a clinical appraisal

of seven radioiodine tests in humans.
uptake,
rate,

These included:

(2) 24 hour u p t a k e , (3) 24 hour excretion,

(5) extrarenal disposal rate,

(7) thyroidal

iodide clearance.

(l) 6 hour

(4) accumulation

(6) conversion ratio,

and

These workers stated that all seven

were r e la t i v e l y accurate for diagnosis of exophthalmic goiter in the
92 pa ti en ts studied.

It was felt that the 6 hour uptake and thyroidal

iodide clearance were the most precise of the tests studied because
they dist in gu ish ed sharply between the patients with exophthalmic
goiter and those without thyroid disease.

However,

it was point ed

out by these investigators that radioiodine tests are of limited
usefulness

in diagnosis of myxedema.

They concluded that the clinical

interpretation of uptake studies is always hindered by the knowledge
that exogenous iodine may frequently be the cause of abnormal results.
More recently,

Jaimet and Thode

(1956) reported that although

the s al iv a- pl as ma iodine ratio and thyroid activity gave a good c o r ­
relation,

a much better indication of thyroid function could be o b ­

tained when the saliva-PBI ratio at 24 hours was used.

This was

found to be almost 1 0 0 % accurate when other radioiodine tests gave
inconclusive results.
for hyperthyroids,

Observed S-PBI ratios ranged from 0.14 to 44

from 150 to 250 for norma]

to over 1900 for hypot hy ro id individuals.

subjects and from 400

31

Wolff
r adi oi od in e

(1951) used the measurement of output half-time of

from the rat thyroid as a m et ho d of studying the various

factors whi ch influence the release of iodine.
3.3 days in control animals.

Propylthiouracil

He found a value of
treated animals had an

a cc el er at ed release with a half-life of 1.6 days.

Hy po ph ysectomized

and thyroxine treated rats had similar values of 24 and 26 days,
respectively.

It was also shown by Wolff that thyrotropin action

had a latent peri od of two to three hours before any significant
effect could be shown on 1-131 release.

Albert and Tenny (1951)

made similar observations using the method of proportional rate of
disappearance of thyroidal
Perry

1-131 as percent per day.

(1951) also reported a method of de termining an index

of thyrcidal hormone release based on the decline of ra dioactivity
in the gland.

Besides showing that secretion is markedly inhibited

by exogenous thyroxine Perry further demonstrated a r el ationship b e ­
tween the amount of thyroxine given and the degree of inhibition of
hormone release.
Singh

(1955).

Such a relationship was also shown by Reineke and

This was accom pl is he d by measuring the r ad ioactivity

of the thyroids of several

groups of animals after each group had

b ee n injected with a different dose of 1-thyroxine rangi ng from 0 to
10 micrograms.

This radioactive count was expressed as a perce nt of

the activity de termined pr ev io us to thyroxine injection.

By plotting

the perc en t of initial activity against micrograms of 1-thyroxine
injected a standa rd curve was obtained by Perry whereby unknown
t h yr oi d active mat eri al s could be assayed.

From the quantities of

t hy roxine re qu ir ed to block the release of radioiodine Perry

found

32

that the daily thyroid hormone demand of a 200 gram rat was a p p r o x i ­
m a t e l y 10 mi cr ograms of 1-thyroxine.
Bas ed upon Perry's work, Henneman,

et a l . (1952) developed

a m e t ho d w her eb y thyroid secretion rate in individual sheep could be
q u an ti ta ti ve ly measured.

This procedure consists of injecting the

animals with radioactive iodine and determining the thyroidal
and output of iodine.

Thjs is done by mak in g radioactive measurements

directly over the thyroid gland.

After peak uptake of iodine has

occurr ed and output is established,
are begun.

uptake

daily injections of 1 -thyroxine

Ev er y third day the injection dose is increased by an

appropriate, increment.

With increasing amounts of injected t h y r o x ­

ine there is a co rrespondingly increased amount of inhibition of the
th yr oi d- pi tui ta ry system r esulting in smaller amounts of iodine
leaving the gland.

The degree of inhibition of the thyroid-pituitary

sys te m is m e as u re d by determining the percent of p re ceding r adioactive
count.

The end-point then is that amount of exogenous 1 -thyroxine

needed daily to prevent

1-131 output;

or,

that point where the r a d i o ­

active count becomes 10 0% of its pr ec eding count.
on rats Reineke and Singh
this method.

(1955) have further extended and confirmed

Since this time it has been used to study various e n ­

vironmental and physiological
Reine ke and Lodge,
a l ., 1956; Lodge,

factors in dairy calves

1955; Lewis, Lodge and Reineke,
1957),

(Lewis,

1956; Pipes,

hybrid mice

(Amin,

et a l ., 1956,

and the influence of senescence on thyroid function

et a l ., 1957).

et

genetic differences in thyroid activity

of several strains of inbred F
1957),

In experiments

(Wilansky,

33

MAT ER IA LS AND METHODS

A.

Experimental Animals

The experimental animals used in this work were obtained
from a herd of goats m ai nt a in e d by the ph ysiology department of
Michig an State University.
b ree d in g of the dairy type.

These animals were

female,

of mixed

They were divided into groups according

to age a nd to wheth er they were pregnant or non-pregnant,
or non-lactating.

lactating

Environmental temperature was not controlled e x ­

cept for su pp le me n ta r y heat

in the colder weather when the animals

were k ep t inside the barn.

The

feeding regimen consisted of p a s t u r ­

age in the summer and hay in the winter.

This roughage was s u p p l e ­

m ent e d throughout the year with a grain concentrate ma nufactured
by Darling and Company.

The manufac tu re rs guaranteed analysis of

iodine content of this ration was reporte d to be 0.008 percent.

B.

Apparatus

Figure 1 shows the apparatus that was used to hold the
animal

in place

for d et er mi n in g radioactive count and also the r a d i ­

ation c ou nting equipment.

This apparatus was quite

allowed m an y changes in adjustment
sized animals.

flexible and

to be made to accommodate various

The ra diation counting equipment consisted of a

s cin ti l la ti o n counter,

Nuclear Chicago count rate meter,

and an Es te rl i ne - An g us recorder.

Model 1620.

The standardization of this equip-

ment was checked each time it was used by means of a Co

60

r ad iation

34

Figure 1

Apparatus and r ad i at i on co un ti ng eq ui pment
used in d et e rm in in g thyroidal r ad io a ct i vi ty
in the goat.

35
Figure 1

36

source.

A spacer m o u n t ed on the tube holder facilitated the d u p l i ­

cation of geometry in successive determinations.

It was

found that

m a x i m u m count was obtain ed when the ring at the top of the spacer
was p l a c e d just behind the thyroid cartilage*.

This was

therefore

used as a convenient landmark in posit io ni ng the tube for each d e ­
t erm ination .

C.

Thyroxine

The 1-thyroxine used was obtained from the Glaxo Laboratories,
Greenford,

Middlesex,

England.

This material was

further purified

b y repeated r ec r ys tal li za tio ns by Dr. E. P. Reineke.

Twenty mg.

of

crysta ll iz ed thyroxine were weighed on an analytical balance and
then dissolved

in a small amount of 0.1N NaOH.

Enough 0.1N HC1 was

then added to make the solution slightly cloudy which signifies the
point where the m o no s od i um salt of thyroxine

is formed.

This s u s ­

pe n si on was then diluted with an appropriate amount of distilled
water to obtain the desired concentration.
sto re d in the r ef ri ge ra to r

This stock solution was

for short periods of time and the s o l u ­

tions for injection were made up from it.

D.

Radioactive

Iodine

The radioiodine used in these experiments was obtained
the AEC l ab oratories at Oak Ridge,
of this carrier-free

Tennessee.

from

Fifty m ic rocuries

1-131 was injected su bc utaneously in the shoulder

region of the experimental animals.

To facilitate the determination

of the percent of injected dose taken up by the thyroid a standard

37

co nt a in in g 1/4 of the injected dose was made up in small
planchettes.

The radioactive

glass

iodine contained in this material

was s ta bi li zed by the addition of 0.2 cc of a casein suspension
c ont ai ni ng an excess of KI and NaHSO

.

The radioac ti vit y contained

in this st an da rd was m e a su r ed each time a determination was made on
the animals and at the same geometry.
background,

Appropriate corrections

for

physical decay and dilution were made on each of these

counts.

E.

Thiouracil

In Experiment 5 a group of five animals was given thiouracil
to compare their thyroid secretion rates with those of normal a n i ­
mals.

Two days after peak uptake occurred thiouracil

begun.

The dose,

given by means of capsules,

k il o g r a m bo dy weight

was 0.1 gm. per

twice daily at twelve hour intervals.

F.

Method

The a pp li ca ti on of the met ho d of Henneman,
Griffin

(1955)

treatment was

Reineke and

to goats and the validity of the acquired data is

r epre se nt ed by the following two figures.

Figure 2 shows an uptake

and output curve of 1-131 and the influence of thyroxine injections
in graded doses on the output portion of the curve.

The data are

p l ott ed with the log of percent of the injected dose taken up by
the gland on the ordinate,
50 uc of

and time in days after the injection of

1-131 or the abscissa.

These data were obtained by maki ng

daily counts directly over the thyroid region until maxim um uptake

38

Figure 2.

A graphic i ll ustration of thyroidal 1-131
uptake and output, and the influence of
thyroxine injections in grad ed doses on
the output portion of the curve.

Figure 2

£
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40

h a d been established.

After that,

counts were made every third day.

These r ad io ac ti ve me asurements were corrected for decay and both
general and bo dy background.
the external

thigh region.

Body background counts were made over
Output half-times were determined from

the data o bt ained from the middle portion of the curve just prior
to the start of thyroxine injections.
to these p oi nt s by the
the curve

meth od

A straight line was

fitted

of least squares. The latter

reflects the influence ofgraded

part of

doses of 1-thyroxine.

When the data contained in this part of the curve are plot te d as
percent of p re c ed in g count on the ordinate against micrograms of
1-thyroxine

injected daily we obtain a curve as shown in Figure 3.

As the dose cf 1-thyroxine is increased the degree of inhibition
of output is also increased so that the percent of previous count
becomes greater.

By using the prediction equation:

Y = a + (b x )
or as in this case

where

x - Y - a
b

Y - 100
a ^
b -

7 0 a43
0 *1233

it is possibl e to extrapolate

to 100 percent previous count.

point repr es en ts complete inhibition of output,

or that dose cf e x o ­

genous 1-thy rox in e wh ic h satisfies the animal's requirement
ro id hormone,

This

for t h y ­

and it is p re su m ed that TSH secretion from the p i t u i ­

tary is not stimulated.

41

Figui'e 3

Graphic il lu stration
r elat io nsh ip between
output and thyroxine
e xtrapolation of the
obtain the estimated

of the dose r es po ns e
thyroidal 1-131
injections, a nd the
data in order to
t hy roid se c re t io n rate.

42

RATE

Figure 3

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THYROXINE

ESTIMATED

DOSE

GOAT M2
SECRETION

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43

E X PE RI ME NT AL P R OCED UR E AND RESULTS

Experiment 1

This experiment was conducted during the month of February
to determine wheth er or not there was a difference in thyroid s e ­
cretion betw ee n y ou n g and aged, pregnant and non-pregnant animals.
T wenty goats were selected for this study and divided into four
groups.

Group 1 consisted of 5 aged, pregnant animals having an

age range of 4 ^ to 5% years.

Group 2 consisted of 5 aged,

pregnant animals with an age range of ZVz to 5^ years.

non­

Five 2-year-

old p regn an t animals were designated as Group 3 and five 2-year-old
non-pregnant animals as Group 4.
After the output of 1-131 had been established an initial
dose of 75 m ic rog ra ms of 1-thyroxine was administered.

This dose

of exogenous thyroxine was increased every third day by a 75 m i c r o ­
gram increment.
Although 20 goats were used in this experiment,
secretion rates were ob tained for only 10 animals.
were aged pregnant and 2 aged non-pregnant;
nant and three young non-pregnant.
it was

the estimated

Of these,

two

three were you ng p r e g ­

Faced with this reduc ed number

felt that more indicative results coulc be obtai ne d if the

data from the p re gnant animals,

both young and old, were placed

one group and compared with the data obtained from all
pregnant animals.

the non-

in

44

The results of this experiment are shown in Tables

I and

II.

The mean es ti ma te d secretion rate,

expressed as m g./ lO O lbs./

day,

for the pregna nt animals was found to be 0.262 +_ 0.026.

For

the n on -pregnant animals a value of 0.277 +■ 0.05 was obtained.
these mean values were subjected to the
ficant difference was

’’Student

When

t" test no s i g n i ­

found to exist.

Once it had been established that under the conditions of
this experiment p re gna ncy had no effect on the estimated secretion
rate the data obtained for all the young animals were combined in
one group and compared with the data from all the aged animals.
These results are shown in Tables III and IV.
rate,

e xp ressed as mg./l OO lbs./day,

to be 0.187 _+ 0.016.
was obtained.

The mean secretion

for the aged animals was found

For the young animals a value of 0.325 _f 0.019

This time the application of the t test revealed a

high ly significant difference,

as shown in Table VI-

Experiment 2

This experiment was conducted during the month of May,

The

object of this study was to determine the relation between l a c t a ­
tion and the thyroid s ec retion rate.
Twen ty goats were again selected for this experiment and
d iv id ed into 4 groups ac co rd in g to age and whether lactating or nonlactating.

All of the lactating animals were approximately 2 m onths

p o s t - p a r t u m at the time the thyroid secretion rate was determined,
most of them havi ng k id de d around the middle of March.
were at pea k lactation at this t i m e .

These animals

Grcup 1 consisted of 4 aged,

45

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VALUES OBTAINED FOR THYROID SECRETION RATES, OUTPUT HALF-TIME,
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49

la ct a ti ng animals r a n g in g from 4 to 6 years of age.
t ain ed 7 young,
Five aged,

Group 2 c o n ­

la ct at in g animals and were all 2 years of age.

n o n - l a c t a t i n g , 5- to 6-year-old animals formed Group 3

and Group 4 was composed of four 2-year-old,

non-lactating animals.

The initial dose of 1-thyroxine administered was 75 m i c r o ­
grams,

this b ei ng increased by a 75 m ic rogram increment every third

day.
The a ni mals were no longer kept inside
time but were left out to pasture.

the barn at this

The mean temperature

for the

duration of this pa rticular experiment as recorded by the U. S.
Weather Bureau

in East L an si ng was 57° F. with a standard deviation

of + 8.4°.
The data obtai ne d for each individual animal
ment are shown in Tables A through D in the Appendix.

in thrs e x p e r i ­
The mean

secretion rate of 0.188 _+ 0.053 for the aged lactating goats was
not signif ica nt ly different
rate of 0.263
Similarly,

(Table VII)

from the mean secretion

+ 0o030 obtained for the young lactating animals.

no significant difference was found between the mean

secretion rates of l ac tating and non-lactating goats when animals
of similar age g ro upings were compared.

However,

the non-lactating

animals do sh ow a higher average rate of secretion.
tained for the aged and young,
were 0.199

The values o b ­

no n-lactating animals r es pectively

+ 0.024 and 0.278 _+ 0.036.

The difference betw ee n these

two mean s is significant at the 10 percent level.

50

Experiment 3

This st ud y was co nd ucted during the month of July to obtain
more information conce rn in g thyroid secretion rates in lactating
animals as compared to n on -l ac ta ti ng animals as well as obtaining
data with regar d to summer temperatures.

The same goats that had

been us ed in Exp er im en t 2 were used in this study and were again
placed

in the same groups.

The animals were at this

time in the

d ec lining phase of lactation.
The m e a n temperature during the
71° F.

+-.4.2°.

time of this

experiment was

Because of the decrease in thyroid function that

was expected at this time of the year the initial dose of 1-thyroxine
was reduced

from 75 micrograms to 25 micrograms.

This injected dose

was increased ev er y third day by an increment of 25 micrograms.
was soon found,

however,

It

that in most of the non-lactating animals

this amount of thyroxine was too small to have any effect on the
inhibition of thyroidal

1-131 output.

For this reason the a d m i n i s ­

tration of thyroxine was stopped for a p er io d of three days in the
animals showing no response.

A n ew series of injection doses was

then begun s ta rt in g w it h 50 micr og ra ms and increasing by 50 microgram increments.
The resul ts of this study for each goat will be found in
Tables E th rough H in the Appendix.
animals,

With regard to the lactating

a re la t io ns h ip similar to that

found in Experiment 2 a p ­

pears to exist betwe en the thyroid secretion rates obtained at this
time for the y ou n g and aged lactators.

As shown in Table VII,

no

51

s i gn i fi ca nt difference was
0.05 +_ 0.016

found when the mean secretion rate of

for the aged lactating goats was compared with the

value of 0.07 _+ 0.0074 obtained for the young lactators.
the seasonal

decline

In contrast
ference was

However,

for both groups was significant.
to the previous experiment a significant d i f ­

found at this time between the mean secretion rates of

l act ating and n on -la cta ti ng goats when animals of similar age
groupings were compared

(Table VII).

The values obtained for the

aged and you ng n on -l ac ta ti ng animals respectively were 0.099
and 0 .177

0.013.

cantly different.

As shown in Table VI,

0.02

these values are s i g n i f i ­

The seasonal decline for both of these groups,

as shov'n by a comparison with the values obtained in Experiment 2,
was also significant.

Ex periment 4

In v ie w of the unex pe ct ed results that were obtained in the
two p re vi o us experiments with lactating animals it was decided that
another study should be made.

The shme animals were again divided

into the same groups and the experimental procedure again repeated
d uring the mon th of August.
this time almost stopped.

Lactation in some of the goats had by
The mean temperature for the duration of

this e x per im en t was 70.1° F.
Since it had been

5.1°.

found in the previous study that the

amount of thyroxine a dm i nis te red to some of the animals was not
adequate,

the dose was therefore increased to 30 ug for the l a c ­

tating an imals and to 60 ug for the n o n - l a c t a t o r s .

This amount was

52

i ncr ea se d every third day by an increment of 30 and 60 ug, r e s p e c ­
tively.

In spite of the increased amount of injected thyroxine,

four of the y ou n g lactators showed no response to the hormone as
judged by thyroidal
in these animals

1-131 output.

The treatment was again stopped

for a per io d of three days and a new series was

st arted at the level of 40 ug a nd increased every third day by an
equal amount.

In this way a good response was achieved and s u c ­

cessful results were obtained.
There were also included in this study 5 immature goats
which had been born the previous spring.

Earlier attempts at

m e a s u ri n g the secretion rate of these kids had not met with much
success.

Because of their small stature in proportion to the a p ­

p ar at us that was used for hold ing the goats in place it proved
difficult to get a true measure of ra di ation over the thyroid region
during successive determinations.

However,

by adding a few new a d ­

justments and along with their increase in size it was possible at
this time to determine thyroid secretion rates in these animals.
The thyroxine dose given was 30 ug and was increased by this amount
every third d a y .
The r es ul ts of this experiment are shown in Tables J through
N in the Appendix.

Again,

similar to the previous two experiments,

no signi fi ca nt difference was found between the thyroid secretion
rates of the yo un g and aged lactating animals.
were r e sp ec t iv el y 0. 17 7 _+ 0.04 and 0.121

0.04.

These mean values
In contrast to

the p rev io us experiment no significant difference was found between
the me an secretion rates of lactating and non-lactating goats when

53

anima ls of similar age groupings were compared.

However,

the l a c ­

tating animals again showed a higher average rate of secretion.
values obtain ed for the aged and young,

non-lac ta ti ng animals

r e s pe c ti ve l y were 0.197 _+ 0.33 and 0.213 + 0.0168.

As may be noted

in Table VI the aged animals show a significant seasonal
No si gn ificant difference appears

increase.

to exist between the average

secretion rate s of these you ng and aged animals at this time.
value of 0.262

The

+_ 0.051 obtained for the immature goats was not

si gn if ic an tl y different
for the young,

The

from the average secretion rate obtained

n on -l ac ta ti ng animals.

Experiment 5

The object of this investigation which was carried out
during the month of October was threefold:
1.

To obtain data en animals of various ages during
fall weather.

2.

To determine if any effect on thyroid activity,
ex is te d during the period of estrus.

3.

To determine whether or not thiouracil treatment
h ad an effect on thyroid secretion rate values.

To accomplish this,

24 goats were divided into 4 groups.

Eight

animals r an gi ng in age from 3 to 6 years wer e - designated as Group 1.
Group 2 co ns isted of six 23^-year-old goats and 'Group 3 contained
five 2 ^ - y ea r -o l d t h io ur ac il -t re at ed animals.
of 5 immature, 8 -m o nt h- o ld animals.

Group 4 was made up

All mature animals were showing

the usual signs of estrual behavior at this time.

54

The r ec or de d temperature during this experimental period
was 50.15° F. _+ 6.6°.

The initial dose of thyroxine administered

to the matu re animals was 75 ug.

This initial amount was increased

by a 75 ug increment every third day.

The immature animals were

given a 40 ug dose and this was increased every third day by 40 ug.
A lt ho ug h 24 goats were used in this study,
could be d e te rm in ed on only 22 animals.

secretion rates

One aged animal died during

the experiment and was found to be heavily parasitized with stomach
worms.

Another animal

during the experiment.

in the thiouracil-treated group also died
However,

no autopsy report was received on

this animal and cause of death was not established.
Figure 4 shows output curves
old animals,

for the two groups of 2-year-

one group normal and the other thiouracil-treated.

The graph is defined by percent of initial count on the ordinate
and time in days on the abscissa.
count.

Peak uptake was taken as initial

The top curve re pr esents an average percent

of 6 animals.

initial count

The bottom line is an average of 4 animals.

Two days

after peak uptake had occurr ed thiouracil treatment was begun.

The

dose given was 0.1 gram per k ilo g ra m body weight twice daily at
12 hour intervals.

The output half-time

days and for the thiouracil
uptake thyroxine

for the normal

group 7.2 days.

is 11.07

Eleven days after peak

injections were begun and the thyroid secretion

rate was determined.
The r es ul ts of this experiment as shown in Tables 0 through
R in the Append ix reveal that a significant difference exists under
the c o nd it io ns of this experiment between the mean secretion rate

55

Figure 4.

Graphic illustration of thyroidal 1-131 uptake
'and output for both normal and t h io u ra c il iz e d
animals, and the effect of thyroxine injections
in graded doses on the output p o r t i on of these
curves.

56

Figure 4

too
80

o— o =

NORMAL
THIOURACIL

TH I O U R A C I L

k.

I
o
°

60

C:

|

40
THYROXINE

20

15

DAYS

57

of the aged animals
immature goats.
v alues de termined
_+ 0.037)

animals.

(0 243 _f 0.03) when compered to the young and

No significant difference was
for the yo un g

found between the

(0.336 +_ 0.018)

and immature

When the mean secretion rate of 0.379

mg./lOO l bs ./day ob ta in ed for the young,

found.

+ 0.04

thiouracilized goats was

compared to that calculated for the young,
ficant difference was

(0.403

normal animals no s i g n i ­

This p ar ti cu la r part of the experiment

will be d is cus se d in greater detail

in a later section.

Although all mature animals were showing the usual signs of
estrual be havior duri ng this experimental period there appeared to
be no significant difference between thyroid activity at this time
when compar ed to thyroid secretion rates obtained during February.
In oider to summarize the data obtained from all
periments the mean values
peak and 48 hour uptake
respective

for secretion rates,

five e x ­

output half-times,

for the various groups of animals and the

time of year are recorded in Table V-.

also give the same data in graphic

form.

Figures 5 and 6

The mean Vfe values for

all normal open goats for the entire year ranged from a low of
6.70 days r e c or de d for the you ng goats during the month of May
to a high of 12.55 days
July.

for the aged animals during the month of

Upon closer e xa mination of the data it may be seen that

during Fe br u ar y and May both aged and young groups of goats showed
a much faster output rate as compared to July, August and October.
During Fe br ua ry and May these values ranged from 6.70 to 7.33 days;
for July, A ug ust and October the range
values was 10.35 days recorded
12.55 days

for these output half-time

for the aged animals in

for the aged animals in July.

August

to

58

Figure 5.

A graphic su m ma ry c o m p a ri ng the mean
thyroid s ec retion rate da ta o bt ai n ed
from all five ex pe riments for the
various groups of n o n - l a c t a t i n g an imals
at various times of the year.

59

OCTOBER

Figure 5

3 t in j. V tA W I
9ND0A
039V

v w w i

AUGUST

3b m

9ND0A

I
------ 1

039V

\

JULY

9N D 0A
039V

MAY

9ND0A

FEBRUARY

039V

9ND0A
039V

O
<3

O'

o

A va j

o
o
o

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a

o
<\i
O'

o
o

C>

o
o

'S 9 1 0 0 / j 3 N !X 0 d A H l- l DAI

o
o
<3

60

Figure 6.

A graphic su mm a ry c o m p a r i n g the mean
t hyroid sec re tio n rate da ta ob ta in e d
at various times of the year for d i f ­
ferent age groups of l ac ta ti n g a nd nonl a c tat in g animals,

61

QNDOA

2.0 V I

9N D 0A

—I

H 2 0 V I - NON

039V
20V1
Q 39V

2 0 V 1 -N 0 N
9ND0A
'1DV1
H
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2 0 V 1 -NON
H
039 V
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039V

JULY

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AU G U ST

Figure 6

2 0 V 1 - NON 9ND0A

:'1D V 1 -N 0 N
H

o>
°0
05

20V1

MAY

2 0 V 1 9ND0A
039V
039V

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05

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05

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a

A V O /'S d l0 0 ! / 3 N IX O d A H I

05

- 7

DIN

62

With regard to both 48 hour and peak thyroidal iodine u p ­
take studies,

the greatest average amount taken up by the open

goats occurred in May.

The mean 48 hour uptake recorded for aged

and yo un g goats during this time was 31.9 and 32.40 percent of the
injected dose, respectively.

The peak uptake values during May

were 44.06 for the aged and 35.68 percent of the injected dose r e ­
corded for the yo un g animals.

The lowest average iodine uptake

values were observed during the colder weather.

In October

the

average amount taken up in 48 hours for the aged goats was 11.40
percent of the injected dose, whereas in the young animals this
value was 9.55 percent.

Average peak iodine uptake

was 15.14 and 14.80 percent of the injected dose
you ng goats, respectively.

for October

for the aged and

These October values were the lowest

re corded for the entire year.

In February the mean percent of i n ­

jected dose taken up ,in 48 hours by the thyroids of aged and young
goats r es pectively was 16.00 and 20.50

Peak uptake values during

this same experimental p e ri od were 20.56 and 25.60 for the aged and
young goats respectively.

In contrast to the open animals the l a c ­

tating goats took up smaller amounts of iodine.
uptake studies reveal

Forty-eight hour

a range in values from a low of 6.90 percent

of injected dose o cc urring in the young lactators during August to

I

a high of 15.20

found in the young animals during May.

With regard

to the influence of season on iodine uptake in lc,ctating animals a
trend somewhat

different is noted when these animals are compared

to the open goats.
corded in May.

In both cases the highest mean values were r e ­

The average peak amounts taken up by the lactators

63

d ur in g M ay was 15.47 and 16.70 percent of the injected dose for the
aged and lac ta ti ng goats,

respectively.

uptake values were observed during July.
this mean value was found to be 9.58;

However,

the lowest peak

Fof" the aged animals

for the young animals 10.41

percent of the injected dose was taken up by the thyroid.

In

August again there was an increased amount taken up as indicated
by the values of 14.05 and 10.53 for the aged and young animals,
respectively.

The reason for the lower values occurring during

the summer is belie ve d to be due to a loss of 1-131 by way of the
mammary gland.

This aspect will be discussed in more detail

in a

later section.
Output rates

for the lactating animals were similar to

those observed in the open goats.
curred in May.

The greatest output rate o c ­

The mean t1/^ values r ec orded during this month for

the aged and young goats r es pectively was 7.59 and 8.96 days.
July and August higher values were observed,
range of 13.40 days output half-time recorded
tators duri ng August

During

as indicated by the
for the young lac-

to a low of 10.05 days for the young lactating

goats during July.
The si gnificance of these

findings pe rtaining to thyroidal

1-131 uptake and output for both young lactators and nor.-lactating
animals,

as well as aged lactating and open goats,

will be discussed

further in a later section.
For the purpose of comparing the mean thyroid secretion
rate data,

Tables VI and VII have the test statistic,

or student t

64

value,

and degrees of fr eedom re co rd ed at the intersection of the

appropriate com pa ri so n columns.
In order to study the r el at ion sh ip between thyroid s e c r e ­
tion rate and uptake as well
active iodine,

as secretion rate and output of r a d i o ­

correlation coefficients

were calculated.

for these respective values

These data may be found in Table VIII.

As may

be noted in these tabulations, when the data concerning 48 hour
uptake and se cr et io n rate

for the entire year are compared,

relation coefficient value of 0.067 is obtained.
s ignificant correlation.

Similarly,

a cor­

This reveals no

with regard to a comparison

of output and se cretion rate during the entire year,

the correlation

coefficient value of 0.1685 r evealed no significant correlation.
When these data are broken down into sub-groups according to. months
again no co rrelation was observed,
August when an r value of 0.53

except

in the experiment during

(P less than 0.01) was found for 48

hour uptake versus secretion rate.

Similarly,

in comparing output

half-time with secretion rate no significant correlation was o b ­
served,

except during the month of May.

of 0.56

(P less than 0.01) was found.

out by Montoye

(1955)

At this time an r value
However,

it has been pointed

that the p os si bil it y of one significant value

out of several which are not significant may be due purely to
chance.

In this regard,

Wilkinson

(1951) has published binomial

expansion tables whereby it m ay be ascertained just what the
p r o b ab i li t y is of obtaining,
putations,
chance.

in a given number of statistical c o m ­

a significant correlation which is due entirely to

In the case of the present data,

one significant correlation

65

(P less than 0.01)

out of six was observed.

According to W i l k i n ­

s o n ’s findings the p rob ab il ity of this happening simply by chance
is 5.8 percent.

66

IN

ID

, SUMMARY OF THE MEAN VALUES OBTAINED
PERCENT UPTAKE AND THYROID SECRETION

FOR OUTPUT HALF-TIMES, 48 HOUR PERCENT UPTAKE, PE AK
RATES FOR ALL GROUPS AT VARIOUS TIMES OF THE Y EA R

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67

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68

TABLE VI
THE t V A LU ES A ND D EG RE ES O F F R E E D O M O B T A I N E D WHEN M EA N SEC RE TI ON
R A T E S OF VA R IO US G ROU PS O F A NI MALS A R E C O M PA R ED
WITH R E G A R D TO S E AS ON A N D A G E

MAY

FE BR UA RY
Y ou ng
t
D.F.

; t **
Young

Young
t
D.F.

Aged
t
D.F.
+

13.50

-

Aged
t
D.F.

9

1 .15

10

-

42

0)
Uh

+.+ + +
Aged

13.50

9

-

-

0, 42 0

-

9

+
1 .15

10

-

1.8

1.8

9

-

8

-

9

*
-

Aged

-

0. 42

9

* ** *
>2
3
•■3

-

-

May

Y oung

Young

6.43

**
10

-

2.64

****

_

Aged

-

3.44

* ***
9

3.20

-

10

-■ ............
* +* *
Young

4.42

10

-

1.63

8

-

-

-

-

12

-

1.44

<c
Aged

0 .0 4 9

10

1.14

13

4-------Oct.

1
Young

1

0.42

10

1
Aged

-

-

* + * + P ro bab il it y O.Ol
***
"
0.02
**
"
0.05

0.10

1.65

12

-

-

69

TABLE VI -- Continued

JULY
Young
D>,

t

AUGUST
Age d
D.F.

t

* ** t
6,43

10

t

J oung
D.F.

OCTOBER
Aged
D.F.

t

* ***

j

4.42

10 1

t

Young
D.F.

0,42

Age d
D.F.

t

12

i

i
j
i

*+**
3.44

9

1.65

12

j

**

1

2.64

8

1.63
______

** **
3.20

_

**

*

i
- j 0.049 10
i

10

9

1.69

i
;

*

3,78

9

-

-

+

1 .69

+

S
! 1.14

i
13

i
1
|

*

10

j
i
i

*

*

*

*

j

*

j

-

2.54 10

1
1

!

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7.16

8 j -

10

-

*

+

3.78
* **

8 : 1.44
t
i
__________ __

*

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i 4.00
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13

1

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0.433 9

_

8

4.97

10

-

:

1

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9

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13

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10

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*

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_

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*

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13

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13

*

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2.66

14

-

14

t VALUES

AND DEGREES OF FREEDOM
OF ANIMALS ARE COMPARED

OBTAINED WHEN MEAN SECRETION RATES OF VARIOUS
WITH REGARD TO AGE, SEASON AND LAC TAT ION

GROUPS

70

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72

GENERAL DISCUSSION

As p ointed out pr eviously the method employed in this study
has the advantage of allowing the quantitative measurement of th y ­
roid secretion rate in individual

intact animals.

Since it is not

necessary to slaughter or in any way change the animal physiologically
it is therefore possible to follow thyroid activity in the same
animals over a long p eri od of time.

A further point in favor of

this method is its relative ease of technique along with a minimum
of animal manipulation.
which should be kept

However,

there are certain precautions

in mind when obtaining data by this method.

In the present study a dose of 1 -(thyroxine equal to approximately
1/3 to 1/4 of the animal's daily secretion rate had to be a d m in is ­
tered initially and increased by that increment every third day.
For this reason,

the usual time required for actual measurement of

secretion rate was either 9 or 12 days.
this experimental

It was felt that during

time a great deal of change in thyroid activity

could p os si bly occur.

Since this m ethod is one which involves s u b ­

stitution its degree of accuracy is dependent upon a minimum of v a r i ­
ation in thyroid secretion during the experimental period.
hy pot hetical

example,

As a

if the first two points on the response curve

were obtained during a period of low thyroid activity,

these points

repre se nti ng percent of previous count would be high.

Likewise,

the last two points were taken during a time of high activity,
percent

of previous count would be relatively lower

if

the

than would be

73

exp e c t e d had the secretion rate remained constant.
this the degree of slope

As a result of

for the curve would be lessened to such an

extent that an abnormally high secretion rate would be calculated
which woul d not at all reflect the true thyroid activity.

However,

in view ol the good agreement between the data obtained in the
present study,
this method,

as well as the data of previous

investigators using

when compared with that obtained by other techniques

it appears that the hypothetical example cited above is a relatively
rare circumstance.

The more probable situation which exists is that

the v ariations in activity are of short duration and are such that
periods of high activity are compensated by periods of low activity
so that a radioactive measurement taken every
an average between these various

third day represents

cha_nges.

It should also be mentioned that a certain amount of d i f f i ­
culty may be encountered in obtaining a good correlation for these
dose response curves as a result of variable activity during the
experimental period.

As an example of this,

if thyroid activity

should be higher than normal as a result of an untoward e n v i r o n ­
mental change,

or some such variable

previous count based on 1-131 output

factor,

then the percent of

from the gland would be low.

The next value m a y be based on a return to normal

1-131 output but,

because the percent of preceding count is contingent upcn the
p rev iou s low value obtained,

This subsequent value will be represented

by a point which is abnormally high on the curve.

The problem is

further compounded by its inherent perpetuation resulting in a l ­
ternately low' and high points throughout the rest of the curve.

A

74

straight line fitted to such a curve would lie along the average
between the high and low extremes and give a good estimate of s e ­
cretion rate but the variation about the regression line would be
large.

This same type of curve may also result from improper t e c h ­

nique on the part of the

investigator when measuring the amount of

ra dioactivity over the thyroid.

Because of this, positioning of

the scintillation counter and the duplication of geometry during
successive determinations is of paramount importance.

In the present

study very gcod correlations were obtained.
A further point in regard to the technique concerns the
theory upon whi ch it is based.
depends upon the loss of 1-131
( L e r m a n , 1940;
1941; Perry,

Taurog,

1951).

loss of 1-131,
stream
release

The decline in thyroidal radioactivity
from the gland as hormonal

Wheat and Chaikoff,

1945; Perlman,

The rate of hormone secretion,

et a l .,

and therefore

depends upon the amount of pituitary TSH in the blood

(Taurog,

Chaikoff,

and Bennett,

from the pituitary,

1946;

Astwood,

1949).

TSH

in turn, depends upon the plasma con-

coneentration of thyroid hormone
Therefore,

iodine

(Uotilla,

1940; McGavick,

1951).

since the administ rat ion of exogenous hormone results

in a greater concentration of thjroid hormone in the plasma, an
increase in inhibition of TSH release would result
cedure.

from such a p r o ­

The degree of inhibition would be proportional

to the

amount of hormone in the blood from the exogenous source plus the
amount from the glandular source minus the amount metabolized by
body tissue.

These relationships may be theoretically pictured

m a t h e m ati cal ly as:

75

B + C - D
where

A = pla sma concentration

(1 )
of thyroxine

B _ amount of exogenous hormone administered

and,
blood,

C -

amount of hormone

released by the gland

D =

amount of hormone

metabolized and lost by excretion

since C- is proportional

to the amount of pituitary TSH inthe

it may be stated that
C = Ke

where

(2 )

e = amount of TSH in the blood stream
K - constant re pre senting the proportional relationship
between amount of hormone released by the gland and
blo od TSH levels;

and further,

e is inversely proportional

hormone in the blood.
A
where

to the amount of thyroid

This may be represented by the equation
(3)

e

k = constant r epr esenting the proportional relationship
bet we en blood thyroxine and blood TSH levels.

With these equations in mind,

it follows that as A is increased by

an amount of B of exogenous hormone,
TSH level

the value

is p rop or tionately decreased

"e" for' the blood

(3); and as

Me" is decreased,

the amount of blood thyroxine C is also proportionately decreased
(2).
amount

Therefore,
B

the smaller the value for C,

requ ire d to decrease it.

the smaller need be the

It is on this basis that the

un kno wn amount C can be measured simply by substituting for it the
k n o w n amount B.

The point of complete substitution can be determined

76

on the basis of no loss of 1-131 from the gland,

as indicated by

the measure of ra dioactivity in the thyroid gland.
practice this point

, In actual

is arrived at b y extrapolating the data obtained

from adm ini st eri ng several graded doses of thyroxine.
these graded doses equal

For each of

to B in our equation an amount C is still

b e i ng secreted by the gland.

In effect,

what is being measured by

this metho d is the metabolic demands of the body on the thyroid.
In view of the foregoing equations an alternate method of calculating
the secretion rate might be arrived at.

This would involve a slight

mo dif ication in the procedure so the.t only one injected dose level
would be required.

The percent decrease in the value of C as a

result of this injected dose would equal the percent of the secretion
rate injected.

The percent decrease in C w ou l d equal the difference

in percent output as determined before and after thyroxine treatment.
In other words,

the secretion rate is calculated by multiplying the

injected dose level by the quotient arrived at as a result of d i ­
viding the percent output before thyroxine treatment by the d i f f e r ­
ence in percent output before and after treatment.

This treatment

is of course theoretical and would require substantiation by e x p e r i ­
mental trial.
As m ay be noted in the tabulation cf results,
amount of radioactive iodine taken up by the
48 hour uptake,
experiments.

thyroid,

the peak
as well as

reveals a great deal of variation within and between

Although no conclusions as to the causes of these d i f ­

ferences can be drawn from this study the major factors involved a p ­
p ear to be season and whether the animals were lactating or not.

77

The data obtained in these experiments indicate the highest peak
uptake occurred during the late Spring and Summer months and in­
volved the n on -lactating animals.

Of these, the aged non-lactators

had the greatest uptake in all three of the Summer experiments.
With regard to the comparison of iodine uptake in lactating
and ncn- lac ta tin g animals,

the results can be explained on the

basis of the perm eab ili ty of the mammary epithelium to iodide.
cause of this iodine permeability,
pool

Be­

the size of the blood iodide

is reduced considerably in these animals by way of secretion

into the milk.

Therefore,

the thyroid of the lactating animal does

not have access to as much iodine as the non-lactator and consequently
not as much is taken up.
The

findings regarding seasonal variations in thyroidal

iodine concentrations are somewhat similar to those reported in
cattle by Seidell and Fenger

(1913) and Lodge

tigators noted that the highest
was

(1957).

These inves­

iodine content of cattle thyroids

found in the later Summer and the mi nimum iodine content in the

late Winter and Spring.

In this last respect the data obtained in

goats show a m arked difference.

Lodge found that the minimum iodine

uptake occurred in S pr ing (April - June), whereas in the present
study the greatest amount of iodine was found to be taken up by the
goat thyroid during the month of May.

Although it may be stated

that in general both goat and cattle thyroids take up more iodine
in Summer than in Winter, under the conditions of these experiments,
except during the month of August,

there is no correlation between

78

iodine uptake and secretion rate in the goat, as may be noted in
Table VIII.
In v i ew of the widespread use of thyroidal
an indication of thyroid activity,
are quite pertinent.
tool

1-131 uptake as

it is believed that these data

The use of such uptake studies as a diagnostic

is based on the assumption that the more active gland retains

the most iodine.

The data herein reported,

by the p reviously m entioned investigators,
opposite to be true.

Lodge

"that during the Summer,

as well as those obtained
seems to indicate just the

(1957) points out that such data suggests

when the actual secretion is low, a large

amount of iodine is collected by the gland and stored for future
use."
high

Likewise,
the lower

during the Winter months when secretion rate is

concentration of radioactive iodine mep.sured in the

gland represents the amount taken up minus the amount of hormonal
iodine secreted.

In ether words,

uptake minus iodine output.
rem ai n constant,

thyroidal

Therefore,

iodine equals iodine

if thyroidal

iodine were to

output would equal uptake and a decrease- in thyroidal

iodine would indicate an output greater than uptake.

Likewise,

an

increase in storage of thyroidal iodine would be the result of an
uptake greater than output.
"steady state",

This is in contrast

as reported by Dougherty,

to the so-called

et a l . (1951).

Evidence

obtained by these workers tends to show that an increase in secretion
rate is c ompensated for by an increase
versely,

in iodine uptake and, c o n ­

a decrease in secretion rate is accompanied by a decrease

in iodine being taken up by the thyroid.

As mentioned before,

the

results obtained under the conditions of the present experiments

79

seem to favor the theory that the amount of thyroidal
equal to the algebraic sum of uptake and output.
measurable p e r a met er of thyroidal
pendent upon two variables,

iodine is

In either case the

iodine at any given time is d e ­

namely uptake and output.

Uptake in

turn may be influenced by any circumstance which will alter the
bloo d iodide pool and this may account
in individual animals.

for the large variations found

An example of this is the lactating goat

which has already been discussed.

Other cases may be individual

differences in enterohepatic circulation and renal excretion.
genous iodine

Exo­

from known and unknown sources would also be expected

to affect the blood iodide pool.

Considering these factors,

the

large amount of variation in uptake within and between experiments
and its lack of correlation with the measured secretion rate is not
too surprising.
In revie wi ng the data concerning the thyroidal output halftime of iodine it will be noted that the greatest rate of release
occurred in the Winter and Spring.

The values obtained for Summer

and Fall

except the immature,

for all groups of animals,

a range of 10.05 to 13.4.

fall within

This difference between Winter and Summer

is to be expected since the thyroid is undergoing greater activity
during the Winter and therefore r eleasing a greater amount of h o r ­
monal iodine.

However,

if this explanation is accepted there are

certain incongruities to be noted in the Summer and Fall data.

Al­

though the thyroid secretion rate is significantly greater in the
Fall

than in the Summer the output half-time values recorded for

both of these periods are approximately the same.

Likewise,

the

80

secretion rate values
ferent,

for Fall and Winter are net significantly d i f ­

although a great deal of difference is found between the

output ha lf-times

for these two periods.

This situation not only

exists between various times of the year but also within experiments
as well.

This may be noted in Table VIII.

lack of correlation,

except

The reason for this

for the one instance during the month

of May, betw een thyroid secretion rate and output cannot be fully
explained on the basis of the present findings but it is strongly
felt that more

indicative results could be obtained if more c o n ­

trolled environmental

conditions as well as animals of more uniform

breed were to be used.
to this question,

A further point to be discussed in regard

however,

concerns the recycling of iodine and the

variability of iodine intake.
the experiment

Directly bearing on this subject is

involving the thiouracilized goats.

A comparison

between the output half-times of the thiouracilized and nonthiouracil

treated animals reveals a difference of approximately the

same magnitude as found between Winter and Summer t)fe values.
theless,

no significant difference was found between the mean s e ­

cretion rates recorded for these two groups.
Tables P and Q in the
normal,

Never­

Appendix,

the mean secretion rate for the

young goats is 0.336 _+ 0.018.

animals the value is 0.379
which is abnorm al ly high,
thiouracil iz ed group.

As may be noted in

+■ 0.042,

For the thiouracil

treated

A secretion rate of 0.502,

was calculated

for goat number 16 of the

It should be mentioned here that during the

last part of the experiment this particular animal appeared to be
quite ill and died one week after the termination of the experiment.

81

When the data obtained from this goat is eliminated the mean sec re­
tion rate for the t hiouracilized group is 0.338

+ 0.015.

The difference in tV2 values between normal and thiouracil
treated animals,
different,

even though their average secretion rates were not

could be attributed to two causes:

n on-thiouracilized animals,

1) In the case of the

a certain amount of recycling of 1-131

as well as 1-127 would be expected to take place.

This would be r e ­

flected in a. slower decline of radioactive count in the thyroid and
result in a longer output half-time.
pro b a b l y the more

important,

2) The second case, which is

is the difference in composition of the

thyroid iodine pools over a period of time.
animal

In the thiouracilized

the or ganification of iodine is blocked and therefore this

iodine pool would be expected to diminish over a period of time.
However,

the proportion of non-radioactive iodine to radioactive

iodine in the pool would remain constant and the thyroid hormone
released from the gland would therefore be labeled with the same
amount of 1-131 at one instant as at a later time.

But in the case

of the normal a n i m a l , iodide uptake and organification is unimpeded.
Therefore,

the size of the iodine pool should remain relatively c o n ­

stant over a short pe riod of time, but its composition with regard
to 1-127 and 1-131 would be changing.

The change would be such that

1-131 in the pool is continually being diluted by 1-127 and the
pr opo r t i o n of non-radioactive iodine to radioactive iodine would be
growing larger.

1-131 labeling of the hormone then wou]d become

i ncr easingly sme.ller.
not as much radioactive

The end result of such a situation is that
iodine leaves the gland over a given period

82

of time because of the diluting effect on the pool by 1-127.
also wou l d be refle cte d in a longer output half-time.
case, although there is a difference in t l/2 values,

This

In either

the same amount

of hormone wou ld be expected to leave the gland if all other things
are considered equal.

Since the method employed in this study is

one of substitution and acts through inhibition of thyroid s t im u­
lating hormone

from the pituitary,

it would be expected that similar

secretion rate values would be calculated.
Although the circumstances involved in obtaining the data
from normal

animals in the other experiments are admittedly not

comparable to those in the study just cited,

the possible

importance

of individual variations in recycling and iodine intake and their
effects on output half-time may be readily' appreciated.
Seasonal

differences in thyroid secretion rates have been

observed p r evi ou sly in both goats
sheep

(Hennemar,

et a l ., 1955).

(Schultze and Turner,

1945) and

The seasonal changes were noted in

goats by means of the goitrogen technique,

whereas the values o b ­

tained for sheep were determined by the same method as used in the
present study.

The mean secretion rates reported by H e n n e m a n , et a l .

for open two-ye ar -ol d ewes ranged from a high of 0.20 milligrams
daily during the mont h of March to a low of 0.04 milligrams
month of July.

The latter value was found to be significantly lower

than at any other month of the year.
observed in September
different

for the

(0.14 mg. ).

The next lowest secretion was

This was

from the value recorded for March.

found to be significantly
No quantitative figures

conce rn ing seasonal variation were included in the publication of

83

Schultze and Turner but

it was pointed out that the dosage levels

of thiourea and thyroxine which caused the thyroid

weight to return

to normal

resulted in much

in other similar animals in warm weather

greater thyroid weight wher. employed in the Winter.

On this basis

it was co ncluded by

them that a much higher secretion rate

in these animals in

cold weather.

also been reported.

Reineke and Turner

existed

Seasonal studies in chicks have
(1945) observed the summer

secretion rate in yo ung chicks to be approximately one-half the Winter
level.
The results
and Figure 5),

tend

parison to sheep,

of the present study,

it will be noted

to confirm the previous observations.

In c o m ­

goats of a comparable age and approximate weight

appear to have a much higher rate of thyroid activity.
secretion rate

(Table V

The mean

for the two-year-old goat was found to range

high of 0.336 ± 0,018 mg.

from a

1 -thyroxine per 100 pounds per day during

the month

of October to a value of 0.177 _+ 0.013 for July.

to sheep,

the secretion rate in goats

for July was

Similar

significantly

lower than any other time of the year studied except August.

Whether

the app roximately 50 percent reduction in thyroid secretion in summer
is due pri marily to the depressing effect of high temperature,

in ­

creased light exposure or a combination of both cannot be determined
on the basis of present

findings since these factors were not c o n ­

trolled .
For the purpose of comparing the mean secretion rates of all
non-lactat.ing animals according to age groups and for various times

84

of the year,

Table VI lists the t values and degrees of freedom at

the intersection of the appropriate comparison columns.
In c ons idering the data concerning the relationship of age
and thyroid activity it was

found that in February,

there was a highly significant

July and October

difference between the mean secretion

rates of the young and aged gop.ts.
secreting thyroxine at a lower level

Although the older goats were
in all experiments,

the reason

that no significant difference was found in May or August cannot be
fully explained at this time.

It is possible that this may be a t ­

tributed to changing environment during these times resulting in a
greater variabi li ty of response

in individual animals.

flected to some extent in the larger standard errors
these times.

Similar

Turner

(1948) observed a pronounced

decrease in secretion rate of older hens.
Long,

to age in cattle.

found at

findings of variations in age groups have been

reported for other species.

iodine technique,

This is r e ­

Using the protein-bound

et a l . (1951) have observed differences due

Henneman,

et a l . (1955) have reported a s i g ni fi ­

cant difference due to age when 2-year-old ewes were compared to
4-yep.r-old ewes during various times of the year with the exception
of July and September.

However,

in all cases the older ewes were

secreting thyroxine at a lower level than the younger animals.
In the study on the effect of pregnancy on thyroid secretion
the results obtained are in agreement with those obtained by H e n n e ­
man,

et al,

(1955).

These

investigators

found no significant d i f ­

ference in daily secretion rate between pregnant and non-pregnant
ewes.

It has also been reported by Monroe and Turner

(1948)

that

85

no signi fic ant

differe nce

contrast,

(1951)

Rugh

bett er p r o t e c t e d
greater output

from normal exists in pregnant rats.

In

o bse rve d that the l actating mouse thyroid was

from 1 —131 r a d ia ti on damage pr e s u m a b l y because of

from the gland.

Pr ev i o u s studies involving lactating animals have shown
that t h y r oid ac ti v i t y is si gni fi c a n t l y higher in these animals
when compared

to n o r - l a c t a t o r s .

secretion rate of 0 , 20 mg.

of 1-thyroxine daily for open 2-year-old

ewes and a value of 0.32 mg.
the month of March.
May.

H e n n e m a n , et a l . (1955) reported a

for l actating 2-year-old animals during

A similar significant difference was noted in

In contrast to these observations the results of the present

experiments,

as record ed in Tables A through M (Appendix),

show that

the n o n - l a c t a t i n g goats had a si gnificantly higher secretion rate
July than did the l a c t ati ng animals of co rresponding ages.

in

In May

and Au gust there was no significant difference between comparable
groups but

the n o r - lac tat or s again revealed a higher secretion rate.

Although these results elude explanation at the present

time,

ex­

pe rim en ts u t i l i z i n g adrenocortical hormones and their effect on
thyroid act i v i t y as m e a s u r e d by this substitution method might prove
to be a pr o d u c t i v e area of investigation.

Very few findings appear

in the literature r eg a r d i n g the probl em of the effect of lactation
on thyroid secr et ion rate.

To this author's kno wledge there are no

data con ce r n i n g se cr eti on rate in l actating domestic animals which
were o bta i n e d d uring the summer mo nths with which to compare the
pre se nt
be

findings.

theorized

Confr ont ed with this lack of information,

it may

that d uri ng the higher temperatures of summer the

86

p r o c e s s of l a c t ati on m a y place an added stress on the animals which
would shift the endocrine balance,

possi bly via the adrenals in such

a w a y that t h y r oid s e c re tio n rate is reduced.
ci rcumstances that the

It may be under these

feeding of thyroid materials offsets this

condition and in this way they exert their beneficial

effects.

87

SU M M A R Y AND CON CLUSIONS

The s u b s t i t u t i o n technique of H e n n e m a n , Reineke and Griffin
(1955)

for m e a s u r i n g t h y r oi d secretion rates in individual

intact

animals was a d a p te d to the study cf thyroid activity in dairy goats.
The a pp l i c a t i o n of this m e t h o d to these animals is described and
discussed.
The perc en t of the injected dose of radioactive iodine taken
up by the thyr oid was also investigated in goats of various ages,
during p r e g n a n c y and lactation and at various times of the year.
It was

found that although no correlation with thyroid secretion

rate existed,

with the exception of the experiment during August,

there was a d e f i n i t e l y increased uptake of iodine during the summer
months.

It was also noted that

take up less

the thyroids of lactating animals

iodine than n o o - l a c t a t o r s .

The implication of these

findings is discussed.
Output h al f-t ime s were determined for the same animals.
Again,

no c o r r ela ti on with sec retion rates was noted except

month of May.
pools.

These data are di sc uss ed in relation tc thyroid iodide

In con nec t i o n with this,

uraci li zed and non-th iou ra cil

the experiment con cerning thio­

treated animals

o bse rve d in these two groups of animals
release of

1-131 was different,

substitution

for the

is examined.

It was

that although the rate of

s ec retion rates determined by the

technique v/ere the same.

88

The thyroid secr et ion rate values obtained
v arious ages,

for goats of

d uri ng p r e g n a n c y and lactation and at various times of

the year are herein r ec ord ed and discussed.

The observations made

in the p r e sen t st u d y that season and age affect thyroid activity
are in line wi th the

findings of previous investigators.

pregnant animals do not differ
gard to thyroid s e c r eti on
ings.

In sharp contrast

That

from non-pregnant animals with r e ­

is also

in agreement with previous

to reports

in the literature

find­

it was

found

in these e x p e ri men ts that non-lacta ti ng goats secreted thyroxine
at a h ig h e r level during summer months then lactating animals.

Dif­

ferences d u r i n g the remainder of the year were non-significant.
Based on the present
is given.

However,

findings no explanation

for this occurrence

it is theorized that the adrenal cortex may be

implicated.
Co n c l usi ons which may be drawn from this w o rk include:
1. Higher tem pe ratures effect a lower
on a b ody weight basis in the goat
temp era tu res result
2.

level of hormone secretion

thyroid and,

conversely,

lower

in a higher level of secretion.

The rel a t i o n s h i p b e t w e e n age and thyroid activity is such

that lower levels of hormone secretion on a body weight basis occur
in the older goat as compared to the younger
3.

Altho ugh thiouracil

output rate

treatment

increases the thyroidal

1-131

the thyroid rate as measured by the substitution t e c h ­

nique is not sig ni f i c a n t l y different
goats.

animal.

in thiouracilized than in normal

89

4.

P r e g na ncy

in the goat does not effect a change in thyroid

ac ti v i t y as judged by hormone secretion on a body weight basis.
5.
able

R ad ioactive

iodine uptake studies do not constitute a r e l i ­

indication of thy roi d activity in the goat.
6.

Release rates of radioactive iodine

not always depend abl e

from the thyroid are

in de ter m i n i n g thyroid activity in the goat.

90

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103

AP PE N D I X

104

F O R M U L A E USED IN C A LCU LAT ION S

With r e g a r d to statistical

tr eat men t an d computations,

has a l r e a d y b een p o i n t e d out that straight lines w e re
all curves b y the m e t h o d of least squares.
lating output half-times,

the

formula used

it

fitted to

In the case of c a l c u ­
for fitting the str aig ht

line was
Y = a + (bx)
wh e r e

Y - log of perc ent of i n j e c t e d dose taken up by the
thyroid
x = days after output of 1-131 has been e s t a b l i s h e d
a = int ercept
b = slope

The d e t e rm ina tio n of

b

b =

was o b t a i n e d by the

Sxy

(Sx)(Sy)
-______ n________

Sx2 -

whe re

The

formula:

(Sx)2
n

N = the n umber of o b s erv at ion s
S = s u m m a t i o n of

formula for d e t e r m i n i n g

a

:

a - Y - (bx)
wh ere

Y
x

- mean value of Y
- mean value of x

The c a l c u l a t i o n cf outpul

h alf-time

(t1
^) was obtai ned by the

formula:

tVz = 0. 693
-B
To d e t er min e

-B

:

-B = 2.3

T
w h e re

A

- initial activi ty

1 g

A
_t_
A
o

r

105

A

t

T

- a c t i v i t y at an a r b itr ar y time

interval after A

_ the a r b i t r a r y time interval bet we en A

The va lues

for A q and A^ w er e taken

o

and A

o

t

from p oints situa te d on the

fitted line.

In c a l c u l a t i n g the es timated secretion rate,

the

formula

again u s e d to fit a straigh t line was:
Y = a + (bx)
where

Y =,p er cen t of previo us count
x = daily 1 -thyroxine dose in micrograiris

As was m e n t i o n e d previously,
used to pre di ct

a different

form of this equation was

the dose at which 100 pex-cent of previous count o c ­

c ur red or the amount of 1 -thyroxine which would have been required
to c o m p l e t e l y inhibit

1-131 output

from the thyroid.

A coe ff i c i e n t of correlation was determ ine d on each of the
es t i m a t e d se c r e t i o n rate curves using the
r2 =

a Sy + bS(xy)

formula:

- N(Y2 )

SY2 - N(Y2 )
where

The

r r co rr e l a t i o n coeffi cie nt
Y = p e r ce nt of pr ev i o u s count
x - dai l y thyr ox ine dose in micrograms
a :intercept
b
- slope
N -- number of observa tio ns
Y = m ean per ce nt of previ ous count
S = summation of

"r" value

for each individual

the t ab ulated res ult s in the

det ermination will be

following section.

found in

106

The m ea n estimated, se cre tio n rate

for each group of

a n i m a l s wa s d e t e r m i n e d and the s ta nda rd error of the mean was
calcu la ted u s i n g the formula:

S.E.

where

=

S x 2 - (Sx)2
N
N - 1

x = e s t i m a t e d s e c re tio n rate
N = n u m b e r of o b ser vat ion s

To det erm ine

the existence of significant differences b e ­

twe en the m e an s of an y two groups,

the following calculation was

made:
ml

"

m2

(S.E.1 )2 + (S.E.2 )2
wh ere

T =. test statistic
m^

- m e a n of group 1

m2

- m e an of group 2

S.E.^

- s t a n d a r d error of group 1

S*E._

= s t a n d a r d error of group 2

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