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Xerox University Microfilms
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- s
«G7 Deutsch, Marshall E 1921-
1951 Studies on the thyroid."1
•D4- iii,129p. tables.
Thesis (Ph.D.) - N.Y.U.. Graduate
School, 1951.
Bibliography:p .59-71,126-129«
1.Thyroid gland. 2.Dissertations.
Academic - N.Y.U. - I95l«
C80I4.73
V Ul f
Xerox University Microfilms, Ann Arbor, Michigan 48106
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.
STUDIES ON THE THYROID
Marshall E. Deutsch
April 2, 1951
A dissertation in the department of Physiological
Sciences submitted in partial fulfillment of the
requirements for the degree of Doctor of Philosophy
at New York University..
The author wishes to acknowledge his indebtedness to
Professor I. Greenwald who' initiated these studies,, and
under whose direction these investigations were carried on.
Grateful acknowledgement is also due to Professor
A. S.. Keston for guidance in Section II of this work.
i i
Zus'X'i
Contents'
Seotion I.. Effects of Thiouracil-Administration on Mice 1..
Historical Introduction 2.'.
Background 2..
Physiological effects on lower: animals and plants 4..
Physiological effects on birds 6;.
Physiological effects on rats 9*-
Physiologlcal effects on mice 23..
Physiological effects on other animals 27^-
Physiological effects on humans 31..
In vitro effects 33..
Present Studies 36..
Experimental 36'..
Results 39..
Discussion of results 50..
Summary' 55 ••
Bibliography 59.,
Appendix 72..
Section II.. The Presence of Thyroxine in Thyroglobulin 84..
Introduction 85..
Historical background 85..
Isolation of thyroxine by chemical methods 88;.
The isolation of thyroxine following enzymic 91.-
hydrolysis
Critique of chemical and enzymic procedures 92.".
Demonstration of the presence of thyroxine by 97».
ultraviolet absorption
Use of radioactive iodine to demonstrate the presence98;.
of thyroxine
Methods for the determination of thyroxine in hydro-102*".
lysates
Present Studies 107;.
Experimental 109..
Thyroglobulin preparation 109..
Determination of amino and ammonia nitrogen con— 111.,
tents of hydrolysates
Blau hydrolysis and chromatography 111.
Preparation of radioactive thyroxine 114.
Isotope dilution experiments 117-.-
Results 120..
Amino and ammonia nitrogen of Blau hydrolysates 120..
Chromatography of Blau extracts 121..
Isotope dilution 121.
Discussion of results 122..
Summary 124.,
Bibliography 126;.
iii
\
Section I
I
Effects' of Thiouracil-Administration on Mice*
Aided by grantsrto Dr.. I.. Greenwald from the Committee
on:. Research in Endocrinology of the National Research
Council,, and the United States Public Health Service..
1*.
Historical Introduction
Background.
2-Thiouracil (hereinafter called thiouracil) was first
described, by Wheeler and Bristol (294) in 1905. It was then
obtained by condensation of thiourea containing some pseudoe-
thylthiourea (later found to be unnecessary) with ethyl sodium
fDrmylacetate.
In 1908, Wheeler ana Lidale (295) gave some details of
the properties possessed by thiouracil, as well as an improved
technique for its synthesis. In their paper, the# reported a
decomposition point of about 340° and a solubility of 0.0533
gfcam per 100 grams water at 17° when starting with a hot
saturated solution, 0.0598 gram per 100 grams water when
starting at room temperature. Differences of opinion as to
the solubility of this compound will be discussed along with
the methods used in the present study.
Very little further study of the chemistry of thiouracil
was made until after Astwood discovered its remarkable biolog
ical properties in 1943 (14). The sequence of events leading
to this discovery is quite interesting and will now be discussed
briefly.
In 1928, Chesney, Olawson, and Webster (41) observed goi
ters in rabbits used for studies of syphilis and raised on a
3-
diet of fresh cabbage daily, oats three times a week, and hay
once a week. Subsequent studies by Vebster and Chesney (292)
and other authors (176) shov.red the goit-rogen to be a oomponent
of the cabbage in the diet, and indicated that other plants of
the genus Brassica (111, 176) were also goitrogenic.
Finally, in 1$42, Kennedy (146), while investigating the
goitrogenic effect of rape seed, discovered that allylthiourea
produced thyroid enlargement when administered by stomach tube
to rats..
In the same year, Richter and Clisby, studying the thio
urea derivatives lethal to rats, developed a tolerance to
phenylthiourea in some of these animals by administering sub-
lethal doses for a few days, and then, gradually increasing the
doses over several months (237)• Autopsy of animals so treated
revealed thyroids which were hyperplastic and had histological
features considered by the authors to be characteristic of
exophthalmic (sic) goiter..
The observation of Kennedy and Richter and Clisby led
Astwood (14) to study the effects of a large number of deriva
tives of thiourea on young female rats. He found that thiouracil
was the most effective of the 106 compounds (including some not
chemically related to thiourea) which he tested for goitrogenic
activity. A maximal response (gross and histological) was pro
J
duced by 0.02 or 0."ii thiouracil in the drinking water or 0.10
-■
or 1.00$ in the food of the animal for ten days. Alkali was
used to obtain the higher concentrations in the drinking water.
4.
The higher level in the food led to the formation of coincretions
in the urinary tracts of about one-third of the animals, but
those who survived this complication and received the drug for
five months "showed no symptoms or signs other than those attrib
utable to a state of hypothyroidism" (14).
Astwood"s discovery led to further studies of the chemical
and physical properties of this compound (47, 156, 201, 241, 248,
250, 302) as well as to publication of more than three hundred
papers on its physiological effects.
The salient chemical properties of thiouracil, which, like
its parent pyrimidine "base", is an acid, are its reducing pov.-er
(47, 201, 260, 302) and its ability to form complexes with cop
per (155). Thiouracil has also been shown to be bound by pro
teins (302)..
Physiological effects on 3*wer animals and plants.
Thiouracil has been shown to influence many types of living
beings. The growth, on synthetic media, of many species of bac
teria (including Eschirlchia coll) was inhibited by it (57, 259,
310) and this inhibition*As reversed by uracil (259, 510).
However, thiouracil, in common with other reducing, agents, has
been reported to stimulate the oxidation of glucose and of
1
fructofuranose-l,6-aiphosphate by Eschirlchia coll (43). Low
concentrations (1/3500 M) of thiouracil inhibiWthe increased
oxygen-consumption ordinarily resulting from addition of certain
substrates (not including; glucose) to suspensions of grevrer's
yeafct, while higher concentrations (1/350 to 1/175 M) temporarily
increases/ the oxygen-uptafcfe of yeast (221) in the presence or
absence of glucose.
5
Thiouracil has been reported to prevent germination of
cress seeds (276). Uracil reversed this effect.
The oxygen-consumptlon of an annelid (269) and of an
insect embryo (31) have been shown to be stimulated by thio
uracil. The drug has been shown to inhibit the development
of fertilized sea-urchin eggs (267, 268).
In the phylogenetic scale, thyroids first appear in the
chordates (103). The most primitive chordate on which the
effects of thiouracil have been recorded is Mustelus laevls,
a species of dogfish in which the force-feedinm of the dru’g
induced hyperplasia and hyperemia of the thyroid, resistance
to anoxia, and markedly less loss in weight than was observed
in controls (during fasting) (270). A mechanism of action
which involves Inhibition of thyroid secretion is compatible
with the results.
The naturally-occurring metamorphosis of several species
of amphibian larvae was delayed or prevented by thiouracil in
their culture fluid (53, 95, 96, 167, 181). Thiouracil also
interfered with the production of metamorphosis by Injected
thyrotropic hofcmone (126), but die. not Inhibit the stimulation
of metamorphosis by thyroxine (126, 235). Thyroidectomy like
wise inhibits metamorphosis, and the conclusion reached by
Hughes and Astwooc (126) is that thiouracil prevents the pro
duction of thyroid hormone.
The effect of thiouracil in inhibiting the pigmentation
of amphibia (129, 140, 167) may have been due to formation of
copper complexes (140).
In the adult frog (183), injection of thiouracil into a
