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LD3907
•G7 Cohen, Herman, 1915- 1
1951 The Iso latio n and dem onstration of a
•C55 non-estrogenic u terin e stim ulating and
estrogen augmenting substance from
pregnant m ares’ urine*
34p. ta b le s,d ia g rs•
Thesis (Ph.D .) - N.Y.U., Graduate
school, 1951*
B ibliography; p .31-34.
1 .E strogen. 2 .D issertatio n s, Acadc Ac -
N.Y.U. - 1951. I.T itle . II.T itle:N on*
estrogenic u terin e stim ulating;and
estrogen aug menting substance.
Official File
Xerox University Microfilms, Ann Arbor, Michigan 48106
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.
THE ISOLATION AND DEMONSTRATION OF A NON-ESTROGENIC UTERINE
STIMULATING AND ESTROGEN AUGMENTING SUBSTANCE JROM PREGNANT
MARES’ URINE
Herman Cohen
Spring 1951
A. dissertation in the Department of Biology submitted in partial ful
fillment of the requirements for the degree of Doctor of Philosophy at New
Yorlt University.
TABLE OF CONTENTS
I Introduction ............ pages 1 - 3
II Material and Methods 3 - 5
III Results ...................... 5 -1 7
IV Discussion................. 18 - 27
V Conclusions ............... 27 - 29
VI Addendum 30
VII Bibliography . 31 - 34
VIII Tables and Figures
INTRODUCTION
The presence of substances capable of augmenting the affects of
androgens, although possessing no androgenic activity themselves, was
demonstrated by Freud and co-workers (1933, 1935), who obtained such
substances from extracts of testes and human urine* The occurrence
of similarly acting materials in the extracts of human urine, capable
of augmenting the effects of estrogens was also postulated and demon
strated by various workers. Emmens (1938) described the presence of
substances in the phenolic fraction of normal human female urine which
in themselves are non-estrogenic but when given orally, increased the
injected potency of estriol, Emmens (1939) also demonstrated that
in normal human female urine, fractionation .of the urine into a
phenolic, neutral, and acidic fraction and assaying each fraction
separately, will yield a total unitage which is less than that of the
original starting material and also less (about 50 per cent) than
that obtained when the fractions are recombined. He also reported
that the acidic fractions were capable of augmenting the effect of
both estriol and estrone. Similarly, Pincus and Pearlman (1941) ob
tained data indicating that normal men and women exorate non-ketonic
materials which enhance the activity of urinary estrogens and that
these materials are fractionated by the Ccbon and Marrian (1936) pro
cedure. Smith et al. (1939) reported that pregnandiol and 2,7-
dihydroxynapthalene enhanced the estrogenic effect of estrone on the
vaginas of spayed rats, but the extent of the potentiation was not
described.
Sheinerman and Gershtein (1947) have reported that injection of
-3-
asoorbic acid with folliculin would increase the uterine weight of
immature mice about three times as much as folliculin alone, which ob
servation could not be confirmed (Bates, 1949)* Schiller (1946) pre
sented data showing that the estriol fraction obtained from pregnancy
trine of humans was more active than similar amounts of crystalline
estriol in causing withdrawal bleeding in the castrate monkey, and
attributed this to the presence of enhancing substances ini the crude
extract not present in the crystalline estriol* However, these two
preparations were equally potent in the rat assay.
Inasmuch as such substances had been demonstrated in human urines,
it was deemed of interest to examine the extracts of pregnant mares*
urine which are used therapeutically to see; if such augmenting sub
stances are present therein and to attempt to characterize and deter
mine their mode of action. Such an investigation seemed to be of
particular interest inasmuch as it has been shown that crude estro
genic extracts are pftp- clinically more active than are similar
quantities of crystalline hormone in oil solution (Rakoff, 1949;
Allen, 1944)*
Our attention was further focused on the problem by observations
made during the course of extraction of estrogens from pregnant mares*
urine. At this time i t was noted that biological and chemical assays
were invariably at variance, Hbwever, If these crude extracts were
separated into ketonia and non-ketonio fractions then the correlation
between biological and chemical assay of the ketonic fraction was
excellent, whereas the poor correlation* still remained when the non-
ketonic fraction was examined. In all these oases of studies
on the non-ketonio fraction the biological assay was usually two to
■3-
three times as great as the chemical assay (Cohen and Bates, 1947;
Bates and Cohen, 1950)• These discrepancies led to a systematic
fractionation of the noxfc-ketonic materials with concomitant studies
as reported below*
ACKNCffT^nMRKTO
1) The author is indebted to Dr. H. A. Charlpper far his constant
encouragement, guidance and critical advise during the course
of this investigation, and for his valued advice both as a
teacher and friend.
2) I wish to express my gratitude to Dr» Robert W. Bates of the
Squibb Institute for Medical Research for his invaluable aid
and for his encouragement throughout the course of this work.
3) 1 am very much indebted to S. R. Squibb & Sons for their kind-
ness in allowing the facilities of their laboratories and equip
ment to be used during the course of investigation.
METHODS AND MATERIALS
Commercially prepared concentrates or fractions of pregnant
mares' urine* were used. These concentrates were prepared by the
usual solvent fractionation methods and were ostensibly phenolic in
nature. These phenolic fractions were separated into ketonio and
non-ketonic fractions by means of Girard's P reagent (Girard and
Sandelescu, 1938).
The non-ketonic fractions were subjected to this treatment
twice more in order to assure removal of all the ketonio estrogens.
The non-ketonic fraction was then dissolved in toluene and frac
tionated into a 0,1N NaOH soluble fraction, a IN NaOH soluble frac
tion and a neutral fraction as illustrated in Figure 1. The term
"neutral" as used here is to be interpreted as meaning the apparent
ly non-phenolio fraction remaining in toluene after extraction with
0.1N and IN NaOH.
Bioassays were performed by a modified Kahnt- Doisy method
utilizing adult ovariectomized rats. Each extract was assayed at
three dose levels using 20 to 40 animals per dose. About 150 rats
per dose were used to determine the effect of the augmentor on
crystalline estrone. For bioassay purposes the extracts were dis
solved in corn oil and the injections given in two doses of 0.1 ml.
each at 5 hour intervals. Forty-eight hours later vaginal smears
were examined for signs of estrus. As a standard, crystalline estrone
dissolved in corn oil was used at three dose levels. For studies
on the effect upon the uterus, 21 day old ovariectomized female rats
*Used in the preparation of Amniotln (Squibb).
were employed. Injections were given once a day in 0.2 ml. volume
of com oil, for three days. On the fourth day the animals were
smeared to determine absence or presence of vaginal cornification.
The uteri were then excised, liquid expressed and weighed on a
torsion balance to the nearest tenth milligram.
RESULTS
1) Demonstration of Augmentation -
The non-ketonic fraction obtained from preparations 71509 and
70722 was fractionated according to the scheme in Figure 1. Each
fraction thus obtained was bloassayed. They were then recombined
and bioassayed. Results are presented in Table I. The data pre
sented in Table I demonstrate^ the presence of substances in preg
nant mares' urine which, although estrogenically inactive by them
selves, are capable of augmenting the estrogenic activity of the
other estrogenic fractions. It oan be seen that the 0.1N NaOH
fraction contains 50 per cent of the total starting estrogenic
activity while the IN NaOH and the neutral fractions contain about
3 per cent and 0.3 per cent, respectively. When these are recom
bined and assayed, the full estrogenic potency as obtained with the
starting material is recovered.
2) Localization of Augmentor -
From the above data (Table I) it was inferred that the aug
menting factor or factors are located either in the IN NaOH or the
"neutral" fractions, which fractions by themselves have little or no
estrogenic activity. In order to determine in which of these two
—6—
fractions the augmenting factor is present, the following experiment
was carried out.
The 0.1N NaOH, the IN NaOH and the "neutral" fractions were
bioassayed individually. Bioassays were then obtained upon
combinations of the 0,1N NaOH fraction with the IN NaOH frac-
tion and of the 0.1N NaOH fraction with the "neutral" fraction.
The results are presented in Table II, These data show that
the augmenting materials are present in the "neutral" frac
tions, inasmuch as with the "neutral" fraction which has no
estrogenic activity itself, the estrogenic activity of the
0,1N NaOH fraction is increased by about 90 per cent. No
increase in activity was obtained when the IN NaOH fraction
was injected in conjunction with the 0JLN NaOH fraction.
3) Mode of Action of Augmentor -
Having localized the fraction wherein the augmenting materi
als are present, it became of interest to determine whether the aug
menting action was systemio or whether it was due to the presence of
substances which delayed the absorption of the active estrogens at
the site of the injection. To that end the following experiment was
carried out.
The 0.1N NaOH fraction, dissolved in corn oil, was injected
subcutaneously over the shoulder of the assay animal in two
injections of 0.1 ml. each at 5 hour intervals, and increasing
amounts of the "neutral" or augmenting fraction, in 0,1 ml,
com oil, were injected subcutaneously over the hip at
similar intervals. The results obtained are presented in
