Estrogens and progesterone in the posterior vena cava and estrogens in the urine of cattle
by Pran Nath Varman
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE in Animal Science (Animal Physiology)
Montana State University
© Copyright by Pran Nath Varman (1964)
Abstract:
Cannulation of the posterior vena cava of four Jerseys and one Holstein (non-gravid) and one Jersey
(gravid) was performed. Blood samples were collected every other day until the tubing plugged* Urine
was also sampled at the same time* The samples of blood were analyzed for the estrogens and
progesterone and of urine for the estrogens* Two peaks in the total blood estrogens occurred in all
cows sampled* In the majority of the cows the first peak occurred during the period of days 6 to 8 and
the second during the period of days 12 to 17 after heat* Similarly, the quantities of the excretion of
estrogens in the urine reached two peaks* In most of the cows the first occurred during a period
between days 3 and 8, whereas the second peak occurred between days 15 to 20, after heat* The levels
of progesterone reached a peak level on day. 17 Of the estrous cycle* During thirty-four days
postconception only one peak of the total estrogens in the blood and urine, corresponding to the first
peak observed during estrous cycle, was found to occur. The possibility of the interplay of the
gonadotropic hormones, progestins, estrogens and the estrogen-inactivating mechanism has been
postulated.
) ESTROGENS AND PROGESTERONE IN THE POSTERIOR VENA CAVA
AND ESTROGENS IN THE URINE OF CATTLE
by
PRAN NATH VARMAN
A thesis submitted .to the Graduate Faculty in partial
fulfillment of the requirements for the degree
of
MASTER OF SCIENCE
in
Animal Science
(Animal Physiology)
Approved;
- ------H e a d e Major Department
.—
^ ______ ________
Chairman, Examining Committee
'.Dean9 Graduate Division
MONTANA STATE COLLEGE !
Bozeman„ Montana
December, 1964
-iiiACKNOWLEDGEMENTS
The author expresses his sincere appreciation to all the members
on the staff of the Animal and Range Sciences Department for their
cooperation and encouragement throughout the period of his stay in
Bozeman,
Special reference must be made to acknowledge the generous help
and valuable guidance given by D r 0 Ervin P 0 Smith during the entire
course of this work and in preparation of the thesis.
Thanks are also
due to D r 0 J 0 C 0'Boyd for awarding the author a research assistanship
in the form of a grant, which made it possible for him to come to the
United States and pursue graduate work at Montana State College 0
Sincere appreciation is expressed to Professor F 0 S 0 Willson,
D r 0 D 0 W 0 Blackmore, D r 0 El-Negoumy and D r 0 L 0 G 0 Young for helpful
suggestions during the research program.
The author is grateful to D r 0 Graeme Baker, who allowed the
author to work in the Chemistry Laboratoryj to D r 0 M 0 W 0 Hull fgr
performing the surgical operations and cannulation; to D r 0 R 0 R 0 Roehm
and the Home Economics Department who made the D 0U 0 Spectrpphotometer
available and from time to time provided the necessary guidancej and
to Jack Taylor for taking the photographs 0
Sincere appreciation is extended to the Department of Veterinary
Science and Animal Husbandry and "The Ministry of Agriculture", Punjab
State, India, for the opportunity which they gave to the author to come
to the United States for higher studies.
;
t
I,.....'.i
=>iv~
L a s t , but not the least, the thanks are due to the parents and
fnendj? an Ipdaa who made it possible for the author to pursue the
post-graduate studies.
Perhaps without their sacrifice, it would have
/■
■
not been possible to reach the United States of America,
z
TABLE OF CONTENTS
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INDEX TO TABLES
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ABSTRACT o o a o o o o o o o o o o o o o o o o o o a o o
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INTRODUCTION
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LITERATURE REVTEWo
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DEFINITIONS OF ESTROGENS AND PROGESTINS = , » , »
10
Ho
Estrogens
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Progestins
,
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METABOLISM OF ESTROGENS AND PROGESTERONE>
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Biogensis> 6 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
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Ho
Estrogens
0
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Progesterone =
0
0
0
0
0
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0
0
0
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0
0
0
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6
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Interconversion of Estrogens
Co
Inactivation of Estrogens and Progesterone
I,
Estrogens>
0
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0
0
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0
6
6
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Progesterone O o o o - o o o o e o o o o o o o
Bound Progesterone »
EXCRETION OF ESTROGENS0 v
0
0
0
0
0
0
0
6
0 0 0 0 0 0 0 0 6
0
0
0
0
0
0
0
7
8
9
Conjugated and Bound Estrogens O O O O 0 O
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o o o o o o o o o o o
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EXTRACTIONit ISOLATION AND MEASUREMENT OF ESTROGENS
AND PROGESTERONE 0 0 0 0 0 0 » » « » » » 0 1 1 0 0 0
A.
Extraction of Estrogens
Bo
Isolation of Estrogens»
Co
6
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19
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By Paper Chromatography
2«
Adsorption Column Chromatography 6 0 6 0 6 0 6
20
3«
Thin Layer Chromatography » /« « »
20
6
6
6
6
6
Quantitative Measurement of Estrogens
O
6
0
6
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6 - 0
O
O
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E»
Isolation Of Progesterone
Fo
Quantitative Measurement Of Progesterone
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DISCUSSION AND C O N C L U S I O N .................... ..
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LITERATURE CITED o
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SUMMARY o o o o o o o o o o o o o o o o o o o o o o o o
A P P E N D IX o
25
0
6
Levels, of Estrogens in Blood Plasma and Urine
During the Estrous .Cycle.,. 0 » » » » 0 » » » » ». » .»
Levels.of Estrogens.in Blood Plasma.and Urine
During First 34 Days After Conception 0 0 6 0 0
21
21
O
Extraction -Of Progesterone FyomrBlood And
Other Tissues »' » » » . » » » » » » » » »
RESULTS o o . ' O o
!I.
6
Io
EXPERIMENTAL PROCEDURE.
I.
15
0 6 6 6 0 0 0 6 0 6 6 6 6 0
■Kober Reaction.. « » » « -» » »' »
D.
15
6
0
0
0
0
o o o o o e o o o o e o o e o o o o o o o o - o o
70
81
INDEX TO TABLES
Showing the R values of progesterone and
contaminating substances contained in the
plasma e x t r a c t .on thin layer chromatography „ „
„
A measure of the quantities, of estrogen ..secretion
.at different .periods .of the estrous..cycle* ., .
.Cows ..no* I. to. 5 & i & 0 - 0 0 - 0 0 6 6 6 0 6 6 • 6 0 0 0
0
A measure of the quantities of estrogen, secretion
at 'different .periods., of..the. estrous.. cycle during
po&tconception,period*...Cow,.no* 6 0 0 j-Aa O 6 6 6 .0
6
A measure of the quantities of estrogen excreted
at different.periods of the estrous cycle from the ,
uri n e .. Cows no* I to 5 0 0 . 0 0
0
0
O.e 0 0 6 0 0 0
A measure of the quantities of estrogen excreted in
the-urine, at ..different, periods, of the estrous .cycle
during postconception.period* .Cow.no* 6,
'0 0 6 0 0 0
Plasma progesterone at various states,“of the estrous'
cycle 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6 0
-vili-INDEX TO FIGURES
Figure
Io
Page
The occuranee of a double peak in the levels of
estrogens during menstrual cycle of a female ■
chimpanzee o o o o o o o o o o o o o o o o o o
15
2o
Standard curves of pure estrogens for Kober1reaction .
4-3
3,
The levels .of .estrogens, (micrograms, per, milliliter)
.
in-.bloo4 -plasma. during,..estrous... cycle, of ..cow., no 0.Io e„ o. o «
46
.
4o
5o
The levels ,of .estrogens (micrograms per,.milliliter)
• in'blood plasma...during..estrous .,cycle, of cow. no, 2 » » « o o
The levels of estrogens (micrograms per milliliter)
in blood plasma during., estrous .cycle., of :
COW -no o -3 and .4 o . O O - a . O . O - O O . o O O - O O . o O - O . O O
O
0
O O
47
48
6,
The l'evels..of. estrogens, (micrograms,..per .milliliter), ............
in blood. p.lasma._during..estr6us1..cycle..of.cow.;nOo S o . o ^ . o ,
.49
7o
The. levels ,.of ,estrogens ...(micrdgrains-. per .liter) excreted
in urine during...estrous. cycle, of. cow ..no o. I .o,o .o ..o o,. o.o
52
The levels of .estrogens ..(micrograms, per. liter)'excreted .
in- urine .during, estrous cycle of .cows .No.,.2 and 3. ...
..
53
The l!eyels,,of..estrogens,, (micrograms,..per.liter) excreted
in urine during, estrous cycle, of ,,cows. no,. ..4. and.. 5.. ... .... .
54
8o
9.
IOo
The levels, of.estrogens .(micrograms per milliliter) in
■ - - blood plasma, of cow no. 6 during, posteonception. period.,. . .
Ho
55
The. levels of estrogens (micrograms per .liter) excreted
in the urine, of cow no. 6 during postconception period
.=
56
12o
Standard curve of progesterone.for.ultraviolet assay «
..
57
13.
The levels of progesterone (microjgrams per milliliter)
in blood plasma during estrous cycle . . . . . . . . . . .
58
-ix™
• ABSTRACT
Cannulatipn of the posterior vena cava of four Jersqys and one
Holstein (non-gravid) and one Jersey (gravid) was performed6
Blood
samples were collected every other day until the tubing plugged.
was also sampled at the same time.
Urine
The samples of blood were analyzed
for the estrogens and progesterone and of urine for the estrogens.
Two peaks in the total blood estrogens occurred in all cows
sampled.
In the majority of the cows the first peak occurred during
the period of days 6 to 8 and the second during the period of days
12 to 17 after heat.
Similarly, the quantities of the excretion of
estrogens in the urine reached two pea k s ,
In most of the cows the
first occurred during a period between days 3 qnd 8, whereas the
second peak occurred between days 15 to 20, after heat.
The levels of progesterone reached a peak level on day. 17 Of-the
estrous cycle.
During thirty-four days postconception only one peak of the
total estrogens in the blood and uri n e , corresponding to the first peak
observed during estrous cycle, was found to occur.
The possibility
of the interplay of the gonadotropic hormones, progestins, estrogens
and the estrogen-inactivating mechanism has been postulated,
)
INTRODUCTION
It has been recognized for many years that one of the larger
problems concerned in the physiology of reproduction of animals
(especially the cattle) is low fertility rather than sterility.
Many
factors contribute to low fertility and thus cause great economic loss
to the animal industry.
Disorder of the endocrine glands is probably
one of the most important factors,
Another problem being faced by research workers interested i n '
the reproductive physiology is inconsistent results of synchronization
of the estrous cycle of domestic animals.
Similarly the physiologists
engaged in the study of ova-transplantation in cattle have not as yet
completely succeeded.
It seems evident that there is a lack of knowledge concerning the
physiological levels and the interactions of various hormones concerned
with reproduction.
This lack of knowledge may be responsible for the
Veterinarians not being able to satisfactorily diagnose or treat some
of the endocrine disorders of the animals.
This may also very well be
responsible for the unsatisfactory responses in synchronization and
ova-transplantation,
The levels of the estrogens circulating in the blood„ as well as
being excreted in the urine of normal women during menstrual cycle,
have already been established.
Whereas there has been no report which
has shown the day-to-day changes in the levels of these hormones during
the estrual cycle of domestic animals.
In contrast to relatively higher concentration of estrogens in
a=2the urine 8 the quantities of these hormones in the blopd obtained from
the jugular vein of the cattle are extremely Iow0
It is known that the posterior vena cava drains bipod from the
hind portion of the body and also receives blood from the ovaries 0
uterus (also placenta in pregnant individuals) and the adrenals„
Collection of the blood from this vein before much dilution and inacti­
vation of the sex hormones has taken place can possibly be advantageous„
Considering the problems mentioned above9 the purpose of this
study was
s
(1)
To cannulate the posterior vena cava at a site close to
all the points of secretion of the sex hormones„
(2)
To determine the day-to-day changes in the levels of the
endogenous estrogens and progesterone in the blopd and
estrogens in the urine of cattle during the estrous cycle.,
(3)
To determine the relationship of the secretion and
urinary excretion of the estrogens <,
REVIEW.O F .LITERATURE.
Io DEFINITIONS OF ESTROGENS AND PROGESTINE
A* Estrogens:
Estrogens s, a Greek W o r d 9 is made up of two words 9 Oistros0
meaning "mad desire" and "Gen" referring to "to beget" (Stedman6s
Medical Dictionary)„
It i s „ therefore6 evident that an estrpgenic
compound refers to any substance which produces estrus whether derived
from the ovary or not.
In general9 estrogenic compounds may also
produce growth of secondary sexual characteristics* changes in
vaginal m u c o s a 9 and proliferation of the endometrium=
A hormone is
defined as a physiologic* organic substance liberated by living cells
of a restricted area of the organism which diffuses or is transported
to a site in the same organism where it brings about an adjustment
that tends to integrate the component parts and actions of the
organism (Zarrow9 1962)„
Estrogenic substances found in animals are
referred to as natural estrogenic hormones because these compounds do
fit the preceeding definition of hormones„
Chemically 0 estrogens are steroids and are characterized by an
aromatic Ring=A and a hydroxyl group at carbon atom-3=
Estrane is
considered to be the parent compound of the estrogens„
The major
'
estrogens of mammals are estrone* IVs^ and 17B=estradiol0 and estriol
(Fieser and Fieser 1959)=
The nomenclature of thef-two" isdmdrs of estradiol refers to the
"cis" or "trans" position of the hydroxyl group-at carhop-17 position=
“4“
The isomer with "cis"-hydroxyl group is designated by the prefix "B"
while that with "trans" hydroxyl group as the VJ' (Turner, 1961)„
Progestlns s
Another major group of hormones of reproduction is known as
Progestins0
"Pro" (Greek) means "in favor of" and gestatio (Latin)s
from gastatuss meaning "to bear" (Stedman’s Medical Dictionary).
This
generic term was suggested for substances regardless of chemical
purity, which cause changes in the endometrium, adapting it for the
■■
■■
reception of the fertilized ovum (ova in.multiparous animals).
Progestins also, may partially, regulate the periodicity of the sexual
cycle, stimulate the mammary glands, inhibit uterine musculature and
relax the pelvic ligaments (Turner, 1961).
Progesterone, also a steroid, is the most potent natural
progestin.
Theoretically, allopregnane and pregnane are the parent
substances for the progestins.
II. METABOLISM OF ESTROGENS AND PROGESTERONE
A. BIOGENESIS
Estrogens
s
The estrogens have been isolated and identified from various
tissues and endocrine glands of the body.
These hormones, in the
female, are produced by the ovary, placenta and adrenal cortex and in
the male by the testis and the adrenal cortex.
Like other steroid
hormones and unlike nitrogen containing hormones', -estrogens are not
stored in the organs concerned with their b i o genesis■( O ’Donnell and
-5Preedy„ 1961)„
It is generally thought that estradiol-17B is the principal
estrogenic hormone produced by the ovary and is the mother substance
of estrone and estriol (Boscott6 1 9 6 2 6 Harrow and Mazur9 1962)»
To
determine the sites of biogenesis of estrogens„ extraction and identi­
fication of these hormones from various tissues have been performed„
H o w e v e r 9 Nyman e t . a l 0 (1959) have pointed out that the presence of
a steroid in an endocrine organ is no proof of its synthesis at that
site* nor does the ability of the tissue to metabolize a steroid
prove that the products ,are released into the blood stream in
physiologically significant amounts*
Human ovaries obtained from patients primed with FSH are able
to convert acetate into estrone* estradiol* and cholesterol (Ryan and
Smith* 1961a)*
Similarly* Heard e t * a l * (1956)* and Werthessen e t * a l *
•(1953) Jiave reported the conversion of acetate to the estrogens in
pregnant mare and sow ovaries respectively* but Hollander and Hollander
(1958) during in vitro studies with dog ovaries could not demonstrate
the conversion of acetate-l-Cl4 to C11^estradiol-ITB and -instead some
radioactive cholesterol was isolated*
Conversion of acetate to choles­
terol in the rat has also been reported (Caspi e t * a l * 1962),
Bloch (1945) pointed out that the animal organism is capable of
synthesizing the steroids which it normally requires,
It was*
therefore* thought that the compounds possessing the cyclopentanophenanthrene structure arise either individually by synthesis or by degrade=
—6—
tion from a common precursor0
Cholesterol has been suggested to be
the precursor of the steroid hormones (Bloch, 1945, Ryan and Smith,
1961c, and Werbin e t o a l 0 1957)»
Huffman e t o a l o (1940) proposed that progesterone could function
as an obligatory or main intermediate in normal estrogen formation»
Ovaries from already primed humans are able to convert p r ogesterone-C^
into radioactive estrone and estradiol (Ryan and Smith, 1961b)6
Although estrogens have been isolated from the human placenta^ Ryan and
Smith (1961b) were unable to detect any in vitro conversion of pro=
gesterone to estrogens in human placental system.
Very recently Aasted
and Stakemann. (1963) have reported that the estrogens of human pregnancy
are produced neither by the placenta nor by the fetal adrenal aloxie
but by
complex procedure involving both tissues.
It has, therefore,
been suggested that fetal adrenals produce a steroid precursor which is
metabolized into estrogens by the placenta,
Nathanson and Towne (1939) demonstrated that the use of testos­
terone propionate in human female castrates leads to vaginal cornification and increased urinary estrogen levels, decreased follicle stimulat­
ing hormone titres and increased urinary androgens„
The in vivo conver­
sion of testosterone propionate by. ovariectomized and andrenalectomized
w o m e n , to estrone and estradiol-17B has been reported by West e t , a l ,
(1956),
Estrone and estradiol are promptly synthesized when testis
of the gonodotropin primed stallion are perfused with horse blood and
sodium acetate (Nyman et; al, 1959),
This may be due to the presence
-7of a relatively high proportion of L e y d i g 's cells in stallion testis0
Similarly pregnant mares have also been found to be able to convert
testosterone into the estrogens (Heard e t „ a l 0. 1956), and for the con­
version of testosterone to estrogens in human ovarian tissue (Bagget
e t a a l a 1956, Wotiz e t a a l a 1956), in placental tissue (Ryan, 1959a)
and in rat ovaries (Stitch e t « a l a 1962),
Ryan (1959b) demonstrated that 16ot-hydroxyestrone (a metabolite
product of androstenedione) is metabolized by the placental system to
form estriola
It was suggested that aromatization of an already 16-
hydroxy lated androgen may also give rise to estriola
It is known that 19-nortestosterone when administered to humans
is converted to estrone and the magnitude of conversion is similar to
the one observed on the administration of testosterone«
These findings
indicate that the absence of the angular methyl-group ( 0 1 9 ) does not
significantly influence the extent of the conversion (Engel e t a a l a
1958)a
Progesterone s
Allen and Wintersteiner (1934) were the first to isolate pro­
gesterone from extracts of corpus Iuteum0
It is generally believed
that progesterone is synthesized by all the steroid-producing glands
via the acetate^cholesterolepregnenolone^progesterone pathway (Samuels
1955) a
Although there is no direct evidence, a general agreement exists
that the cells of the theca interna in the graafian follicle are
” 8”
responsible for the estrogen production«
The granulosa cells s" after
they have hypertrophied to form the corpus Iuteum9 are responsible for
the production of progesterone (Short 1961)0
Teledy eto a l » (1963)
have reported that in dogs 9 during estrous cycle 8 no progesterone could
be detected in ovarian venous blood when the corpus luteum was not
presento
Similarly, Huang and Pearlman (1962) have indicated that the
r a t ’s corpus luteum in contrast to h u m a n ’s does not form estrogens and
■.
'
instead is responsible for the formation of progesterone0
It is
interesting to note that cow adrenals are capable of secreting as muph
as Io5 mgo progesterone per hour (Balfour and Comline 1957)«
' Bo INTERCONVERSION OF ESTROGENS
Following the isolation of estrone from the urine of pregnant
women by Doisy e t o a l 0 (1929)9 this ketonic estrogen was considered to
be the hormone of the ovarian follicle =
In 1936
„however 9 MacCorquodale
e t o alo were able to identify ITekestradiol as the principal estrogenic
hormone of the liquor folliculi of the swine ovary 0
From time to time
various workers have demonstrated the interconversion of estrogens»
The conversion of eO-estradiol into estrone in man (Heard and Hoffman
1941) and into estrone and B-estradiol in guinea pigs and rabbits
(Fish and Dorfman 1941) has been demonstrated*
of estrone to estriol and estradiol-17^,
Similarly the conversion
in rats (Wotiz e t * a l * 1958b)
and in humans (PearIman and Pincus 1943) has been demonstrated*
It
has been suggested that both estradiol-17bC and estriol may very well be
the metabolites of estrone*
Intestinal microorganisms have also been
-9shown to be able.to reduce estrone to estradiol (Beck 1950)6
The conversion of estradiol=17B to other estrogens zIiag also been
VS
reported by many w o r k e r s 0
Engel et, al, (1959) demonstrated that the
human fetal liver slices are capable of converting estradioL=17B into •
estriolo
The conversion in man of estradiol=17B into 16-Ketoestradiol-
17B (Levitz e t 0 a l e 1956) ? and 2«=methoxyestrone (Kfaychy and Gallagher
1957) have also been demonstrated0
Ce INACTIVATION OF EgTROGENS AND PROGESTERONE
Ie'ESTROGENS
The maintenance of circulating estrogens at a physiological
level is dependant to a considerable extent on the inactivation qf the
estrogens by metabolic conversions o
*
A highly effective mechanism for
»
«
■
the capture of the free estrogens is in existence in the body tissuesD
It has been demonstrated in rats (Oakey e t 0 a l 0 1962) and in humans
(Fishman et„ a l 0 1960) that within 30 minutes after the administration
of e x o genous'estrogens9 abqyt 98 percent of the total free hormone
cannot be recovered from the blood 0
If an ovary is transplanted into the mesentery„ or estrogen
pellets are implanted intrasplenically„ the hormone would be inacti­
vated by its passage through the liver prior to its entrance into the
systemic circulation0
This shows that the liver is a very important
i' ■"
organ for the inactivation of the estrogens (Coppedge e t i -a l 0 1950)„
Inactivation of estrogens may take the form of conjugation and
elimination from the body 9■and metabolism to known and some unknown
=10=
compounds„ and/or oxidation (Eccles e t c a l p 1962)6
Very little is
known about the nature of the inactivation products, of gstrogens but
the evidence exists that estbbnq and estradiol are .attacked by an
oxidative enzyme system (HellersI 19’ij-O)„
The liver is thought to form non-steroid estrogen metabolites
as degradation products of estrone (Jellinck9 1959)0
Similarly*
Werthessen etc a l 0 (1950r) have demonstrated that estrone on incubation
with blood medium loses its IV=Ketosteroid group as well as its estro­
genic activityo
The possibility of the presence in blood of an enzyme
capable of inducing oxidation of estrohd tp a form whose nature is
unknown has therefore been suggested.
Conjugated and Bound Estrogens:
The term conjugated means a probably chemical union such as
sulfuric acid or glucuronic acid with a hydroxyl group on a steroid
molecule to form an ester.
Whereas binding means any association of a
steroid with a protein (Antoniades e t . a l . 1957).
In 1938* Schachter and Marrian reported the isolation of
potassium salt of estrone sulfate from the urine of pregnant mares.
Similarly the occurance of estrone-hydrogen sulfate in the urine of
pregnant women (McKenna et. al. 1961) and estriol in women (Levitz et.
a l . 1958) has also been established.
Conjugation of diethylstilbestrol
with sulfuric acid was suggested to be occurring in rat* cat nnd dog
but not in rabbits (Starnes and Teague 1949)* whereas its conjugation
with glucuronic acid in the rabbit had already been demonstrated by
-11Mazur and Shorr (1942).
Zimmerberg (1946)sindicated that the conjuga­
tion of diethylstilbestrol is perhaps the preferred method of inactiva­
tion.
Oxidation of this synthetic estrogen occurs only when the liver
cannot handle it by conjugation.
Diczfalusy (1953) reported that a large proportion of estrogens
in the pregnancy blood or plasma occurs as conjugated, presumably
glucuronides and sulfates.
Both conjugated and unconjugated radio­
activity in extracts of various protein fractions of plasma have been
demonstrated by Antoniads et_. a l „ (1957),
Similarly Purdy et, al.
(1961) found that plasma obtained two and one-half hours after the
administration of C1^ e s t r a d i o l to women* contained C14-estrpne sulfate
as the principal radioactive metabolite.
It wag, therefore* syggested
that estrone sulfate may be ah important transport form of estrogens
in human plasma.
DeMeio et. al. (1958) have shown that Mg++ and
adenosine triphosphate are essehtial for the biosynthesis of the
sulfates of estrone and estradiol-17B.
The presence of estrogenic lipoprotein complex in the circula­
tion of several species has been reported by many workers.
Estrogen-
serum protein binding in the presence of surviving rat liver tissue
occurs selectively with albumin (Szego and Sidney* 1956) and this
mechanism is correlated with the functional state of the liver (Szego9
1953),.
It has also been reported that the enzymetically catalyzed
association of estrone or its metabolites with albumin is inhibited or
a b o lished'by increasing the concentrations of cortisol.
This shows a
“12—
competitive phenomenon exhibited by the estrogens and certain cortico­
steroids in vivo (Szego and Sidney9 1956)»
Reigel and Mueller (1954) have indicated that the Iiver8 to a
much greater extent than other tissues (uterus 9 submaxillary glands and
kidneys)8contains an enzyme system which catalyzes the formation of a
protein-bound estrogen»
This, system requires an electron-ddhating
Substrate9 an electron-transporting coenzyme (TPN) and Oxyg e h 9
suggesting thereby an oxidative or perioxidative step in the binding
process,
Erlanger e t , a l , (1959) h a v e estimated that at legst twenty
estrone residues are linked to each molecule of bovine serum albumin,
B-glucuronidase 9 an enzyme which can hydrolyze estrogenglucuronides9 has been found to exist in the liver and spleen of calves
(Bernfeld e t , a l , 1953)j spleen of calf (Bernfeld and Fishman 1953)j
ox spleen (Mills 1948),
The major portion of the glucuronidase
activity of the bipod is concentrated in the leucocytes and platelets,
B-glueuronidase activity has also been demonstrated in Saliva9 gastric
ju i c e 9 spinal fl u i d 9 urine and tears (Fishman e t , a l , 1948),
2, PROGESTERONE S
The inactivation of progesterone is brought about either by its '
conversion to other metabolites or the binding with serum proteins.
Short (1961) has indicated that if an assumption is made that the
progesterone molecule can undergo reduction at C ^ 8 Cj. and
^ theoreti­
cally, twenty-six possible progesterone metabolites can be formed.
Eleven of these are already known to occur in human tissues qr urine.
"13”
The generally accepted scheme for representing reduction of
progesterone involves arrangement of the metabolites in order of
increasing state of reduction (Taylor 1955)»
The scheme indipates that
progesterone is reduced initially, at the double bond in Ring=A to yield
5ot and 5B"pregnene"3s20"dione which undergo reduction ft C™3 to the
three pregnanolones9 Sp^hydroxy-5o<"pregnan”20"one 9 SB-hydroxy-So^
pregnan"20?phe and 3o(=hydr6xy"5B"pregnan"20-one0
The latter is then
reduced at C^20 to give BB-pregnane-Sots 2OcXrdiol (pregnanedipl) „
Bound-Progesterone g
There has been no report which has shown that progesterone
exists in the body in a conjugated form.
When incubated with whole
blood very little progesterone enters the red-cells and instead its
major portion remains confined to the plasma (Short9 1958a)„ •
Westphal e t 0 a l 0 (1955) observed that when progesterone is added
into human serum albumin or blood serum and the mixture is ^un on paper
electrophoresis9 over 90 percent of the added progesterone migrates
with the protein fraction0 . When it is added to rat serum albumin only
50 percent migrates with the protein fraction9 indicating prpnounced
spedies difference0
It has also been indicated that not more than one
albumin mtilecule associates with each molecule of progesterone (Short
1961)o' Eik-Nes e t o a l p (1954) have reported that the albumin fraction
of plasma proteins is responsible for the solubility of the progesterone
in the blqod-whereas globulins have no measurable effect of such kind.
“14“
!Ho
EXCRETION OF ESTROGENS
Between 50-80 percent of the estrogens in the blood are present
5
as closely bound to plasma proteins,
Presumably this prevents the
bound hormone from being filtered out of the blood as it passes through
the glomerulus of the kidney (Villee 1961)»
Excretion of estrogens in
various excretory products has.been studied by many workers»
It has
been observed that the major portion of estrogens excreted in the urine
is in a conjugated form,
As such the estrogens are firmly united to
such hydrophilic groups as sulfuric or glucuronic acid so that their
solubility in the urine is increased»
The low binding capacity of these
conjugates with serum albumin explains their rapid filtration through
the glomeruli of the kidney into the urine (Eik-Nes e t » g.l» 1954)»
In
cattle the excretion of the estrogens in urine during gestation is
lowest on the 50th day and an increase in the excretion at an accelerated rate occurs during the last part of gestation (Nelson and Smith
1963)o
Similarly the urinary estrogenic content on the IOth day after
parturition has been shown to be equivalent to 251 ug» of estrone per
liter as compared to a value of 642»5 ug» found on the 275th day of
pregnancy (Smith e t . a l » 1956)»
Assay of urinary estrogens in primates has often shown double
peaks (Everett 1961)„
The occurrence of a double peak in the levels
of estrogen excretion in the urine by a female chimpanzee (Fish et» a l »
1941) is shown in figure no» 1»
:- - - !- - - >- - - S- - - !- - - 6- - - 1- - J- - - 6- - - 1- - - »- - - ) - - S - - - 1_ _ _ I_ _ _ c_ _ _
'I
5
10
'
r
15
fr
«
■_
>
»
20
»
)
I
^
<
25
d
-
30
Days Of Menstrual Cycle
.Figure.-Is
T.o.tal. ^ s t r o g e n .excretidn-by _a-female-chimpanzee. ...
(from Fish et^> al» 1941)
. .
The occurrance of such two peaks in normal human female's has
also been demonstrated (Markee and Berg 1944j Pedersen-Bjergeiard and
Pedersen-Bjergaard 1948)0
The first peak has been observed to occur
on day 12 and the second on day 21 of the menstrual cycle„
Nd sudh
peaks have ever been shown to occur in the bovines=,
I V a EXTRACTION* ISOLATION AND MEASUREMENT OF ESTROGENS AND PROGESTERONE
A c -EXTRACTION OF ESTROGENS
It is known that the urinary estrogens exist in a conjugated F o r m 6
To free these hormones some kind of hydrolysis is necessary=,
Stimmel
(1946) acidified urine samples (to Congo red) with hydrochloric acid
and then extracted with Oi125 volume of butyl alcoholi
After £he
extractionj the butyl alcohol was evaporated and the residue was taken
up in 100 m l o of -0„2 N NaOH solution which was further diluted to 400 ml.
= 16™
volume with W a t e r 0
This mixture was then acidified with I O 0O m l 0
of concentrated hydrochloric acid and autoclaved at 15 pound steam
pressure for 3 hours 0
The ffed estrogen Cphtent9 after hydrolysis9 was
then extracted with Pther which was ultimately washed with 9 percent
'
y'
-
NaHCOg splution and finally with water.
'
The recoveries of estrogens
added t'q ap essentially estrogen™free urine (from castrated female)
extract for estrone and Ot-Pstfadiol9 and Pstriol were 80 to H O
percent
'
.
and 57 to 94 percent respectively,
F r i e d g o o d .et, a l , (1948) hydrolyzed the urine samples by using
■
30 volume percent of 6N sulfuric acid (equivalent to 15 volume percent
of concentrated hydrochloric acid)«
The mixture was refluxed for -10 ■■■
minutes and extraction was then achieved with ether.
The quantitative
removal Qf estrogens from the ethereal extract was accomplished through
a reduction in their solubility by the addition of carbon tetrachloride.
The estrogens were then extracted with IN=KOH solution whereas the
neutral steroids were found to remain in the organic phase (pthers
carbon tetrachloride as Is 18),
Potassium hydroxide phase was then
acidified with 6N=sulfuric acid (to Congo red) and extracted with ether
which was ultimately washed with sodium bicarbonate solution and water.
The recoveries of added estrone 9 estradiol and estriol were found to be
‘
91,5-99,9 percent, 91,6-100 percent and 91,0-98,0 percent respectively,
Stimmel (1949) studied the effect of the addition of 4 mg,
percent zinc along with 15 volume percent hydrochloric acid on the
hydrolysis pf urinary conjugated.estrogens.
It was o b s e r v e d t h a t under
-17such treatment estrone was converted to estradiole whereas 15 volume
percent of hydrochloric acid alone and refluxing for 10 minutes gave
the maximum recovery of 90 percent.
The next highest recovery was
obtained with 5 volume percent of sulfuric acid and refluxing for 10
minutes.
Similarly Engel e t . a l , (1950) reported that higher yields of
estrogens are obtained after autoclaving human pregnancy urine with
15 volume percent of 12N sulfuric acid for 5-10 minutes than after
refluxing for 10 minutes with the same concentration of the acid.
In 1 9 5 4 9 Katzman e t , a l , found that pregnancy urine containing
15 volume percent concentrated hydrochloric acid when boiled for 30
minutes gives the highest recovery whereas Preedy and Aitken (1961)
have reported that maximum recoveries are obtained by refluxing the
urine samples with 15 volume percent concentrated HCl for 45 minutes,
Anyway9On hydrolysis with such a concentration of acid for 30 minutesB
the free estrogens are found to be quite stable (Katzman e t , a l , 1954),
Even though acid hydrolysis for extraction of urinary estrogens
is so widely used, the use of some enzyme preparations for this purpose
is another alternative,
Katzman e t , a l , (1954) have indicated that
the presence of chromogenic material in urinary extracts when acid
hydrolysis is used, can be a major obstacle to the use of the Kober
method for the estimation of e s t r o g e n s T h e
h a s , therefore„ been advocated.
use of enzyme preparation
Beer and Gallagher (1955a) have
supported the report of Katzmah et, al, (1954) that glucuronides form
the major fraction and-sulfate esters a very minor fraction of estrogen;=
eoISe=
esters in the urine»
This is perhaps the most probable reason why
only B-glucuronidase enzyme has been widely used (Kinsella et» a l 6 1956;
Beer and Gallagher 1955a and 1955b)„
P-myI a s e 9 which contains
phenolsulfatase-enzyme and "glusulase"* an enzyme preparation which
contains both glucuronidase and Sulfatase9 have also been vised
(Kinsella eta alo 1956; Givner et» alo 1960; Ladany and Finkelstein
1963; Veldhuis 1953),
Mather (1942) studied the distribution of estrogens between
immiscible solvents a
The distributions between benzene of ether and
0a3-M sodium carbonate solution were recommended for separating estriol
from other estrogens0
It has well been established that a large proportion of the
estrogens circulating in the blood are in a conjugated form (Antoniads
> !I
e t a a l a!957; Purdy et» ala 1961)»
It is also known that estrogens are
transported in the blood in the form of an estrogenic-lipo-protein™
complex (Szego and Sidney 1956)»
I t 9 therefore* seems evident: that
i. h
some kind of hydrolysis is essential to set the estrogens free a
Preedy
and Aitken (1961) have pointed out that there is no difference in the
-. ■ '
procedure, for the urinary and plasma estrogen isolations except some
minor differences in the handling", :
Similarly the procedures for extraction of estrogens from ovarian
follicles (MacCorquodale e t » a l » 1936)9 endometrium (Szego and Samuels
1943)* ovaries (Westerfeld et» a l » 1938) and fetal cotyledons
'
(Veenhuizen eti a l l '‘I960)"resemble the procedure used for urine or ■
/
“19blood estrogenso
B 0 ISOLATION AND PURIFICATION OF ESTROGENS
It is possible that the unwanted substances present in the
biological extracts may interfere in the quantitative analysis*
It i s 8
therefore9 advisable to isolate the concerned products in their pure
forms o
Several chromatographic procedures which can successfully be
applied for this purpose are available*
I*
By Paper Chromatography:
Paper chromatographyg which has been used for the separation of
estrogens and other compounds9 is relatively simple*
By this method
homogenous material is concentrated in one position rather than distri­
buted over a series of fractions as in column partition chromatography*
This technique has some disadvantages ass I) large quantities of the
* material cannot be handled so that the plasma extracts containing much
lipids cannot be chromatographed directly9 2) the resolution is poor
( O ’Donnell and Preedy 1961)*
Many solvent systems with various solvent combinations have been
reported (Oakey 1961; Axelrod 1953; Reineke 1956; Veenhuizen e t * a l *
1960; Burton et* al* 1951a)*
Burton et* al* (1951b) observed that
replacement of water by certain polar organic solvents
overcomes the
tailing and that the bulky urine extracts can be applied in a narrower
•zone along the starting line when formamide rather than propylene
glycol is used as the impregnating solvent*
absolute methanol
( 1 1 1
by v o l u m e i s
Formamide„ when added to
considered to be a good stationary
I
="2P=>
phase (Burton e t a a l a 1951b; Axelrod 1953)«
2a
'
Adsorption Column Chromatography;
Adsorption column chromatography has been used very extensively
for isolation and purification- of estrogens in the extracts of biological
fluids and tissues.
The method has the advantage that large amounts of
the material can be processed.
The disadvantages of this procedure
are the difficulty in the standardization of the adsorbent, moisture
content, tailing and relatively large volume of the eluate ( 0 ,Donnell
and Preedy 1961),
Adsorbents such as celite (Bauld 1955; Givner e t , al, 1960),
alumina (Stimmel 1944; Umberger and Curtis 1949) and silica=gel (Beer
and Gallagher 1955a arid 1955b; Leyitz d t , a l , 1956) have been used to
■-prepare the column fpr this technique,
- •• 3,
Thin Layer Chromatography;
^
Lisboa and Diezfalusy (1962) have very recently reported the
;■
use of thin layer chromatography for the separation and characterization
of the estrogens,
The said authors have reported .the Rp V a l ties for
i':’
' '
24 phenolic estrogens in various solvent systems," This method has also
been found to be very useful by Ladany and Finkelstein (1963),
The
method has a favorable resolution, reproducibility and high precision.
Time given for running chromatograms is less than 2 hours.
When applied
■ as a strip line, sufficiently large quantities of the extract,can be
handled,
...
^
•
•
Similarly, countercurrent distribution ‘had also been uded for
J
-21=
the isolation of the estrogens (Engel et_0 a l 6 1950),
Co QUANTITATIVE MEASUREMENT OF ESTROGENS
-
Various methods for the detection of estrogens in paper chromato-
• graphy and thin layer chromatography have been successfully used,
Turnbull's blue method (estrogen spots when treated with solution con=
taining equal volume of I percent aqueous ferric chloride and I percent
aqueous potassium ferricyanide9 give blue coloration) which in fact is
i
a general method for the detection of phenols3 has proved to be of parti­
cular importance ( O ’Donnell and Preedy 1961),
Axelrod (1954) has
cautioned that even corticosteroids* androgens9 urinary pigments and
«
".,V 1
impurities can give positive results with this .method0
The technique
of dipping the chromatographic strip in 15 percent fuming sulfuric
acid for one minute and then observing
under an ultra-violet-lamp for
fluorescence* has also been used (Axelrod 1953)«
The fluorimetric (Goldzieher e t 0 alo 19j52 and 1 9 5 4 1 Veldjhuis 1 9 5 3 1
Slaunwhite e t c, a l « 1951% Nako and Aizawae Nocke 1961% Purdy -eto a l 0
1961)* enzymatic (Hurlock and Talalay 1957) and bioassay techniques
(Allen and Doisy 1923% Emmens 1950* Freud 1939* Lauson e t o a l 0 1939e
Evans and Varney 1941) have also been quite extensively used for the
quantitative measurement of estrogens0
Kober color method which has
been extensively used for the estimation' of estrogens is reviewed in
the following Sectiqn0
'Kober R e a ctions
In 1931* Kober discovered that when estrin is heated with con=
'
-22centrated sulfuric acid and phenol it gives rise to a yellow color0
On
addition of water and reheating the mixture„ the yellow color is con­
verted into a highly specific pink color
(W x 9 522
mu).
Anyway9 the
pink color thus produced is not very stable and the impurities contained
'
in the biological extracts containing estrin produce an intense brown
color which can mask the color due to estrin.
Venning et. al. (1935)
ewe# ivmonis
indicated that the light absorption of the test mixture should be
measured not only at 522 mu but also at 420 m u e the latter being the
wavelength of maximum absorption of brown color.
By doing so the light
absorption due to brown component at 522 mu could therefore be calculated
from its absorption at 420 mu and thereby by subtraction, the absorption
at 522 mu due to the pink component is obtained.
It has also been st^dwn
that there is no other substance* except estrogensg in the iirine. of
humans which can produce a pink color with the Kober reagent (Venning
e t . a l . 1935).
The compositions of the reagent and its amount used*
the preheating time to produce yellow color * amount of water added to
the yellow mixture and the reheating time are the factors which can
influence the test results.
Bachman and Pettit (1941) found that the treatment of the final
•"
■■
crude color product with hydrogen peroxide * as originally suggested by
Kober (1938)* reduces but does not eliminate the absorption due to the
impurities.
The said authors also pointed out that the methpd for the
correction of absorption *•due to the brown color * proposed by Venning
et. al. (1935)* does not permit reliable determination of the estrogens
-22abecause the spectra of the individual brown colors from different
samples vary too much.
In 1950, Allen suggested a correction formula which permits the
analysis of mixed absorption curves provided the absorption curves of
the contaminating substances closely approximate a straight Iine0
This
has been found to be true in the case of urinary estrogenic extracts
(Allen 1950),
The correction equation is as follows:
CDXx
=
-ODx -
t■•,PPb
,
Where ODx = observed density ,at wavelength "x" as the absorption
maximum
OD q and OD^ ■= the observed densities at wavelengths "a" and "b",
"a" and "b" must be equidistant from "x"
CDXx = calculated density due to substance "x" having an adsorp=
tion maximum at wavelength "x"
The actual amount of "X" is then calculated from CDXxo
Bauld (1954) observed that a ;depression of about 15-40 percent
in the final color produced by Kober reaction can be caused dye to the
presence of solvent residues.
The three possible types of inhibitions
which can be responsible for interference in the color development are
a s 'follows:
a) failtupe to form the initial yellow complex (inhibition type I)
b) failure to convert the yellow complex fully into the pink
(inhibition type II)
c ) instability .of the pink complex (fading).
“23=
The type I inhibition in the case of estriol is due to diminished
reducing power of the reagent.
The second stage of the Kober reaction9
involving conversion of the yellow complex into the pink o n e ? involves
oxidation (Nocke 1961) which if excessive causes fading.
The Kober
reagents used by Bauld (1954) for estriolg estrone 6 dnd estradiol con=
twined
percent* 66 percent and 60 percent V/v of sulfuric acid
respectively.
The modifications suggested by Bauld (1954) were incorporated
into Brown's method for the urinary estrogens (Brown 1955).
According
to this method the concentration of sulfuric acid in the reagents is
the same as used by Bauld (1954).
added into each reagent.
Quinol at the rate of 2 percent is
It was suggested that addition of quinol to
the estrogen fractions^before evaporating the solvent* can prevent any
oxidation of the hormones.
Chromogenic impurities from the crude urine
extracts were separated by methylating the estrogens in alkaline solu=
tion with dimethyl sulfate and then oxidizing the contaminants with
hydrogen peroxide and extracting estriol methyl ether with benzene and
the estrone= and estradiol=methyl ethers with light petroleum.
This
was based on the fact that methylation of the estrogens does not limit
the estimation of the hormones by the Kober reaction (Marlow 1950).
The
appropriate quinol=sulfuric acid reagent (3 ml) for the particular estro=
gen is added to the estrogen fraction.
minutes,
Preheating is done for 20
After cooling* I ml* 0.5 ml and 0.2 ml water is added to the
tubes .containing estriol* estrone and estradiol respectively.
The tubes
-24ar.e then reheated for 10 minutes«,
The readings for light absorption
are taken at 480g 615e and 552 mu for estriol and estrone and 4809 518*
and 556 mu for estradiol.
In 1 9 5 6 8 Bauld modified the method used by Brown (1955),
The
composition of the reagents was kept the same whereas their amount
added to the estrogen and quinol residue* the heating periods and the
final concentration of the acid were modified.
The appropriate reagents
.?•
as described by Brown (1955) (2,6 ml for estradiol=17B and estriol and
3,0 ml for estrone) were added.
Preheating in a boiling water bath
was followed by cooling in water bath at 15° C,
At this stage 50+ 5 mg,
of quinol was added and the dilutions were carried out as follows: ■
estradiol-17B with 0,7 ml of the reagent* estriol with 0,7 ml of water
and estrone with 0,3 ml of water.
Reheating time allowed was 15
minutes and after cooling the absorbancy was measured at 480* 512*
and 544 mu for estrone and estriol and at 480* 515* and 545 mu for
estradiol^lTB,
The method was used later on'by Givner et, al, (1960)
and was found to be quite satisfactory,
A similar method was reported by Hunt (1957),
The Kober reagents
for this procedure contain sodium nitrate* quinone and hydroquine,
No
quinol is"added after the preheating interval'and the final concentra­
tion of sulfuric acid is 60 percent.
This method has successfully been
used by Nelsdn and Smith (1963),
:
-
■,
'
'
i
- D, EXTRACTION,OF PROGESTERONE FROM BLOOD AND-OTHER TISSUES
As already stated* the progesterone in the blood exists as
“25”
bound to the plasma proteins 0
It I s g therefore 9 evident that to iso­
late the hormone its binding with the protein part should be broken«
Zander and Simmer (1954) and Oertel etc al« (1959) have pointed put
that the proteih-steroid binding is broken down even with the organic
solventso
It is also known that pretreatment of plasma with a solution
of sodium hydroxide breaks the .progesterone-protein binding apd while
extracting with ether prevents any emulsion formation (Short
1 9 5 8
a
and 1958b j Sommerville and Deshpande 1 9 5 8 1 Sommerville et» a l 0 1963),„
Lombardo e t c a l e (1955) have reported the possibility of the application
of dialysis for the extraction of progesterone from the b l o o d 6
E o 'ISOLATION OF PROGESTERONE
The paper chromatography method for progesterone with petroleum
ether and 80 percent methanol system as recommended b y Bush (1952) has
been used quite extensively (Gomes e t 0 a l 0 1963| Raeside and Turner
'
1 9 5 6 1 Short 1958a and 1958b)»
•
:■
In 1961 Short reported that decalins
m e t hanolsw a t e r s methy!cyclohexane 5 acetic-acid swater and methycyplohexane
water §methanol systems can also prove to be quite satisfactory 0
Simi­
larly 6 methanolsbenzene system has been used (Oertel e t 0•a l 0 1959) with
satisfactory resolution.
The adsorption "column chromatography with silica^gel (Sommerville
:
etc alo 1 9 6 3 ) g aluminum-oxide (Noall et. a l 9 ,1953$ Loy eto alo 1957) and
f
thin layer chromatography (Lisboa 1963$ Futterweit e t o al. 1963) have
also* successfully$ been applied.
z
=26=
F o QUANTITATIVE MEASUREMENT OF PROGESTERONE
Progesterone has been described as the least polor of all the
naturally occurring steroid hormones and no other
B-unsaturated oxo-
steroid has as yet been identified in vivo with the same Rp value as
•progesterone in paper chromatographic system.
all steroids possessing the
(Short 1961)o
It is also known that
^ S - k e t o n e group absdfb ultraviolet light
Progesterone being a member of this group of steroids
and at the same time having a definite characteristic Rp-value can ,
easily be identified,
I
Progesterone can also be identified from its color reaction.with
many reagents,
Dinitrophenylhydrazine reacts with
B-unsaturated ketone
group of progesterone to give an orange-red color., whereas m-diriitrobenzene reacts with the
2 0
-keto carbopyl to give a brown purple color
(Raeside and Turner 1956),
Because of its unsaturated ketone grouping progesterone exhibits
a characteristic absorption maximum in ethanol at 240 mu (Short 1961),
This single characteristic for its quantitation has been used by many
workers (Raeside and Turner 1 9 5 6 1 Gomes ,et, al- 1963J Loy C t v a l ,
1957)
even though Zaffaroni and Burton (1951) have pointed out that the other
lipids contained in the extract also absorb in the ultra-violet spectrum.
And when the hormone is separated from the contaminants with paper
chromatography 9 and eluted, some background material from the chromato­
gram itself can enhance the absorbance.
It has, therefores been
emphasized by Lombardo (1955) that the readings-of the densities of
I
=27=
the eluate of an unused area of the chromatogram must be taken into
consideration 0
Samuels (1947) has indicated the solution of progeste­
rone in ethanol should possess a well defined peak at 240 mu and there
should be very little or no absorption at 280 mu.
To correct the effect of the interference due to contaminants in
the final progesterone elu a t e „ the readings are generally taken at 230
m u 8 240 mu and 250 mu.
The corrected absorbance due to progesterone is
then calculated by applying the Allen's correction formula (Gqmes e t ,
al, 1963; Loy et, al, 1957),
Oertel et, al, (1959) have advocated
the use of sulfuric acid=ethanol reaction.
absorption is obtained at 295 mu,
By this method the maximum
However 9 this method* due to its low
sensitivity* has not proved to be very useful (Short 1961),
A gas chromatographic technique for the identification and
measurement of progesterone has been reported by Futterweit et, al,
(1963,),
EXPERIMENTAL PROCEDURE
......
aurmu^fiKm
;
■ Five Jerseys and one Holstein Iactatiiig cowj from the d#iry herd
at Montana State College^ Bozeman 9 were used in this study*
Laparotomy
-I'
- ■from the right si<Je of the animal was performed and the posterior vena
cava was cannulated at a distance of about one inch backward from the
level of the right kidney using Silastic Silicone rubber tubing*
f* 1
The
tubing was very kindly donated by Dow Corning Corporation 9 Midland 9
Michigan*
The internal and external diameter of the tubing were 0*1325
cms* and 0*2625 cms* respectively*
was about 72*5 cms*
The total length of the tubing
The length of the tubing inside the vein was
approximately 37*5 cms* whereas about 30*0 cms* portion outside the vein
was anchored to the abdominal'w a l l *
The remaining 5*5 cms* was kept
outside the body and a syringe adapter was fitted onto this end of the
tubing*
Except when the blood was actually drawn this end was always
kept sealed with a metal plug*
An iron ring with an internal and
external diameter and thickness of 9 cms* g 7*5 cms* and 0*1 cms*
respectively 9 and having a spongy pad fixed on the under surface was
sutured to the skin*
An aluminum lid of the same size as that of the
ring was then bolted onto the ring*
The lid could thus very easily be
removed at the time of the sampling of the blood*
About 250 ml* blood was collected with the help of a syringe
every other day*
Five ml* of sodium citrate solution in distilled
water (0*5 g,*/ml») was used as an anticoagulant*
After the collection
of the sample* the cannula was flushed with about 5-10 ml* heparin
solution (200 I 0 U* per ml* normal saline)*
Heparin solution was
-29injected dyery other day even though no samples were collected*
To
prevent the growth of fungus or other micro-organisms in the heparin
solution
Terratnycin■ was included in the stock solution (I m g 0 / m l * )„
Urine samples were also collected immediately after the blood
was drawn *
The samples were immediately brought to the laboratory and
the blood was centrifuged at a rate of 3000 rpm,
If the samples could
not be analyzed immediately the plasma was stored in the freezing
chamber of the refrigerator at a temperature of about -10 C?to -S 0 G e,
The urine samples were stored at a temperature of about +5 C?*
Sample collections were made ■during the e'strdus cycle of four
Jerseys and o n e .Holstein cow and during the first 34 days post-conception period from one Jersey cow,
c
The urine samples were extracted using the method outlined by
Nelson (1959) which is a modification of the methods of Friedgood (1948)
and Stimrnel (1946)*
no *
The procedure followed is outlined on flow sheet
io
As recommended by Preedy and Aitken (1961) the extraction.pror
cedure for estrogens from the plasma was the same as for the urinary
estrogens except for the following changes
;
":
t
.
.
C: -
1) About 30 m l * plasma filtered through glasswool was diluted
with distilled water to make 500 ml.
2) To avoid any bumping during hydrolysis with 15 volume percent
of concentrated hydrochloric acid, the acidified diluted
plasma was heated very gently to boiling till the bumping
-30FLOW SHEET NO. I
250 ml. Urine
Filtered through glasswool and hydrolyzed
for 18 minutes with 15 volume percent
concentrated H C l . After cooling extracted
Pooled Ether
Washed 4 x
and then 2
Evaporated
Extract
30 ml. 9 percent NaHCOg solution
x 30 ml. of distilled water.
ether to dryness under vacuum.
Aqueous Phase
(Discarded)
v
Residue
Dissolved in 6 ml, ether and then added 108 ml,
CCl4 . Extracted the phenolic phase with 3 x 50
ml, N KOH solution and I x 15 ml. distilled water.
't
"
-------------Pooled KOH and Water-Fractions
Washed I x 20 ml. ether, acidified to pH 3,0
with 6 N HgSOy.
Extracted 3 x 80 ml. ether,
--------------
*
—
-------
—
Ether Extract
Washed 3 x 30 ml, 9 percent NaHCOg solution and
then 2 x 30 ml. distilled water.
Ether Extract
Extracted 5 x 20 ml. 2,5 percent NagCOg solution
and then 2 x 30 ml, distilled water.
VEther
Phase
Evaporated ether to dryness under vacuum.
Dissolved the residue in 10 ml. 95 percent
methanol.
Ether-Carbontetrachloride
(Discarded)
&
Aqueous Phase
(Discarded)
Aqueous Phase
(Discarded)
*
Aqueous Phase
(Discarded)
'V
Extract stored in the refrigerator ready for chromatography.
” 31“
stopped (about
1 0
minutes) and then the boiling was con­
tinued for 20 minuteso
The volume of the hydrolyzed’plasma
left after boiling was on an average, about 405 m l 0
3) Extraction from the hydrolysate was performed three times
with
1 0 0
tively,
ml,,
2 0 0
m l , , and
2 0 0
ml, ether each time respec­
The emulsion formed with ether and plasma was broken
by centrifugation,
4) Ether extract was washed 3 x 50 ml, 9 percent NaHCO 3 solution
and then 2 x 50 ml, distilled water,
5) Washing of the ether phase to remove estriol With 2,5 percent
NagCO^ solution was limited to three times instead of 5 times
as in the case of urinary extract.
Almost all the yellow pigments contained in the ether extract
were left behind in the ethers carbon-tetrachloride fraction.
While
extracting the phenolic phase (estrogens) with IN-KOH solution from
ethers carbon tetrachloride, some emulsion formation was observed which
broke itself on standing for a few minutes,
■
- •■
The method for the extraction of progesterone from plasma used
was the modification of the methods of Short (1958) and Sommerville
et, al# (1963),
The procedure adapted by the author is outlined on the
flpw sheet number II,
All of the three., estrogenic compounds obtained from the urine
jand blood were separated from!contaminating substances descending paper
chromatography.
The procedure used was the same as developed by
-32FLOW SHEET N O 6 II
Plasma
Diluted with equal volume of distilled
water; added KaOH solution (0,2 g, per
m l , ) to make 1.5 g, NaOH per 100 ml,
diluted plasma.
Extracted 3 x 1/3 volume ether.
Emulsion broken by centrifugation,
I
I
Pooled Ether Extract
Washed 2 x 1/10 volume distilled
water.
Aqueous Phase
(Discarded)
Ether Phase
J Ether evaporated under vacuum.
Aqueous Phase
(Discarded)
Residue
Dissolved in 20 ml, petroleum ether (30° - 60° C),
Rinsed the flask 5 x 20 ml, freshly prepared 70
percent methanol each time before transferring to
separatory funnel for extraction of progesterone
from petroleum ether,
r
Pooled 70 percent methanolic Phase
^Eva p o r a t e d under vacuum.
Residue
Dissolved in 5 ml, and then rinsed the flask
3 x 3 ml, 95 percent methanol.
Nz
Ready for chromatography,
4
Petroleum
Ether
(Discarded)
*= 3 3 =
Burton e t « a l 0
(1951) and later on modified by Axelrod (1953) and
Gorski (1959)»
This method had already been used by Nelson (1959) and
is shown as followss
1) Chromatographic paper strips, about 56 cms„ l o n g 8 were cut
from a roll of Whatman filter paper no,
1
»
The.strips were
boiled in absolute methanol for one-half hour, then washed
once with redistilled methanol and air-dried 0
of a hard lead pencil two lines
and
8
9
With the help
each at a distance of
6
cms 0
‘ Cjns0 from one end of the strip were marked =
2) Two m l 0 of the urine extract in 95 percent methanol and 5 m l 0
of the plasma extract in 95 percent methanol yere taken
separately in conical centrifuge tubes (15 m l 0) and the
solvent was evaporated in the presence of nitrogen g as 0
'
3) The chromatographic paper strip, already boiled, rinsed and
air-dried 9 was dipped in sufficient quantity of formamide s
methanol (Isl) which acted as a stationary ph a s e 0
4) The strips were then laid down on a thick sheet of blotting
paper=
Within about one m i n u t e , the end of the strip with
marked lines was picked up with the help of a clean pair of
forceps and placed in the solvent trough assembly so that the
6
cmso mark would rest on the glass r o d 0
5) The residue was dissolved in three drops of ethyl-acetates
methanol ( 5 si) solution and applied in the form of a narrow
band along the starting line ( B c m s 0 mark).
This was repeated
= SUe3
twice with 2 drops of ethyl~acetateSmethanol 0
tions were made with the help of a lt) 0
The applica­
micropipette 0
Sufficient time was allowed between the two consecutive
applications to prevent the spreading of the applied
materials 0
6 ) To keep the amount of the stationary phase approximately
constant.from strip to strip the time spent in applying the
estrogenic material over the chromatographic paper? strips
was kept constant (30 minutes at room temperature )
0
The
strips were then ready to be hung in the developing chamber.
7) The developing chamber made from a 30 x 30 x 60 c ms 0 glass
tank'kept in a wooden box to give good insulation was used.
The inside of the tank was lined with filter paper 9 the
lower end of which was always kept dipping in about 3.5 cms.
deep mixture of equal volumes of benzene and normal-hexane.
Two filter paper strips were also hung separately near the
side walls and their lower ends were kept dipping in formamide 9
contained in two small glass jars.
The glass lid of the tank
was made to remain sticking firmly by applying starph-glycerql
paste.
The developing chamber was p laceji in a cold r o o m 8 the
temperature of which was being maintained et
8
1 0
Q-t Io C 0
) The solvent trough assembly with the strips hanging down,,
with their
6
cms. mark on the glass rod of the assemblys was
placed on tjie supporting frame inside the developing tank.
=SSea
Benzene and n-hexane
( I s l ) which served as the mobile phase *
was kept equilibrated with formamide in a brown bottle 0
The
amount of the solvents put in the trough and the number of
the strips hung from each';trough were kept constant*
9) One blank strip treated exactly in the same manner as the ones
V-
having the unknown estrogenic material and another having pure
estrogens as a reference strip* was also included*
10) The strips were taken out of the tank after a 24 hour period
and hung for drying*
11) A representative strip* 1,33 cms* wide* was removed from one
side of the 4 cms, wide strip and then dipped in a freshly
prepared solution of equal volumes of
2
percent (weight/volume)
each of ferric chloride and potassium ferric cyanide*
The
strips were allowed to.remain in the developing reagent for
about 30=45 seconds *
them for about
2
These were then washed by immersing
minutes successively in each of four beakers
containing 5 percent HCl * 5 percent H C l * distilled water and
distilled water respectively*
''A
It was observed that washing
■ : lV'
the developed strips in this manner removed most of the back=
ground material and the blue spots * due to an interaction
.
b e t w e e n ■the estrogens and the developing reagent * could be
more clearly visualized* .
Under the conditions described for the paper chromatography the
distances gravelled from the starting line by all the three estrogens
=>36“
%
were as follows
Estradiol“17B
= 5o7
+ O 036 c ms 0
(7 observations)
Estradiol”17oC
=
+ 0=72 C m g 6
(7 observations)
Estrone
= 29o9
8 = 6
+ 1°14 C ms 0
(7 observations)
The reference strips having pure estrogens also helped in locating
the spo t s o
It was noticed, however 9 that the estrogens from the urinary
extracts moved slightly faster than the pure estrogens on the reference
stripso
This may be due to the extract being overloaded with the
contaminantso
The corresponding areas from the undeveloped strips were
eluted with absolute methanol„
The pieces of the chromatographic paper
having individual estrogens were folded with the help of a pair of
forceps to g i v e 'them the shape of a funnel 0
They were then inserted
and kept near the mouth of the 15 ml, capacity conical centrifuge tubes 0
About 10-12 m l o methanol was allowed to falls, drop by drop, from a
buret over t h e m 0
and its elution
9
While cutting the piece of the chromatographic paper
great care was taken not to let any paper fringes
fall into and remain in the eluate 0
The purification of the plasma extract for progesterone was done
by using standard thin layer chromatographic technique 0
The procedure
used for preparing the slurry, process of coating the plates, drying
and prewashing them is outlined in the appendixo
The procedure used for
applying the unknown samples, developing the chromatogram and elution
was as follows s
I) Two starting points were marked, each at a distance of 1„5
■=37”
c m s 0 from the right and left edge and.about 1»5 cms, from
the lower edge of the already prewashed and activated layer*
These points were marked with the aid of a template and were
'
used to apply the pure progesterones hereafter called the
reference spots 0
The distance between these two points was
marked off into four equal parts*
To stop the solvent from
overrunning the chromatogram* the layer was divided at a
distance of 15 cms* by making a mark at right angle to the
direction of development*
2) The methanol from the plasma extract was evaporated in the
presence of nitrogen gas*
3) The residue was applied to the starting point as a narrow I.
cm* long band in the center of one of the four areas ,already
marked*
This was accomplished by dissolving the residue in
three drops of benzene and then rinsing the tube with two
drops of benzene each time*
A microsyringe* (10 ul*) was
used for applying the samples*
4) The plates were placed in the tank already saturated with the
solvent system which was a mixture of
ml* ether and
2
1 2 0
ml* n=hqxanee 80
ml* glacial acetic acid (hereafter referred
to as System " A " ) *
5) After developing* the reference spots were detected and marked
*Microliter #701
Hamilton Co* *■Inc* * Whittier* California
= 38"
by noting the absorption of ultraviolet light by progesterone.
The corresponding areas of the unknown samples were then
scraped off into a buchner funnel fitted with a fritted disc
of fine porosity,
A filter-vac was used to place the funnel
over a filtering flask.
was used for the elution.
About 10=12 ml, of absolute methanol
Vacuum was then applied to suck
the eluate down into a 15 ml, conical centrifuge tube,
6
) The methanol from the eluate was evaporated in the presence
of nitrogen gas and the residue was once again applied on to
another Chromatogram 9 exactly in the same manner as previously.
I
described.
The developing system used was a mixture of 160
ml, b e n zene$ 40 ml, absolute methanol and 2 ml, glacial acetic
a c i d 9 hereafter referred to as System "B",
7) The elution of the corresponding areas was done with 10=12
ml, absolute methanol as it was explained previously,
8
) Methanol was evaporated and the centrifuge tubes containing
the residue were kept in the freezing chamber of the refri=
gerator until they were read on the spectophotometer,
9) After scraping the corresponding areas to the reference points
'■ as explained a b o v e 9 the rest of the layer was sprayed with
50 percent sulfuric acid and then heated in an oven at a
temperature of about 150° C, for 15 to 30 minutes.
Pure
progesterone and the contaminants were located by visualizing
the fluorescence give out by these compounds under the
•=39™
ultraviolet light and the distance they had traveled was
measured.
The Rp values of pure progesterone and the contaminants contained
in plasma extract separated by thin layer chromatography are shown in
Table No, .1, .
TABLE .NO, I
Table Showing the Rp -Values of Progesterone and Contaminating
Subst ances„ ..Contained...JLn„The ..PlasmeL Extract ,.Qn..
Thin Layer Chromatography
---------- s ^ E T m —
Compounds
Progesterone .
Contaminant I,
Contaminant II
Contaminant III
Contaminant IV
"
■1
* System "A”
**System "B”.
0,1063
0,1992
0,2612
0,4685
6,7958
s
’
... System "B"-*.. •'
+ 0,0009
+ 0,0003
+ 0,0003.
+0,0075
+ 0 „ p 0 1 7 '.i'
■
1
.
.
.
: ..... ,
0,61 + 0,0005
0,3867 ± 0,0029
0,8267
«»===360<
=
>
======
,
-U^nr
.
.
Hexane + Ether + glacial acetic acid (120:80:2)
*
Benzene +■• Absolute Methanol-.+ .glacial, acetic acid
(160:40:2)....
Estimation of estrogens was done by using the Kober Color
i
Reaction as modified by Hunt et, al, (1957) I m d is outlined below":.....
-
'
:
■
1) The methanolic eluate of the individual component obtained
'
. ;. "...
v
from the paper chromatogram was evaporated to about 2 ml, by
.
•
- -s
’ ' 1
- e*
. ^
■ j [
/ 'i1
heating in a hot water bath (not over 60° C) in the presence
1 ' ' ■ ..• .: I.
of nitrogen gas.
V'
. .
One tube containing an eluate of an equal
piece of blank chromatographic paper was also included,
2) Two-tpnths ml, of hydroquinone solution (see appendix) was
added to the eluate,
The evaporation was started again until
-40the crystalline residue was Idft behind 0
3) Kober reagent for estradiol (gee appendix) was added into the
tubes containing the estradiol samples„
Similarly 3 ml, of
the reagent for estrone were added to the tubes containing
the unknown samples of estrone.
One tube containing reagent
blank was also included,
4) The t u b e s 9 capped with corks having aluminum foil wrapped
around them were placed in a boiling water bath and allowed
to remain there for 20 minutes (preheating period),
Thq
tubes were shaken twice within the first five minutes without
taking them out of the water bath
8
tp ensure that the
crystalline residues had gone into solution,
5) After 20 minutes
9
the tubes were transferred to a cold water
bath (15° to 18° C) and kept there for 7 minutes,i
6
) Seven-tenths milliliter of the Kojaer reagent for estradiol
■
was added to each of the preheated tubes containing the
unknown estradiol samples and the reagent for estradiol.
Similarly 0,3 ml, of distilled water was added to each of
the preheated tubes containing unknown samples of estrone and
the estrone reagentg so that the final concentration of the
sulfuric acid was 60 percent,
7) The tubes were capped once again and shaken very gently to
iTiix the-contents thoroughly 0
Care was taken not to" let the
contents come into contact with the aluminum foil or the cork
« 4 1 «
Itself 0
8
) The tubes were then placed in the boiling water bath and
shaken once during th^ reheating period of 15 minutes
(Reheating )
0
9) After completion of the reheating t i m e 8 the tubes were
transferred back to the cold water bath and allowed to remain
there for 7 = 10 minutes=
10) The tubes were then left at room temperature for about 10
minutes=
11) Estrone samples were read at 5 44 9 512, and 480 mu and 17e>^>
and 17B=estradiol at 5 48s 515, and 482 mu=
Sulfuric acid
of 60 percent concentration was used as the blank=
12) A l l e n ’s correction equation was used to obtain the corrected
absorbancy=
Any absorbance due to the filter paper blanks
was also taken into account before the quantities against the
corrected absorbance were read from the standard curve=
To estimate the quantities of progesterone the re s i d u e 'obtained
after the evaporation of the eluate was dissolved in I.ml= of 95
percent ethanol=
The tubes were shaken on an electric shaker to get a
homogenous solution and then the absorbance read on a spectrophotometer*
at 2 3 0 8 240 a n d -250 mu=
blank=
Ninety=fivg.-percent ethanol was used as a
-The corrected absorbance at 240 mu was-obtained by applying the
A l l e n ’s correction, equation= ..
•*Beckmari Model "DU" Spectrophotometer
=42=
A standard curve for pure progesterone with the quantities
ranging from 0 to 10 micrograms per milliliter of 95 percent ethanol
was preparedo
The quantities in the unknown samples were obtained from
the standard curve„
Three samples out of the ones W h i c h 9 by ultraviolet assay 9 were
found to be positive for the presence of progesterone were checked by
gas chromatography&c
The gas chromatograph was set up as follows:
A four foot pyrex colum with outer=diameter of
6
mm„ was packed with
3o8 percent SE=30 on siliconized 60/80 mesh Diatoport=S 0
The column
temperature at 245°. C 9 flash heatdr at 310° C 0 and detector at 330° C 0
flame ionization with helium flow of 60 m l 0 per minute were maintained. *
*F S M Model 400 Biornedical0
RESULTS
Standard curves with pure estrone, ITKr and 17B-estradiol for
the Kober reaction, prepared in the beginning of the experiment and then
checked after about one year, are shown in Figure N o 0 2 0
The absor­
bancy obtained for the range of the estrogens used to establish the
standard curves obeys the Beer's Law,
ITB-Estradiol
Estrone
Micrograms
Figure No, 2 Standard Curves of Pure Estrogens For Kober Reaction
-44=
I=
The levels of the estrogens in the blood plasma and urine during
the estrous cycle §
It was noted that each of the five cows exhibited a higher level
of the estrogens at one time before and one time after thp midcycle
than any of the other periods sampled 0
There was a considerable
variation among the cows as to the time when these high levels were
reachedo
The periods of the cycle when individual cows showed the
higher levels are as follows §
A0
Estrogens in the blood plasmas
The fluctuations in the levels of the estrogens in the blood
■
;
plasma during the estrous cycle of five cows are shown in figure n o 0
3 to
6
o
It is evident that three out of five cows (cows numbered 2„ 3
£ 5) had the first high level of 17e4estradiol during the period
between days
6
£ S 9 whereas cows numbered I and 4 exhibited their first
high levels during the periods between days 3 £ 5 and 9 £ I l 9 respec­
tively o
The second high level of 17©6?estradiol occurred during a
period between days 15 £ 17 in cows numbered 2 and 5 and during a
period between days 18 £ 20 in cows numbered 3 and 4 0
had the second high level of IToGestradiol between days
Cow numbered I
1 2
£ 14, which
was earlier than the other four cows„
Similarly the first, high level of 17B-estradiol of three out of
C-
'
-
■
five cows (cows numbered I 9 2 £ 5) was noted during a period between
days
6
£ S 9 whereas for cow numbered 3 this high level was noted between
-45days 3 6 5 =
Three out of the five cows (cows numbered I 9 3
I
6
5) showed
the occurrence of the second high-level during a period between days
1 2
6
1 4 9 whereas cow numbered
days 15
6
17„
showed the second high level between
2
Cow numbered 4 exhibited only one high level of 17B-
estradiol which occurred between days 9
6
11«
The levels of estrone in four out of five cows (cows numbered 2 9
/
!
39 4
5) reached the first high level between days
6
6
6
, whereas those
8
of fhe cow numbered one reached the first high level between days 3 6 5 =
Similarly
sthree
out of five cows (cows numbered 2 9 3
second high level of estrone between days 15
two cows (cows numbered 1 6 4 )
level between days 18
6
2 0
6
5) had the
1 7 s whereas the other
6
had a gradual increase reaching the high
=
Total estrogens of three out of five cows (cows numbered 2 9 3
;
-■
■
-
6
/
5) were observed to reach the first high level during a period between
days
6
6
8
=
Cow numbered one had the first high level between days 3
5 and cow numbered 4 had this high level between days 9
6
11=
6
Similarly
the second high level of the total estrogens of two cows (cows numbered
2
6
5) was noted between days 15
6
17$ whereas this high level of cows
numbered 3 and 4 was observed between days 18
6
20»
The fifth cow (cow
numbered I) had the second high level of the total estrogens between
days 12
6
14=
The data obtained regarding the levels of the estrogens in blood
plasma obtained from the posterior vena cava and in the urine during
estrous cycle; during the first 34 days after conception; and the levels
-46of progesterone in blood plasma, are shown in Figures No. 3 to 13.
* -------- -X It^-Estradiol
-© 17B-Estradiol
Estrone
Total Estrogens
O T cO
0-2
3-5
6 - 8
9-11
12-14
15-17
18-20
Days After Heat
Cow No, I
Figure No, 3:
The levels of estrogens (micrograms per milliliter) in
blood plasma during estrous cycle.
0 =35
-47■X IV0^-Estradiol
■€>IVB-Estradiol
♦ Estrone
- Total Estrogens
12-14
9-11
Days After Heat
Figure No, 4:
15-17
18-20
The levels of estrogens (piicrograms per milliliter) in
blood plasma during estrous cycle.
&
17o£Estradiol
Q-
"S ITB-Estradiol
#-
-# Estrone
W 5
-- Total Estrogens
------- 0-2
3-5
•----------- ------6 - 8
9-11
12-14
______ .
-
15-17
18-20
Days After Heat
Cow No. 3
Days After Heat
Cow No. 4
Figure No. 5:
The levels of estrogens (micrograms per milliliter) in
blood plasma during estrous cycle.
-49Xr
^
O-
-G ITB-Estradiol
0
-# Estrone
-
I-Zr^-Estradiol
o.go
Total Estrogens
\
\
/
/
/
LO
1
\
\
/
2
\
3
4
5
6
Days After Heat
Cow No. 5
Figure No. 6:
The levels of estrogens (micrograms per milliliter) in
blood plasma during estrous cycle.
7
-50B.
Estrogens in the urines
Figures no, 7 to 9 show the variation in the levels of the
estrogens excreted in the urine during the estrous cycle of five cows.
It was noted that the first high level of IV^restradiol of two
put of five cows (cows numbered 3 6 4 )
days 9
11,
6
occurred during a period between
The other three cows (cows numbered I s 2
first high level during the periods between days 0
6
6
8
, respectively.
5) showed the
6
2, 3
6
6
5, and
Four out of five cows (cows numbered I 9 2, 4
had the second high level of this hormone occurring between days 18
20, whereas cow numbered 3 had it between days 15
6
5)
6
6
17,
The urinary excretion of 17B-estradiol by two out of five cows
(cows numbered 1 6 3 )
reached the first high level between days 3 6 5 ,
whereas that of cows numbered 2 and 5 reached the high level between
days 0 6 2
and
numbered I, 2
days 18
6
15
6
17,
15
6
17,
2 0
6
6
6
8
respectively.
Three out of the five cows (cows
5.) had the second high level during the period between
, whereas cow numbered 3 had this high level between days
Cow numbered 4 had only one high level occurring between days
Simil-Sbly two out of five cows (cows numbered 2
6
3) had the first
high level of estrone occurring between days 3 6 5 ,
whereas cows
numbered I and 5 had this high level between days
6
respectively.
6
8
and 9
6
11,
The second high level of estrone for cows numbered I and
5 occurred between days 18
6
20, whereas cows numbered 2 and 3 had this
high level between days 12
6
14,
Cow numbered 4 had only one high
-51level of estrone occurring during a period between days 15
Three out of the five cows (cows numbered 2, 3
6
6
17«
5) had the first
high level of total estrogens occurring between days 3 6 5 ,
whereas
cows numbered I and 4 had the first peak of total estrogens between days
6
6
8
and 9
6
11, respectively.
(cows numbered I, 2, 4
6
Similarly, four out of five cows
5) had the second high level of total estrogens
during the period between days 18
6
2 0
, whereas the second peak occurred
in cow numbered 3 between %days 15
6
17,
-52X:---------
KITo^-Estradiol
/
I
©----------© 17B-Estradiol
/
•----------• Estrone
/
----------- Total Estrogens
Z
/
/
/
/
/
/
/
/
/
/
/
o
O-
CO
200
/
C
C•
0-2
3-5
6-8
9-11
12-14
15-17
18-20
Days After Heat
Cow No. I
Figure No. 7s
The levels of estrogens (micrograms per liter) excreted
in urine during estrous cycle.
-53"* IT0^-Estradiol
SiITB-Estradiol
■0
Estrone
-- Total Estrogens
9-11
12-14
15-17
18-20
12-14
15-17
18-20
Days After Heat
9-11
Days After Heat
Cow No. 3
Figure No. 8;
The levels of estrogens (micrograms per liter) in urine
during estrous cycle.
-54X
17o(-Estradiol
S 17B-Estradiol
Estrone
Total Estrogens v
9-11
12-14
15-17
18-20
Days After Heat
Cow No, 4
9-11
12-14
15-17
18-20
Days After Heat
Cow No o 5
Figure No, 9;
The levels of estrogens (micrograms per liter) excreted
in urine during estrous cycle.
— 55—
II „
Estrogens in Blood Plasma and Urine During First 34 Days After
Conception:
Figure No. 10
6
11 show the levels of plasma estrogens and the
estrogens excreted in the urine during the first thirty-four days of
pregnancy by cow no.
6
.
The levels of 17B-estradiol in the blood
plasma were higher on day
1 1
post-conception than on any other day.
The levels of the total estrogens in the plasma show a small peak on
day 7 and then a higher peak on day 11 after conception.
The levels
began to increase on day 19, reaching another peak on day 26 and then
declined.
The levels on day 34 were equal to the levels on day 19.
Similarly the levels of urinary estrogenic excretion reached a
peak on day 9 after conception.
The levels started to increase on day
16, reaching a peak on day 31 and then declined once again.
On day 34
after conception the levels are equal to the ones on day 15.
o
-X
1
% 6 -Estradiol
4) 17B-Estradiol
-# Estrone
Total Estrogens
Days Post-conception
Figure No. 10:
The levels of estrogens (micrograms per milliliter) in
blood plasma of cow no, 6 .
-56—
X ---a—
X
ITe^rEstradiol
---- ITB-Estradiol
O ---- -----# Estrone
Total Estrogens
Micrograms
__________
Days Post-conception
Figure N o 0Il;
The levels of estrogens (micrograms per liter) excreted
in the urine of cow no„ 6 during post-conception period.
-57Figure no. 12
shows a standard curve of pure progesterone prepared for
ultraviolet assay.
Quantity of Progesterone —
Figure No, 12;
micrograms per ml, 95 percent ethanol
Standard curve of progesterone for ultraviolet assay.
“58—
The pooled data for progesterone are shown in Figure no, 13,
It was observed that the level of progesterone in the plasma of the
posterior vena cava blood was low during the first 9 days and then
began to rise until it reached the maximum on day 17 of the cycle,
The
level of progesterone on day 19 had decreased and was equal to the
level on day 13,
Days After Heat
Figure No, 13:
The levels of progesterone (micrograms per milliliter)
in blood plasma during estrous cycle.
0059”
DISCUSSION AND CONCLUSIONS
The levels of estrogens in the blood and urine of the higher
primates during a menstrual cycle have been established (Everett, 1961)„
But, to the best of the author's knowledge, there has been no report 1
which has shown the day-to-day changes in the levels of these estrogenic hormones during an estrous cycle=
Most workers have tri^d to
study the fluctuation of estrogens in the urine on the day of estrus
and during gestation.
Various attempts to determine the estrogens in
the jugular venous blood of animals during the estrous cycle have not
I
succeeded (O'Donnell and Pre e d y , 1961),
One report describing the
fluctuations in the levels of progestins during the estrous cycle of
■»
bovines has been published (Gomes et, al, 1963),
It has been indicated that the adrenals and the uterus (placenta
in the case of pregnancy), in addition to the ovaries, are capable of
synthesizing and perhaps secreting estrogens and progestins,
To
.
understand the fluctuation in the secretion of these hormones in vivo., ■
at least two alternatives as mentioned below are possible:
I,
To separately, collect- and. analyze the blood coming from all
the possible sources of their syntheses and secretion,. Then
by simple addition the information regarding the total of
these hormones entering the circulation % n the body could
be obtained,
2,
To collect the blood samples' from a location near to the
.site of secretion of these hormones where the blood being
*-60“
drained away from all these sources has already combined.
The second alternative seemed more advantageous»
The posterior
vena cava drains blood from the hind portion of the body and also
receives blood coming from the ovaries, uterus (placenta during preg­
nancy ) and adrenals„
It was for this reason that the posterior vena
■
cava was selected as a point of sampling of the bl o o d ,
In as much as it is known that considerable variation occurs in
the levels of estrogens and progesterone among individual animals a it
is thought to be advantageous to obtain as many samples as ppssible
from each animal during any experimental period.
To collect as many
blood samples as possible from each animal during an estrous cycle, '
cannulation is perhaps the only alternative.
In the beginning of this'
study, a polyethylene tubing was used to cannulate the posterior vena
cava of a cow.
The cannula with such a material could not be kept open
for more than 11 days,.
It was found that a cannula made from the
Silastic Silicone rubber tubing was definitely better than the one
made from polyethylene tubing,
As there was no precedent of such a
cannulation experiment,'the author had to face many problems to keep
the cannula open for as long a- period as possible.
The longest dura­
tion for which the cannula could be kept open was 43 days.
It was
observed that the formation qf a blood clot inside the tubing was
responsible for its being plugged up.
During the present study it was found that the estrogens in the
blood, as well.as in the urine of 1cattle^ during an estrous cycle show
“■61“
■great fluctuations«
It was noted that in most of the cows these hor=
mones reach high levels once before and once after the midcycle»
Three
out of the five cows had the first high level of the total estrogens
in the blood plasma occurring between days 6 6 8 0
Similarly9 three
out of the same five cows showed the occurrence of the first high level
of the total urinary excretion of the estrogens during a period between
days 3 S 5 0
The second high level of the total estrogens in the blood
plasma occurred between days 12 & I H 9 15 6 17 9 and 18 & 20 for. O n e 9
two and two cows respectively»
Similarly four out of the five cows had
the second high level of tot^l urinary excretion of the estrogens
between days 18 S 2 0 o
It is evident that in the majority of the cows the first high
levels of
Yhbrand
ITB-estradiol and estrone in the- blood plasma
occurred between days 6 £ S 0 . Similarly in the majority of the cows the
second high levels of the same hormones in the blood plasma were
observed to occur between days 12 £ 17«
The quantities of these
estrogens excreted in urine by the majority of the cows reached the
first high level during a period between days 3 £ 8 and the second
high level during a period between days 15 £ 20„
It is known that a considerable portion of the estrogens is
excreted in the urine0
It was 9 therefore s thought advisable to collect
the urine samples at the same time=
The urine-collecting-bag9 as has
already been used by many workers ($mith e t „ a l o 1956| Nelson and
Smith 1963) 9 was avoided to prevent the cannula from being .disturbed,,. .
\
“62=
The method of extraction and estimation of the estrogens in the urine
was almost the same as had already been used and found satisfactory by
Nelson and Smith (1963)„
As recommended by Preedy and Aitken (1961)
the method used for the extraction of estrogens from the blood plasma
was the same as for urine except some minor changes„
Although it is the first time that the occurrence of a double
■
I
peak in the levels of estrogens during the estrous cycle of cattle is
being reported it is not surprising because the occurrence of such two
peaks in the levels of estrogens in the primates has already been
established (Fish e t 0 a l o 1 9 4 1 | Markee and Berg 1948; Everett 1961)..
It is also interesting to note that the individual animals showed
considerable variation as to the time of the peaks during the cycle.
It has been reported that 17B=estradiol is the most potent
estrogen whereas Iy0^ e s tradiol is the least (Boscott 1962).
It has
been indicated that estrone $ Iy0^ e s t r a d i o l 8 Ie=Ketoestradiol=IyBsi
2=methoxyestrone and some other estrogenics as well as non=estrogenic8
compounds may very well be the degradation products of 17B=estradiol
(Beck 1950; Engel e t . a l 0 1959; Heard and Huffman 1941; Pearlman and
Pincus 1943; Wotiz e t . a l . 1958; Levitz e t . a l . 1956; Kraychy snd
Gallagher 1957).
Purdy et. a l . (1961) have suggested that estrone may
perhaps be the major estrogenic hormone present in the systemic blood.
Whereas l%^=estradiol and estrone represent the major portion of the
estrogens excreted in the urine (Nelson 1959),
-
A mechanism
63-
ofinactivation
is known to exist in the body of an
individual which helps to maintain the circulating estrogens at a
physiological level (Oakey et, al. 1 9 6 2 j Fishman et„ al« i 9 6 0
eto alo 1950 Ecfcles e t 0 a l 0 1962),
iCoppedge
It seems evident that such a
mechanism may be playing an important role to prevent the animal from
coming in heat during the period when the first high levels of estro­
gens in the blood plasma were observed0
The occurrence of the first
high levels of estrogens in the plasma was in general not followed by
the first level of the urinary excretion of the estrogens ( 1 % & and
17B-estradiol and estrone together)0
It might be possible that during
this period the estrogens were either metabolized to some weak estro'
V• '
Ii
genic or eyen rton-estrogenic compounds or the major portion of the
estrogens was excreted in the feces0
On the other h a n d 8 the second
i'
high level of estrogens in the blood plasma was 9 in'general9 followed
by a high level, of the excretion of the estrogens in the ur i n e „
The
gradual increase in the levels of the excretion of the estrogens in
the urine 8observed after the second peak of the estrogens in the blood- •
had already occurred e might indicate that the magnitude of the inacti­
vation mechanism at this stage also rises gradually 0
The inactivation
mechanism might g therefore„ reach a peak during the same period when
the second peak of estrogens excreted in the urine was observed.
According to the modern concept (Deane 1952) it is believed that
a feedback mechanism is responsible for controlling the production and/
or release of-the"hormones concerned with estrus.
It is generally
“64thought that the level of estrogens during postestrous and diestrous
periods is very I o w 9 whereas the level of progestins during the
diestrous period is very high.
During the last part of phe diestrous
the level of progestins falls down.
This fall* along with the low
level of estrogens9 lowers the threshold for the release of the
;
-
follicle-stimulating-hormone which in turn stimulates the development
of the vesicle follicles.
Growth of the follicles is accompanied by •
an increase in the levels of estrogens.
An increase in the levels of
estrogens at this stage when the level of progestins is very low is
believed to bring the animal in heat.
The present St u d y 9 in contrast to the concept that during an
-
'
■.
estrous cycle of cattle the levels of estrogens rise only once 9 has
indicated that the total estrogens in the b l o o d 9 as well as excreted
in the u r i n e 8 show two pe a k s .
Although no report has shown that the
levels of the secretion of gonadotropic hormones from the hypothysis
of cattle show a double p e a k 8 the occurrence of such two peaks in the
swine has already been reported (Robinson and Nalbandov 1951).
two peaks were observed on days 8 £ 18 of the estrous cycle.
These
No such
information is available to show the fluctuation in the levels of
gonadotropic hormones in the cattle.
It has recently been reported'by Gomes et. al. (1963) that the
levels of progestins in the ovarian venous blood of cattle during an
estrous cycle start increasing on day 6 and reach a peak level on day
"15 after heat. ■ The results from the present study„ although from a
=65”
small number of animals 9 have indicated that the levels of progesterone
in the blood from the posterior vena cava reach a peak on day 17 of the
estrous cycle«
An hypothesis as to how the first peak of the total estrogens in
the blood and the urine occurs and why the animal during such a period
does not come in heat is presented as follows
5
If the secretion of the gonadotropic hormones reaches two peaks
in cattle, as have been shown in swine (Robinson and Nalbandpv 1951)
and if the first peak occurs during the postestrous period, growth of
the vesicle follicles during this period is possible=
The development
of the follicles at this stage would be accompanied by the synthesis
,
'?
:■
and release of
estrogens=
If the levels of progestins start
on day 6 of the estrous cycle (G'om'dsH'et,= al.= 1963), then the
■.
increasing
further
growth-of the follicles decelerated and stopped and then they become
atretic=
At this stage the estrogen inactivation mechanism might be
.very active so
that the higher levels of the estrogens might
immediately
be disposed of
and thus the animal be prevented from coming ip heat=
This may possibly be due to the rise in the levels of progestins and/or
due to some other unknown factors=
The levels of the estrogens in the
blood therefore fall down and are then maintained=
This inhibitory
mechanism due to the presence of very high levels of progestins and
some other unknown factory continue up until the later part p.f the
diestrous period=
If the animal is not pregnant, the threshold due
to prolactin (from the pituitary)-and some unknown factors which are .
"6 Bae
responsible for maintaining the high Jevels of progestins (Turner 1961)*
is lowered,
•This initiates the lowering of the levels of the progestins
during the later part of the diestrous period.
Low levels of progestins
''
'' '•
and/or the occurrence of the second peak of the gonadotropic hormqnes
at this stage* initiate the growth of the follicle vesicles and hence
the second peak of the estrogens in the blood occurs.
During this
period the mechanism of inactivation of the estrogens might not be very
active so that the estrogens could get a chance to bring about the
necessary changes and thus prepare the animal for coming in heat.
Later
on this mechanism becomes very active which might be responsible for the
low levels of estrogens during the estrous and the postestrous period.
The occurrence of a peak of the estrogens during the postconception period similar to the first peak of the estrogens during an
estrous cycle was observed in cow no, 6,
indicates that if the animal "conceives
Data obtained from this cow
.
the threshold due to which the
blood estrogens are maintained at low levels is not lowered and hence
the estrogens do not show the second peak which should have otherwise
occurred.
The absence of the second peak of estrogens may possibly be
used as a diagnostic measure for pregnancy test.
The low levels of
estrogens during the first 34 days after conception are consistent with
the report of Nelson and Smith (1963),
In conclusion* it can be said that the evidence presented indicates
that the quantities of-the estrogens in the blood* as well as in the
urine* of cattle during an estrous cycle reach a double peak.
In most
of the cows the first peak in the blood occurred between days 6 6 8 ,
-67whereas in the urine it occurred during a period between dayp 3 £ S 0
The second peak of the estrogens in the blood occurred on iihys 12''to
17 6 ,whereas the second peak in the utfihe. occurred on days 15 to 2 0 0
The levels of progesterone reached a peak on day 17 of the estrous
cycle«
It is also evident that the levels of the estrogens in the blood*
as well as in the urine* were not high during a period of 34 days postconception*
It seems possible that the absence of the second peak of estro­
gens may be used as an indication that th,e conception has occurred*
SUMMARY
Five Jerseys and one Holstein lactating cows were used for this
Study0
Cannulation of the posterior vena C a v a s, about one inch backward
from the level of the right R i d n ey8 by using Silastic Silicone rubber
tubing was performed0
B l o o d 8 as well as urine samples8 were collected
every other day during the estrous cycle of four Jerseys and one Holstein,,
Samples were also collected during a period of 34 days postconpeption
from one Jersey*
The blood plasma samples were analyzed for the estro­
gens and progesteroneo
measuredo
Excretion of the estrogens in urine was also
The quantitative measurement of the estrogens was done by the
Kober reaction assay whereas progesterone estimation was done by an
ultraviolet as s a y 0
The results have indicated that the measurement of estrogens and
progesterone in the blood from the posterior vena cava of cattle during
an estrous cycle is possible*
The quantities of the total estrogens in the b l o o d 8
as well
as urine of cattle during an estrous cycle 8 reached two peaks 0 In the
majority of the cows the first high level in the blood occurred between
days 6 6 8
whereas the first peak in the urine occurred during a period
between days 3 6 S 8 after heat*
The second peak of the blood estro­
gens occurred between days 12 6 17 whereas the second peak of the urinary
estrogens occurred during a period between days 18 6 2 0 8 after the heat* ■
The levels of progesterone in the blood of the cows were found to
reach a peak on day 17 after heat.
Corresponding to the two peaks of the total estrogens found to
“69“
occur in the blood and urine during an estrous Cycle9 only one peak was '
observed to occur during a period of 34 days postconception <,
Low levels of estrogens in the b l o o d s as well as in the urine of
cattle during the early days of the postconception peri o d 9 are consistent
with the reports by the previous wo r k e r s „
APPENDIX
”
71
"
The purification of the plasma extract for progesterone was done
1
'
by using standard thin layer chromatographic technique.
..
'
'•
■
The.procedure
'
used is outline below s
1)
Three 20 x 20 cms, glass plates of equal thickness were placed
on the aligning tray.
Right and left hand ends of the row of
plates were completed with two 5 x 20 cms, end=plates0
2)
The spreader (Stahl type) was adjusted to give I mm, thick
layers and then placed on the right hand e^B-plate,
3)
Silica-Gel-G (Brinkman Instrument Inc,* batch no, 7731/T6722B
grain size maximum 5-20 u) and distilled water* 50 g, and 80
ml, respectively8 were mixed in a mortar.
Grinding was carried
out very gently and without taking the pestle out of the
slurry.
This prevented the formation of any air bubbles.
Time spent in preparing the emulsion was limited to one minute,
4)
The suspension was immediately transferred to the spreader,
V- " ,
When the slurry started to come out* the process of coating
was begun.
The spreader was then drawn very smoothly across
the plates towards the left end-plate.
The spreader* the end-
plates and the mortar were immediately washed and dried,
5)
The coated plates were left in the laboratory for air-drying
and "setting" for 30 minutes and then were stacked in the
drying rack.
Activation of the Silica-Gel-G layers was carried
out by heating the plates and keeping them in a vertical
position (in the rack) in an oven at 102° - IlO0C, for 30
w72™
minutes»
6)
The adsorbent sticking to the edges and about 2 Inm0 of each
edge of the layer was wiped off with the thiiriib .and finger.
7)
The prewashing mixture9 consisting of Chloroform (3,65 ml.)
;
.■
■
:■
..
.
- and iridthanol (35 m l . ) was poured in the tank and mixed well
by getitle shaking.
8)
Six 5.3 x 20.0 cms. Beckman paper wicks were placed in a verti­
cal position with their lower ends dipping in the prewashing
solvent system to saturate the tank.
9)
When the solvent mixture had reached the top end of the wicks 9
two plates for prewashing were lowered down and placed in
vertical position lying against the opposite walls of the tank.
10)
After developingg the plates were air-dried and heated once
again in the oven at 102° to 110° C. for 30 minutes.
A 2.5
cms, wide strip of Silica-Gel-G was removed from the end of
the plate towards which the solvent was traveling.
The drying
rack with the activated plates was stored in an improvised
desiccator9 having calcium ,chloride as the.desiccant.
-73
TABLE NO, II
A Measure Of The Quantities Of Estrogen Secretion
At Different Periods Of The Estrous Cycle
(Micrograms per ml. plasma)
Period
I
2
I
O - 2
3 - 5
6 - 8
9 - 11
12 - 14
15 - 17
18 - 20
2
2
5
8
11
14
17
20
3
4
5
6
7
O
3
6
9
12
15
18
I
2
3
4
5
6
7
O
3
6
9
12
15
18
2
5
8
11
14
17
20
4
I
2
O
3
6
9
12
15
18
2
5
8
11
14
17
20
5
3
I
2
3
4
5
6
7
I
2
3
3
4
5
6
7
O
3
6
9
12
15
18
Cow
No.
2
5
8
11
14
17
20
4
5
6
7
a*
Days After Heat
No Sample
-
-
IVjt
Estradiol
a*
0.052
0.006
b**
0.064
0.049
0.038
17BEstradiol
a*
Estrone
a*
0.016
0.018
0,019
0.098
0.090
0.060
0.028
0.014
0,031
0.073
0.075
0.075
0.093
0.098
0.118
0.062
0.017
0.035
0.024
0.085
0.032
0.123
0.115
0,016
0.043
0.023
0.019
0.020
0.012
0.020
0.039
0.015
0.034
0.019
0.022
0.024
0.015
0.024
0.087
0.053
0.015
0.016
0.019
a*
0.016
0.021
a*
0.021
0.023
0,062
a*
0.032
0.024
a*
0.009
0.017
0.016
a*
0,023
0.032
a*
0.019
0.033
0.013
0.016
0.025
0.025
a*
0.031
0.040
0.021
0.045
0.024
0.016
0.068
0.129
0.093
0.034
0.086
0.078
0.087
0.018
0.106
0.081
0.014
0.023
0.010
0.014
0.001
0.012
0.005
0.015
0.019
0.037
a*
b** Undetectable
a*
Total
a*
0.096
0.038
0.050
0.235
0.214
0.173
0.265
0.148
0.357
0.258
0.047
0,101
0.057
0.059
0.042
0.048
0.040
0.078
0.101
0.124
a*
0.045
0.056
0.097
a*
0.071
0.077
a*
0,118
0.202
0.127
0.095
0.135
0.119
74
TABLE NO. Ill
A Measure Of The Quantities Of Estrogen Secretion
During Postconception Period
(Micrograms per milliliter plasma)
Cow No. 6
Days PostConceptions
I
5
7
9
11
13
15
19
21
26
31
34
1706Estradiol
17BEstradiol
Estrone
Total
0.006
0.002
0,021
0.013
0.021
0.003
0.014
0.007
0.005
0.008
0.005
0.002
0.032
0.012
0.027
0.020
0.033
0.015
0.006
0.011
0.014
0.008
0.008
0.006
0.034
0.012
0,016
0.027
0,028
0.005
0.013
0.001
0.005
0.023
0,011
0.007
0,072
0.027
0.064
0,060
0,082
0.023
0.033
0,019
0.024
0,039
0.024
0.015
75TABLE NO. IV
A Measure Of The Quantities Of Estrogens Excreted In The Urine
At Different Periods Of The Estrous Cycle
(Micrograms per liter of urine)
Periods
a*
Days After Heat
Cow
No.
17c6
Estradiol
17BEstradiol
Estrone
Total
I
2
3
4
5
6
7
O
3
6
9
12
15
18
2
5
8
11
14
17
20
I
-
a*
31.87
49.12
10.50
24.38
93,75
249.38
a*
46.87
17.25
50.61
73,50
43,50
184.88
a*
50,62
119.25
69,00
61.50
111.00
206,62
a*
129.37
185,62
130.12
159.38
248,25
640,88
I
2
3
4
5
6
7
O
3
6
9
12
15
18
- 2
- 5
8
- 11
- 14
17
- 20
2
40.02
25.80
18,00
21.30
22.42
33.75
36,12
20.18
18.00
15.82
12.75
6.45
12.75
23,55
19,56
63.90
51.90
0.90
27,90
30,00
24,90
79.76
107,70
85.72
34.95
56.77
76.50
84.57
I
2
3
4
5
6
7
O
3
6
9
12
15
18
-
2
5
8
11
14
17
20
3
18,00
31.05
39.00
53.10
7.95
22,05
7,95
1.50
6.60
9.60
8.25
11.92
8.25
11.40
21,90
15,00
12.54
19.50
15.15
13.80
30.90
59.55
54,00
75.24
35.70
49.12
30,00
I
2
3
4
5
6
7
O
3
6
9
12
15
18
-
2
5
8
11
14
17
20
4
a*
4.50
a*
64,05
a*
14.10
56.10
a*
6.00
a*
12,90
a*
15.30
14,40
a*
4,20
a*
9.30
a*
12,90
10,50
a*
14.70
a*
86,25
a*
42.30
81.00
I
2
3
4
5
6
7
O
3
6
9
12
15
18
-
2
5
8
11
14
17
20
5
a*
13,80
10.20
13.65
22.50
21,00
29.40
a*
10.50
13.95
9,60
27.75
24,68
32.25
a*
20,40
18,00
25.10
12.30
16,43
32.25
a*
44.70
42.15
48.45
62.55
62,11
93.55
No Sample
b**
b** Undectable
-76TABLE NO. V
A Measure Of The Quantities Of Estrogens Excreted In The
Urine During Post-Conception Period
(Micfograms per liter of urine)
Cow No. 6
Days PostConception
5
7
9
11
13
15
19
21
26
31
34
17=1Estradiol
17BEstradiol
Estrone
24.00
48.00
43.50
24.60
6.60
b**
12.00
2.40
33.60
5.10
b**
b**
8.25
6.00
9,60
5.10
2,40
19,50
16.50
49,80
24.60
11.25
5.40
5.40
8.10
4,20
13.20
6.00
b**
7.80
5.25
17.10
0.30
Total
55.50
66.90
126,90
54.30
17.85
5.40
13.65
21.90
19.05
35.40
8,70
b** Undectable
TABLE NO. VI
Plasma Progesterone At Various States Of The Estrous Cycle
(Micrograms per milliliter plasma)
Days After Heat
I
9
11
13
17
19
Cows
I
I
I
2
I
I
Cow No,
2
62
2
2 6 78
2
2
Progesterone Content
0.004
0.001
0.014
0.031
0,037
0,031
-
77-
PREPARATION OF .CHEMICALS AND REAGENTS
ie
Benzene8 n-Hexane8 Absolute Methyl alcohol and chloroform were
redistilled before use,
iio
Ether anhydrous washed once with equal volume of one percent
' :
(Weight/Volume) ferrous sulfate solution in distilled water and
then once with equal volume of distilled wat e r 8 and redistilled.
Stored in brown bottles in the refrigerator,
iii,
K o b e r 0S reagent for estrones
Added 660 ml, concentrated sulfuric
acid to 422 ml, distilled water.
Added 10 mg, of sodium nitrate 8
dissolved* and then 20 mg, p-guinone added and dissolved by gentle
warming.
Then 20 g, of hydroquinone was added, and dissolved by
warming and shaking.
Reagent was thqn filtered through glasswool
and stored in brown bottle,
iv,
K o b e r 0S reagent for estradiol isomerss
Prepared in the same way as
for estrone except that 600 ml, concentrated sulfuric acid was
added to 480 ml, distilled water,
v.
Purified e t h anols
Refluxed 750 ml, absolute ethanol with 37,5 g,
1 ••
each of zinc dust and sodium hydroxide pellets for 12 hours
continuously and distilled.
Stored in brown bottle in the
refrigerator,
vi,
Hydroquinone solution inpurified ethanols
Dissolved 0,2 gm,
. hydroquinone crystals in 10 ml, purified ethanol.
freshly prepared.
Always used
-78-
Appendix Figure I.
The equipment used for cannulation;
I 0 Silastic tubing
Ho
Metal plug kept In end of tubing
IlIo
Metal cover to protect end of tubing
IV„
Ring sutured to skin to fasten metal cover
V„
Two way valve used in blood sampling
V l o Syringe used to apply negative pressure.
-79-
Appendix Figure 3,
Drawing blood from cannula,
-
80-
Appendix Figure 4,
Thin layer chromatography for four unknown sample
extracts in solvent system "A",
(The areas corresponding to the
reference spot for pure progesterone have been removed for elution
and separation in solvent system "B",)
Appendix Figure 5.
Thin layer chromatography for five unknown sample
extracts in solvent system "B",
(These unknown sample extracts are
eluates from solvent system "A" as shown in appendix figure 4«)
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