THE QUALITATIVE AND QUANTITATIVE DETERMINATION OF

THE QUALITATIVE AND QUANTITATIVE DETERMINATION OF
THE QUALITATIVE AND QUANTITATIVE DETERMINATION OF
FREE ESTROGENS IN DOG PLASMA DURING THE ESTROUS CYCLE AND
PREGNANCY
by
FRED METZLER, JR.
B. A., University of Maine, 1963.
A MASTER'S THESIS
submitted in partial fulfillment of the
requirements for the degree
MASTER OF SCIENCE
Department of Zoology
KANSAS STATE UNIVERSITY
Manhattan, Kansas
1966
Approved by:
Dr. B. E. ULeftheriou
Major Professor/
ii
C-"3-
TABLE OF CONTENTS
INTRODUCTION
1
MATERIALS AND METHODS
6
RESULTS
16
Qualitative Analysis
16
Quantitative Analysis
17
Estrous Cycle
18
Pregnancy
18
DISCUSSION
Qualitative Analysis
30
30
Quantitative Analysis
32
Estrous Cycle
36
Pregnancy
37
CONCLUSION
k2
SUMMARY
li2
ACKNOWLEDGEMENTS
1^
BIBLIOGRAPHY
\g
—
iii
LIST OF TABLES AMD FIGURES
TABUS
Table 1.
Levels and ranges of individual free estrogens during
the estrous cycle.
Table 2.
Combined free estrogen concentrations during the
estrous
23
Table 3.
Individual free estrogen means and ranges throughout
pregnancy and the first week post-partum.
26
Table h.
Levels of combined free estrogens during pregnancy and
the first week post-partum.
28
cycle.————
—~
21
~ — —~ — ~
FIGURES
Fig. 1.
Flowsheet of estrogen solvent extraction procedure.
Fig. 2.
Two-dimensional TLC distribution of estrogen standards.
19
Fig. 3.
Standard estrogen sulfuric acid-fluorescence curves.
20
Fig. U.
Individual free estrogen levels during the estrous cycle.- 22
Fig.
S>.
Combined free estrogen levels during the estrous cycle.
8
—
2li
Fig. 6.
Individual free estrogen levels during pregnancy.
25
Fig. 7.
Combined free estrogen levels during pregnancy.
27
INTRODUCTION
Just after the turn of the century it was noted for the first time
that biologically active extracts of ovaries were of lipoidal nature
(Iscovesco, 1912; Fellner, 1913; Frank & Rosenbloom, 1915).
These find-
ings along with the historic studies of Stockard and Papanicolau (1917),
Long and Evans (1922) and Allen (1922) dealing with changes in the repro-
ductive tract during the estrous cycle gave the study of ovarian hormones
its initial impetus.
This acceleration quickly led to isolation in
crystalline form of the so-called classical estrogens: estrone (Butenandt,
1929; Doisy et al., 1930), estradiol-17P (MacCorquodale et al .
,
1935;
Wintersteiner et al., 1935) and estriol (Marrian, 1930; Doisy et al., 1930).
In turn numerous other estrogens were, and still are being, isolated from
biological fluids and tissues.
Prior to the mid-1950
use of bioassays.
1
s
most estrogen determinations employed the
Biological tests such as the vaginal smear test in
ovariectomized mice (Allen & Doisy, 1923), increase in uterine weight in
intact immature mice or rats (Astwood, 1938) and increased weight of the
oviduct in immature chickens (Dorfman & Dorfman, 19U8) were more than
adequately sensitive when large amounts of test material were available
and these tests were reportedly specific for estrogens.
However, the usual
drawbacks of a bioassay were inherent in all such tests: the large number
of subjects and, therefore, the large quantity of test material required,
test animal variability, poor reproducibility and test material dependency
upon other variable factors.
In addition, the use of a bioassay gave no
indication as to which particular estrogen was initiating the response and
how much of each estrogen was involved.
Resolution of these two problems
was accomplished with the advent of sensitive and specific chemical methods.
In 1930, Kober published a short paper on a colorimetric technique
for estrual hormone (Brunsthormon).
Although Cohen and Marrian
(I93li)
first
attempted quantitation of estrone and estradiol-17£> in human pregnancyurine with a Kober modification, it was not until much later (Brown, 1955)
that appropriate modifications were made and the method used to accurately
measure estrogens in urinary extracts.
In addition to Brown (1955) several
other investigators (Diczfalusy & Lindkvist, 1956; Ittrich, 1958; Roy &
Brown, I960; Roy & Mackay, 1962; Roy et al., 1965) have used variations of
the original Kober reaction for both urinary and blood estrogens.
The sulfuric acid-fluorescence method for determining estrogens was
first reported by Bates and Cohen (19U7) for pure crystalline estrogens
and by Jailer (19U8) for estrogens in pregnancy urine.
Later work by
Bates and Cohen (1950) established the optimal conditions for this
reaction.
Since then numerous estrogen determinations have employed the
sulfuric acid-fluorescence procedure.
Engel et al. (1950) for example
combined a countercurrent distribution purification with fluorometry to
investigate the possibilities of quantitating estrogens in urine.
Dicsfalusy (1953) measured the free estrone, estradiol-17/2> and estriol in
early and full-term human placental tissue.
Mitchell and Davies
(1951a)
followed this study with a similar one of their own which also measured
the conjugated and protein-bound estrogens.
Slaunwhite et al. (1953)
re-evaluated the Bates and Cohen (1950) study and concluded that after
excitation at
Ii36
mu, estrone, estradiol-17°i. and -17p, and estriol
possessed fluorescence maxima between U78 and U87 mu.
shorter than had previously been reported.
These maxima were
Veldhuis (1953) also used
fluorometry to quantitate the free estrogens in the plasma of pregnant
humans.
Veldhuis (1953) reported that non-specific fluorescing material
which interferred with the accuracy of the fluorometric technique came
from the reagents and to a lesser extent from the plasma extracts.
A
method for the routine clinical determination of urinary estrogens was
subsequently developed by McAnally and Hausman (195U).
Preedy and
Aitken (1957) combined acid hydrolysis, solvent partitioning, column
chromatography and fluorometry to measure the total estrogen content in
plasma of humans during late pregnancy.
This method was again applied
to determine the total estrogen levels in fetal and maternal plasma at
parturition (Aitken et al., 1958), levels in both urine and plasma of
pregnant humans (Preedy & Aitken, 1961), the interrelationships of estrogen
concentration in fetal and maternal circulations and maternal urine
(Manner et al., 1963) and the renal clearance of estrogens (Brown et al.,
I960 & 1961i).
Veenhuizen et al. (i960) adapted the use of solvent
partitioning, paper chromatography and fluorometry to identify and measure
the free estrogens in bovine fetal cotyledons.
By combining the technical information gained from the above invest-
igations with improved methods of isolation (eg., thin-layer and gasliquid chromatography) the determination of micro amounts of estrogen in
small volumes (2-5 ml) of plasma has now become feasible.
Thus far the
only investigation of estrogen levels in circulation throughout a complete
pregnancy has been that for the human by Roy et al.
(1965) with additional
data procured from studies by Roy and Brown (I960), Roy and Mackay (1962),
Preedy and Aitken (1957), Diczfalusy and Magnusson (1958) and Veldhuis
(1953).
Similar data for other species are completely lacking.
Circulating
h
estrogen levels in non-pregnant subjects have also been investigated only
in the human (Preedy & Aitken, 19^7; Roy & Brown, I960; Roy et al., 1965)
the levels being considerably lower than those during pregnancy.
The results of some early studies indicated the absence of detect-
able amounts of estrogen in urine of pregnant dogs (Helm, 1931;
Kflst,
1931) and others (Lesbouyries et Berthelon, 1936; Finck, 1936; Stiasny,
1937; Nilsson, 19U8) reported positive findings in urine.
Recently,
Kristoffersen and Velle (I960) have reported finding small (0 - 2.2
quantities of estrogen in the urine of pregnant bitches.
ngA)
Siegel et al.
(1962) have identified urinary metabolites of injected tritium- label led
estradiol-17p in the dog and concluded that while estradiol-17=< was a
major urinary metabolite, estriol was not.
After administrating estrone to dogs, Longwell and McKee (19U2) found
evidence of estrogens in both bile and urine, but none in control animals.
In somewhat similar studies by Paschkis et al. (I9h3 & I9hk) t who injected
androgens instead of estrogens, it was noted that estrogens were excreted
in urine as well as in bile.
19U5, 19U7
excreta.
Another group of workers (Pearlman et al.,
& 19U8) also injected estrogen into dogs and examined the
It was found that most estrogenic material was present in bile,
a smaller amount in urine and an even smaller quantity in feces.
Cantarow
et al. (I9h3) injected dogs with human chorionic gonadotropin (HCG) and
pregnant mare serum (PMS
)
and concluded that relatively large amounts of
endogenous estrogens are excreted in bile.
After an extensive review of the literature only one reference has
been found dealing specifically with estrogens in dog blood.
Paschkis
and Rakoff (1950) have reported that very little estrogenic activity
could be found in blood from either ovarian or femoral veins after inducing
estrus in the dog by injecting PK3.
The trend in chromatography, particularly of steroids, has been to
reduce running tines and to develop simplified methods for separating
minute quantities of material.
This progress has presently culminated
in two basic types of chromatography, gas-liquid and thin-layer.
The
latter has been widely applied to many substances since its introduction
(Stahl, 1958).
Recently Iasboa and Diczfalusy (1962) published
a
paper on
the separation and characterization of a number of estrogens by meari3 of
thin-layer chromatography.
Though initially used as
a
rapid and sensitive
means of qualitative analysis, thin- layer chromatography has since been
applied to steroid analysis as a quantitative tool.
Ladany and Finkelstein
(1963) have separated estrone, estradiol-17p and estriol on silica gel
plates with a benzene methanol (9:1, v/v) and chloroform :ethanol (9il, v/v)
systems and quantitated them by phosphoric acid-fluorometry.
Considering the dearth of knowledge on the circulating levels of
estrogens in infrahuman species it was thought that any information obtained
along this line would be a worthwhile contribution.
It was first necessary
to standardize quantitative procedures employing thin-layer chromatography
and fluorometry so that these convenient and sensitive methods could be
applied to the present investigation.
The dog was chosen as the subject with which to work since a wellestablished purebred colony was available and because little of the repro-
ductive physiology of this popular laboratory animal at the molecular level
is known.
MATERIAI5 AND METHODS
The dogs used in this study were all purebred adult female animals
which were part of
a
colony maintained at the Hamilton Station, Jackson
laboratory, Bar Harbor, Maine.
They consisted of three pregnant and two
cycling basenjis, two pregnant beagles, one pregnant cocker spaniel and
one pregnant wire haired fox terrier.
Plasma samples of about 5
nil
each
were obtained at various intervals ranging from the onset of the estrous
cycle to two- three weeks after bleeding had ceased.
In the case of
pregnant dogs, samples were taken before fertilization (ie., early estrus)
and after parturition as well as at intervals throughout gestation.
Approximately 12 samples were obtained from each animal.
All plasma
samples were immediately frozen and stored at -20°C until extracted.
The chemical extraction procedure for free estrogens was essentially
that of Veenhuizen et al. (I960).
Frozen samples of about
5
ml were
allowed to melt at room temperature and to each was added 1.0 ml estrone-
16-"*C and 0.£ ml estradiol-17(2>-U-^C in benzene (11,100 disintegrations
per minute each).
The sample was then measured to the nearest 0.1 ml.
The volume of tracer added (ie., 1.5 ml) was subtracted and the net volume
recorded.
The sample was then extracted three times with equal volumes of
anesthesia-grade diethyl ether, the ether extracts pooled, dried over
anhydrous sodium sulfate, and evaporated in vacuo in a 33-UO°C water bath.
The lipid-like residue was dissolved in 10 ml toluene and transferred to a
U5 ml ground glass-stoppered centrifuge tube.
The toluene solution was
washed three times by shaking with 5 ml 5% NaOH and centrifuged at 600 x G
for five minutes.
The NaOH phases (bottom layers) were pooled and back-
washed with 5 ml toluene.
The toluene layers were then discarded.
The
pH of the pooled NaCH layers was adjusted to 7-8 with 6N sulfuric acid.
This "neutralized" fraction was transferred to a separatory funnel and
washed three times with l£ ml benzene to remove the estrogens from the
aqueous phase.
The benzene was evaporated under vacuum and nitrogen in a
water bath at 38°C.
The tube was flushed with nitrogen, capped and kept
refrigerated (about 6°C) until chromatographed.
The qualitative study was initiated by subjecting several (wirehaired
fox terrier for the most part) sample extracts to either one- or two-
dimensional thin- layer chromatography (TLC) in an effort to discern the
identity of dog plasma estrogens.
The TLC procedure and solvent systems
are described below.
Extracts from six cocker spaniel samples equivalent to 32.3 ml
plasma were pooled and subjected to descending paper chromatography
according to the method of Veenhuizen et al. (i960).
Whatman No. 1 paper strips were first cleaned by submerging in
boiling absolute ethanol for 30 minutes, and after drying were then saturated
with a $0% formamide solution in absolute methanol.
The sample was taken
up with a chloroform rabsolute ethanol (1:1, v/v) mixture containing a few
drops of formamide and was streaked with a capillary pipette.
The strip
was run in a glass chromatography tank whose atmosphere was saturated with
Another strip spotted with 20-30 ug each of pure estrone,
benzene.
estradiol-17*, estradiol-17fb and estriol was run simultaneously in the same
manner.
An equilibration period of at least two hours was allowed before
adding a fresh 111 (v/v) mixture of Skellysolve Btbenzene saturated with
formamide.
After addition of the mobile phase, chromatography was carried
out for 16 hours at room temperature in subdued light.
Upon completion of
8
Plasma (about 5 ml)
Extracted 3 times with equal
volume diethyl ether
Aqueous layer
discarded
Ether layer
Dry with Na^C^
Evaporated in vacuo at 38°C
Residue dissolved in 10 ml toluene
Toluene extracted 3 times with
NaOH
5 ml
%
NaOH layer
Toluene layer
discarded
pH adjusted to 7-8
with 6N HjSO^
Extracted 3 times with
1? ml benzene
Benzene evaporated in vacuo
at 38°C
Estrogen residue
Dissolve in chloroform :ethanol (1:1, v/v)
Chromatography
Fig. 1.
Flow sheet of estrogen extraction procedure.
chromatography a hair dryer was used to hasten the drying time which took
between 6-12 hours.
The dried standard strip and a 6 mm portion of the
sample strip were treated successively with 1% TeCl^zl% KCr ? 0- (1:1, v/v),
$% HC1 and water (Veenhuixen et al., I960).
presence of estrogens.
Blue areas indicated the
The sample strip was also counted in a strip
counter (Atomic Accessories, Inc. model RSC-5A) in order to locate the
1
estradiol-17(b-li- kc and to see if the estrone-lo^-^C had run off the strip
into a collecting beaker.
Taking the above into consideration, corres-
ponding areas were cut from the sample strip and eluted with absolute
methanol.
Solvent containing the estrone, which had been eluted into a
collecting beaker during chromatography, was quantitatively transferred to
a test tube.
hours.
The residue was rechromatographed as above, but for only six
After drying, the estrone-l^^C on the rechromatographed strip
was located with the strip counter and this information, in addition to
the unlabelled standard strip developed with the FeCl^iKCr^Oy mixture, was
used to determine the area to be eluted.
The eluate from the area on the original chromatogram corresponding
to the standard estriol was rechromatographed on Whatman No. 1 paper for
five hours in an ethyl acetate :toluene (15:85, v/v) system saturated with
methanol (Mellin, 196U).
In addition to the standard strip, a 6 mm strip
from the sample strip was developed as above.
Since it was found that
the standard had remained principally at the origin with some streaking
(to a distance of 11 cm), the standard strip was interpreted with caution.
The 6 mm strip gave some indication of a blue area 1 cm from the bottom of
the strip.
It was decided that instead of eluting just one area, three
10
sections would be eluted separately.
The first section included the
origin and the first 11 cm, the second section comprised the next 10 cm
and the third section contained the last 10 cm of the strip.
These
eluates were labelled E3 »a», 'b', and «c», respectively.
Infrared spectrophotometry was attempted on the estrone eluates,
but was unsuccessful due to insufficient material.
A reaction to form methyl ethers (Bush, 1961) was carried out on
half of the eluate from each area mentioned above as well as on pure
estrone, estradiol-17<*, estradiol-17(2>, estriol and 16-epiestriol standards.
Another fourth of the eluates and standards were acetylated according to
Bush (1961).
Both the methyl ether and acetylated derivatives were two-dimension-
ally chromatographed on thin- layer plates.
For this and all subsequent
two-dimensional TIC the first direction was run in chloroform :ethyl acetate
(2:1, v/v) and the second direction in an ethyl acetate m-hexane (1:1, v/v)
system.
Preperation of TLC plates consisted of coating 20 x 20 cm glass
plates to a thickness of about 0.30 mm with a slurry of silica gel G
(Merck) purified according to Randerath (1963)
.
The slurry was made by
thoroughly mixing 2 ml double-distilled water with 1 g silica gel Q.
Plates were activated for 30 minutes at 120°C and spotted when cool.
A sample for quantitation was taken up in successive aliquots of
chloroform :methanol (1:1, v/v) and each portion applied to the same corner
spot of the plate, employing a stream of nitrogen to hasten evaporation of
the solvent.
Another plate was spotted with about 20
jig
each of estrone,
estradiol-17ol, estradiol-17p>, estriol and 16-epiestriol standards in the
same manner.
11
In most cases the sample extracts contained a substance, or substances which were soluble in organic solvents, but did not readily
This substance had a tendency to diffuse out from the spot
evaporate.
on the plate.
Since this spreading was accelerated when the plate was
suspended in the tank, the equilibration time for the first direction was
generally reduced to around one minute.
The time for the front to go 18-19 cm was about 90 minutes for the
first system and about one hour for the second.
After chromatography, the standard plate was sprayed with 10$
phosphomolybdic acid in absolute ethanol and heated for about 10 minutes
at 100°C.
Such treatment shows estrogens as varying hues of blue.
The
standard estrogen distribution, as well as the origin and front, were
traced on paper.
After scanning the sample plate with a short UV (2537 A)
Minera light lamp to check for possible fluorescing impurities, the standard
tracing was laid over the sample plate.
The origins were aligned and the
short UV fluorescing impurities and the sample plate fronts were added to
the tracing.
In addition, while the paper was still on the sample plate
the standard spots were lightly traced so as to leave impressions of the
spots on the sample plate.
Taking into consideration the relative front
movements and the amount of impurities (note: impurities tended to cause
distortion of normal mobilities), measured areas were marked off around
These areas were generally rectangular
the corresponding standard spots.
in shape and were about 16-21 cm
excluded as much as possible.
serve as a blank.
.
The short UV fluorescing areas were
A clear area was similarly marked off to
It was necessary to have approximately equal areas
eluted from each plate since the blank varied with the amount of gel eluted
12
and made a difference in the fluororaetric readings.
The areas were scraped off the glass plates with a stainless steel
razor blade, transferred onto waxed weighing paper and then quantitatively-
transferred to glass-stoppered centrifuge tubes.
Although Randerath (1963)
suggested using methanol for eluting steroids from silica gel and most
investigators commonly use methanol for eluting paper chromatograms, it
was thought that at least one of the washings should contain chloroform
because of the relatively low solubility of estrogens in methanol.
The
eluti on procedure, therefore, consisted of one washing with 5 ml chloroform;
nethanol (1:1, v/v) and a second with
cedure was used for both washings.
f?
»1 methanol.
The following pro-
About 2 ml of the eluant was added,
the tube stoppered and then thoroughly shaken for 10-1^ seconds on a Clay-
Adams Cyclo-Mixer.
The remaining eluant was used to rinse down the tube
which wa3 then centrifuged for 15 minutes at 1000 x G.
After centrifu-
gation, the eluates were decanted into test tnbes.
The tube contents were dried with either a stream of nitrogen in a
water bath at 38°C or under vacuum with a Buchler Rotary Evupomix at the
same temperature.
To all tubes was added 1.0 ml absolute ethanol.
For recovery rate
determination purposes, 0.1 ml wa3 removed from each of the estrone,
estradiol-17oL and estradiol-17p tubes and placed in separate counting
vials.
To each vial was added 10 ml of aqueous scintillation fluid.
Radioactivity was then counted with a Packard Tricarb model
scintillation spectrometer.
of 10.
33llj
liquid
Window settings were 50 and 600 with a gain
At least two one minute counts were taken, with the average being
used in the calculations.
In addition to the samples, a blank vial and
13
vials containing 11,100 dpm each of estrone-16-^C and estradiol-17£-U- ^C
were counted each time.
In computing per cent recover;/ the background
was first subtracted, the resulting value multiplied by 10 to get the
total sample count, and this then divided by the standard count and multiplied by 100.
The rationale for counting the estradiol -17<* tube was to
check for contamination by estradiol-17p since the two compounds were not
readily separable in the TIC systems used.
If counts were detected in
If counts were detected in the es trad iol-17<*. tube,
the TLC systems used.
a correction of the fluorescence value was later made.
The fluorometric procedure was a modification of McAnally and
Hausman's (195U) with volumes adjusted to suit the cuvettes used (Mellin,
196b).
To each tube was added 0.1 ml absolute ethanol and 0.7 ml 90$
(v/v) sulfuric acid.
TMs
80°C for 20 minutes.
After heating, the tubes were cooled to room temp-
mixture was then heated in a water bath at
erature, h.3 ml (6% (v/v) sulfuric acid added and the tube adequately
shaken.
After the bubbles had disappeared, the contents were transferred
to matched 12 x 75 mm round Pyrex cuvettes for reading in the fluorometer.
A model 110 Turner fluorometer was employed with a primary filter combination of U7B +
2A_
(the 2k filter closest to the lamp) and a secondary
combination of a 2A-12
the sample).
10$ neutral density filter (the latter nearest
Filter numbers were Kodak Wratten designations.
filter passes wavelengths longer than hl$ mp and the
mission peak at
510 mp.
1*36 mji.
b.TB
The 2A
has its trans-
The 2A-12 filter transmits wavelengths longer than
The 10$ neutral density filter was used to reduce the sensitivity
of the instrument by a factor of 10 in order to maintain linearity between
estrogen concentration and fluorescence (Mellin, 196ii).
Wherever possible,
Ill
snd in most cases it wa3, the aperture setting used was 30 X.
A voltage
regulator was used to stabilize the input current and minimize any drift
of the fluorometer galvanometer.
Standard stock solutions of estrone, estradiol~17£>, estriol
(Calbiochem) and estradiol-17<* and 16-epiestriol (Mann Research Laboratories) were made up to a concentration of UO ng/ml in chloroform :ethanol
(1:1, v/v).
The stock solution was diluted to O.U ug/ral which served as
the working standard.
Fluorescence curves for each standard estrogen
were obtained by adding appropriate amounts of the working standard to
test tubes, drying the tubes and running the fluorescence reaction as
described above.
Depending upon the estrogen, five to seven points were
used for each curve.
Quantities of 0.0U, 0.08, 0.16, 0.2lj, 0.3? and 0.U0
ug were used for the estrone and estradiol-17ot curves.
Similar amounts
as well as 0.60 and 0.80 ug for estradiol-!?/*, estriol and 16-epiestriol,
and up to 1.0
jig
for estriol and 16-epiestriol were used since these
steroids exhibit a lower fluorescence.
In all cases standard tubes and
blanks received the same fimount of chloroform :ethanol (1:1, v/v).
In
addition a blank area from a TIC plate run in the usual systems was eluted
as described and small aliquots of this added to each standard and blank
tube.
The estrogen curves were run at least twice and the best points or
average values used.
The standard curves used in all of the calculations
can be seen in Figure 1, page 8.
Calculation of the quantity of each estrogen was done by converting
the fluorometer reading to micrograms of estrogen with the aid of a standard
curve.
To correct for the counting aliquot removed from the estrone,
estradiol-17c* and estradiol-17yb samples, the microgram quantities determined
:
15
for these estrogens were increased by one tenth.
The corrected values
were then divided by the fractional recovery and by the original plasma
volume.
This jug/nl value was then multiplied by 100 to convert it to
the conventional expression of
jag
estrogenAOO ml plasma.
In instances
where radioactivity (ie., estradiol-17/3 contamination) was detected in
the estradiol-17«t fraction, correction for fluorometric readings had to
be made.
By running many fluorometric determinations the average
estradiol-17oi:estradiol-17/& fluorescence ratio was found to be 1.9.
following equation was therefore used to correctly re-distribute the
fluorometric values
2.9 X • initial estradiol-17<x. fluorometer reading
X
1.9 X
estradiol-17/i contamination
actual estradiol-17o<. value
The
16
RESULTS
taking a
Efficiency of the extraction procedure was checked by
tracer count at every step.
Overall efficiency was found to be 90* or
better.
during
Appearance of an oily residue followed the benzene partition
extraction in the majority of cases.
The contaminant prolonged spotting
estrogens and
time, effected normal chromatographic migration of the
interferred with fluorometry.
The implications of these interferences
will be discussed later.
Qualitative Analysis
;
Preliminary investigations employing
tfce
use of one- and two-dimension-
being an
al thin-layer chromatography (TLC) gave indications of there
and
estriol-like compound, or compounds, and estrogens of the estradiol
estrone type.
Two-dimensional TLC of methyl ether and acetylated derivatives of
that
eluates from the paper chromatographic fractions contributed evidence
estrone, estradiol-17«^, estradiol-17p and estriol were present.
Indi-
cations of another polar estrogen (finally concluded to be 1 6- epi estriol)
were also obtained.
Co-chromatography resulted in no other estrogens,
thereby supporting the derivative data.
The standard 16-epiestriol used for spotting standard plates under-
went a chemical change during the course of the experiment; most likely
oxidation of the 16- and 17-hydroxyl group.
The new estrogen migrated
faster on TLC than did 16-epiestriol, but not as fast as 16-keto-estradiol17/3.
On several occasions a spot corresponding exactly with this
16-epiestriol impurity showed up on sample plates.
Whether dog plasma
17
actually contains the 16-epiestriol derivative or whether it was due to
oxidation of 16-epiestriol in the sample extract was not determined.
However, this "accident" did give further proof of the presence of 16epiestriol.
Quantitative Analysis
;
As mentioned previously, contamination did lead to erroneous results
in some cases.
Evaluation of fluorometric data was further complicated
by low recoveries as determined by the tracer recovery method (the loss
of estrogen occurred at 3ome step, or steps following the extraction pro-
cedure).
Recovery percentages as low as
5>-10$S,
and in a few instances
0$, were obtained for both the labelled estrone and estradiol-17/2>.
Com-
bining all recovery percentages, mean recoveries of 16. h and 31.6$ were
arrived at for estrone and estradiol-17/i, respectively.
In general, the
recovery percentage of estradiol-17p>-li-^C was used in the calculations
of estradiol-17oi, estradiol-17f>>, estriol and 16-epiestriol.
In cases
where the estradiol-17(2>-U-^C recovery was below the mean of 31.6$, the
mean recovery was used in calculating estriol and 16-epiestriol.
This was
done because the positions of estriol and 16-epiestriol standards varied
little from plate to plate and there was little chance of missing the two
estrogens when eluting.
In those instances where the percent recovery
was aero and fluorometric readings were obtained, mean recoveries were
employed in the calculations.
There were frequently sample readings below the blank value.
Whether this was solely a cause of differences in amount of gel eluted or
due to a previously undetected impurity in the blank area was not resolved.
The values were recorded as zero.
18
The only difficulty experienced with the standard curves (Fig. 3,
page 20 ) was with the estriol curve.
Regardless of how many times the
standard curve was run, a straight-line curve could not be obtained.
Evidently, the 10$ neutral density filter was not adequate to maintain a
linear relationship between fluorescence and estriol concentration.
Finally 9-10 concentrations were run several times, the points plotted
and the best straight line drawn.
Estrous Cycle
?
The values obtained during the estrous cycle were divided into early
estrous cycle (initial slight bleeding of the vulva; ie., early proestrus),
middle estrous cycle (full bleeding; ie., late proestrus) and late estrous
cycle (cessation of bleeding; ie., estrus).
The values for the early
estrous cycle are from only one sample and, therefore, must be interpreted
with caution.
Mean values for the concentrations of individual estrogens during
the early, middle and late estrous cycle have been listed in Table 1,
page 21 and graphically represented in Fig. U, page 22,
and Fig.
5>,
Table 2, page 23
page 2u consist of the combined estrogen concentration means
during the estrous cycle.
Pregnancy ;
The estrogen levels during pregnancy were observed on a biweekly
basis.
An average of six samples per biweekly period were examined.
Trends in both individual (Fig. 6, page ? 5) and combined (Fig. 7,
page 27) estrogen levels became apparent.
The least prevalent estrogen,
estradiol-17oi, remained at fairly constant levels throughout pregnancy,
ranging from a low of O.U to a high of 3.0
ugA°0 ml
plasma.
Likewise,
19
Fig. 2. Two-dimensional TLC of estrogen standards. First direction
run in chloroform:ethyl acetate (2il) and the second in ethyl acetate:
n-hexane (1:1). Designations: 0, origin; E
lf estrone; E 2 <S estradiol-17*;
E 2 fj, estradiol-17p; E,, estriol; Ek, 16-epiestriol; and Et> 16-epiestriol
'9
impurity.
20
X
ESTRONE
O ESTRADIOL- 17
ESTRADIOLA
17
°L
(S,
ESTRIOL
• I6-EPIESTRI0L
40
FLUOROMETER
60
80
READING
Sulfuric acid-fluorescence curves for the following standard
estrogens: estrone (E^, estradiol-17ot(E «*), estradiol-17p(E
2
2 /2>),
estriol (E3) and 16-epiestriol (E ) . Each point represents the
means
c
'
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Fig. 3.
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U
23
Table 2.
The total free estrogen concentrations (ug/100 ml) in
plasma during the early, middle and late estrous cycle
periods. K • the number of samples per period.
Period
Mean
Range
n
Early Estrous
1U3.0
Middle Estrous
69.2
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6
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29.6
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LATE
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EARLY
Fig. 5.
cycle.
Combined free estrogen levels in dog plasma during the estrous
25
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Fig. 7. Biweekly levels of combined free estrogens in dog plasma
throughout pregnancy.
fi
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28
Table lu
The means and ranges (ug/lOO ml) of the total free estrogens
during the first h biweekly periods, the ninth week of
pregnancy and the first week post-partum. N
the number of
samples per period.
VJeek
Mean
Range
1-2
21U
8.5-h9.6
7
3-a
Uo.5
7.5-67.9
6
5-6
26.Ii
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0.0-38.U
3
1 p.p
59.8
Ul. 9-87.7
h
29
16-epiestriol also held fairly constant (3.2 -
5.ii
ug/lOO ml).
In
contrast though, both estrone and estradiol-17/2> showed a continuous
decrease in concentration after reaching a peak of 8.6 and 13. h jig/100 ml,
respectively, during the 3-k week period.
The most pronounced decrease
was that of estradiol-17/3 from a peak of 13. li jug/100 ml at 3-h weeks to
a low of 0.7
ugAOO ml at
nine weeks.
Peak levels were attained by all
of the estrogens at 3-h weeks except for estriol, but it too was essen-
tially at its peak.
The 3-b week period was again prominent when the
individual values were combined.
23.2
ugAOO ml
An average free estrogen level of about
was maintained throughout pregnancy except for the 3-h
week period when the concentration rose to
li0.5
ng/100 ml plasma.
During the first week post-partum there was a substantial increase
in every estrogen except estradiol-17°<..
The means and ranges of four
samples during this period can be found in Fig. 6, page 2$, Table 3, page
26 and Fig. 7, page 27, Table h, page 28.
30
DISCUSSION
The 9Q£ plus efficiency of the extraction procedure obtained in
this work was in good agreement with that reported by Veenhuizen et al.
(I960).
The oily residue encountered after extraction was finally traced to
the silicone stopcock grease (Dow Chemical Co.) on separatory funnels used
to partition the benzene and neutralized aqueous fractions.
It was also concluded that stopcock grease contamination caused a
distortion in normal distribution of the estrogens on thin-layer plates,
thus contributing to the possible error in eluting and decreasing recovery
efficiency.
The distortion became progressively more pronounced as the
front was approached (ie., greatest possible elution error was experienced
with estrone and least with estriol).
It is also likely that the oily
residue hindered completeness of sample transfer from test tubes to thinlayer plates while spotting.
Although the stopcock grease contaminant caused some fluorescence
upon exposure to short UV, it was not the sole source, for extracts obtained
without stopcock grease also displayed such fluorescence.
The presence of
non-specific fluorescing materials stemming from both reagents and plasma
extracts has been reported by Veldhuis (1953) and Slaunwhite and Sandberg
(1950).
Veldhuis (1953) suggested that the interf erring materials might
be lipids, and such might also be the case in the present investigation.
Qualitative Analysis
;
The Skellysolve B:benzene:f ormamide paper chromatographic system of
Veenhuizen et al. (I960) proved satisfactory for separating estrone,
estradiol- 17*, estradiol-17(* and estriol.
The difficulty encountered in the
rechromatography of the estriol fraction was due to a procedural error.
An
31
interesting phenomenon was observed in the apparent division of estriol
into two spots on the paper chromatogram as determined by TLC of the
aeetylated products (ie., acetylation of estriol fractions 'a* and 'c 1
gave identical products with respect to chromatographic migration).
It
would seem probable that acetylation resulted in a racemization; the
acetylation of the
16<*-
and (b-hydroxyl groups yielded acetate groups
spa cially- oriented in only one direction.
Results from TLC analysis of unsubstituted extracts, aeetylated
and methylated derivatives and co-chromatography with standards showed
that estrone, estradiol-17=<, estradiol-17/» and estriol were present as
free estrogens in dog plasma.
It should be kept in mind that the estro-
gen samples for TLC were initially separated into discrete fractions with
paper chromatography.
Identification of 16-epiestriol was based primar-
ially upon inference.
The initial one- and two-dimensional TLC analyses
indicated an estrogen migrating slightly ahead of estriol, corresponding
most closely with 16-epiestriol.
Also, the standard 16-epiestriol
impurity did exactly match the extra metabolite observed on several plates.
This latter observation suggests the possible presence of yet another
estrogen metabolite.
Whether dog plasma actually contains this 16-epiestriol
derivative or whether it was due to oxidation of 16-epiestriol in the sample
extract was not determined, however.
The present results indicating the presence of estrone, estradiol-17=^,
estradiol-17(J>, estriol and 16-epiestriol in the dog are in good agreement
with the findings of others.
Early workers (Longwell & McKee, 19U2), using
the vaginal smear method in ovariectomized rats for the assay, could not
detect any estrogens in the bile or urine of control dogs.
However, upon
32
administration of estrone nearly all of the total estrogen detected in
the bile extracts was in the non-ketonic fraction.
Between 61.8 - 80.9?
of the total estrogen in urine extracts was non-ketonic.
This suggested
that the dog has the enzyme systems necessary for transforming estrogens.
As a further note, Paschkis et al. (19i»7) reported that after intra-
muscular injections of acetyl-estrone the major portion of estrogenic
material was recovered in the bile, a lesser amount in the urine and a
yet smaller, but measurable quantity in the feces.
The majority of the
estrogenic material in the bile was in the "free" form and the ratio of
conjugated to free in the feces was small while this ratio was much
greater in the urine.
Pearlman et al. (19U7) questioned whether the dog was capable of
transforming estrone into estriol to any extent.
Siegel et al. (1962)
also suggested that estriol is not a major metabolite in the dog.
However,
Siegel et al. (1962) did report finding trace amounts of estriol as well
as other 16-oxygenated estrogens in dog urine.
In contrast, Longwell and
McKee (19U2) found appreciable estrogenic activity in the 0.3 M Na^CO^soluble, benzene-insoluble phenolic fraction (ie., that fraction consisting of estriol).
Pearlman et al. (I9h£) also detected estrogenic activity
in similar material in dog bile after injection of estradiol-17p.
In
addition to traces of estriol and other 16-oxygenated estrogens, Siegel
et al. (1962) also found estrone, estradiol-17«*- and estradiol-17/£> in the
urine and blood of the dog.
Quantitative Analysis
;
In addition to causing distortions in the estrogen distribution
pattern, the extraction contaminants also interferred with the fluorometry.
33
At times the interference was obvious and the value had to be discarded,
but at other times there seemed to be little or no interference.
Since
it could not easily be discerned whether materials visible under short
UV would interfere with fluorometric readings, it was preferable to avoid
the contaminants wherever possible.
It is also possible that impurities
were present which did not show up under short UV, but did fluoresce when
heated with sulfuric acid.
Experience has shown, however, that if the
plate appeared clean under short UV, the chance of there being extraneous
fluorescence after acid treatment is slight.
The very low recovery percentages of the radioactively-labelled
estrone and estradiol-17p> may be attributed to a number of factors.
The
primary factor was most likely an error in estimating the position of
the unknown from the distribution on the standard plate.
While the
fronts on both standard and unknown plates generally reached the same
heights, impurities in the unknown could still cause distortions in
estrogen distribution.
It was concluded that use of
a
"cold" standard
plate to determine the distribution of the unknowns was not always reliable
and that the preferred method would be to use a TLC-plate scanner to
accurately locate the radioactively-labelled estrogens.
Other likely
factors involved were loss during transfer of the sample from test tube
to TLC plate, avoidance of short UV-fluorescing areas even when they
corresponded to the possible estrogen locations, and loss when scraping
gel off the plate.
This latter factor has been considered by some investi-
gators (Nandi & Bern, 1963) as being one of the drawbacks of using TLC for
quantitative studies.
Although in some instances in the present study
estrogens were observed to remain on the glass, the technique was quite
3k
effective if care and
a
sharp razor were used.
It has also been reported that silica gel impurities interfere
with some methods of quantitation.
Nandi and Bern (1963) have noted
this effect in UV specroscopy, Randerath (1962) in fluorometry and
Siegel and Dorfroan (1963) with the Kober reaction.
However, present
evidence indicates that TLC can be U3ed in conjunction with fluorometry.
While the sample blank gives a considerably higher fluorometric reading
than does the reagent blank, the causative factor (s) is not wholly due
to the silica gel.
Methanol-washed 3ilica gel G was added to a reagent
blank with little effect on the degree of fluorescence.
It was observed
that if the sample was eluted from the silica gel soon after the plate
was run, the background fluorescence was higher than if the sample was
eluted one to two days after running.
The longer the plate remained
exposed to the atmosphere before eluting the lower was the background
fluorescence.
However, it was considered inadvisable to permit exposure
of estrogens to the atmosphere for too long a period since under such
conditions oxidation can occur.
Therefore, plates were kept in a light-
tight box for about two to four days before eluting.
It would appear,
therefore, that one or more of the solvents used in the TLC systems and/or
elution were responsible for the extra fluorescence and that with time
the contaminant either evaporates or undergoes degradation to a non-
fluorescing product.
Veldhuis (1953), among others, has also reported
background fluorescence from eluting reagents.
In most cases during the
present investigation, the extra fluorescence could be adequately blanked
out with the instrument.
Though the use of mean recovery percentages in calculating the
35
quantitative data in the instances mentioned previously may not appear
particularly desirable, to have used recoveries as low as 5% for estraol
and 16-epiestriol would have resulted in values even further from the
truth.
In these cases and those where fluorometric readings were obtain-
ed even though the percent recovery wsn essentially zero as determined
by radioactive counting, the use of mean recoveries seemed justified.
While the method of re-distributing the fluorometric values (see
page 15) when radioactivity was detected in the estradiol-17°t fraction
is not unequivocally correct, it does offer a better approximation of
the true values than if no correction were made.
In discussing results of both the estrous cycle and pregnancy it
must be kept in mind that data for free estrogens only partially contribute
to the overall estrogen status of the animal.
Values for the circulating
levels of both protein-bound and conjugated fractions are also required
in order to more completely establish any conclusions.
Circulating estrogen levels in either the non-pregnant or pregnant
subject have only been thoroughly studied in the human (Preedy & Aitken,
1957 l
Roy & Brown, I960; Roy & Mackay, 1962 j Roy et al., 1965; Smith,
1966) and the white-tailed deer (Eleftheriou et al., 1966; Church &
Eleftheriou, 1966).
Despite the quantity of data, direct comparison of
most such results with this present work is difficult due to the use of
different blood components for analysis.
Whether or not there is a sig-
nificant difference in whole blood estrogens as compared to plasma remains
to be resolved.
The works of Smith (1966) and Eleftheriou et al. (1966),
however, do offer results for comparison.
36
Estrous Cycle ;
For the follicular phase of the menstrual cycle in the human, Roy
et al. (1965) have reported the following total (free, protein-bound and
conjugated) estrogen levels in peripheral whole blood: estrone, 0.020 ±
0.011; estradiol-17p, 0.013 ± 0.008; and estriol, 0.025 + 0.012
Their values for the ovulatory phase were 0.070 + 0.025; 0.028
and 0.037 ± 0.023 jugAOO ml, respectively.
ugA^O
ml.
0.017;
It, therefore, appears that
at least in the case of the human, the general belief that estrogen levels
increase as the follicules mature and rupture holds true.
If this is
also the case in the dog, and from bioassay evidence it seems to be so
(Gier, I960), then the progressive decline in free estrogens from proes-
trus through estrus (Fig. 3) would indicate a concomittant rise of the
estrogens in some other form(s).
Whether the other forms are protein-
bound and/or conjugated fractions or an ever increasing binding of the
estrogens at their tissue receptor sites and subsequent metabolism are
possibilities, but ones which require supporting evidence.
If approached from a slightly different angle, the problem of
explaining the decline in free estrogen levels becomes even more perplexing.
The free form is taken to be the biologically active fraction and
since sex-accessory tissue (eg. uterus, vagina) response appears to
indicate progressively increasing amounts of active estrogen during the
estrous cycle, it would appear logical that the present study should
have shown a steady increase in the free estrogen levels.
the case, the following is proposed.
This not being
The tissues in question have been
sufficiently predisposed by the initial high level of free estrogen, and
quite likely by other hormonal agents such as progesterone and the
37
Therefore, higher
gonadotropins, so as to be sensitized to estrogen.
active levels of estrogen would not be required to initiate maximal
tissue responses.
The requirement of a high concentration of estrogen
to change the physiologic condition of the sex-accessory tissues from
an anestrus to proestrus state is not outside the realm of possibility
since it is apparent that the dog is relatively insensitive to estrogens
when compared with the human and the cow.
Assuming the above hypothesis to be true and since the estrogen
production by the ovary continually increases, the extra estrogen would
have to be directed to protein-bound and conjugated forms with, quite
likely, a corresponding increase in excretion rate.
With respect to
the latter however, Kristoffersen and Velle (i960) found very low levels
of estrogen in the urine of the dog.
Discussion of the possible impli-
cations of renal clearances will follow.
Pregnancy ;
It is known that as pregnancy progresses the circulating and
urinary estrogen levels increase.
This is initially due to increased
ovarian activity and later as a function of placental participation.
Roy and Hackay (1962) and Roy et al. (1965) have shown the progressive
increase of total estrogens in the whole blood of women from the 6-7
week period to the ho**1 week.
They did state, however, that the increase
for each estrogen measured was not always continuous.
Their values for
the 6-7 week period were for estrone, 0.138 ± 0.081; estradiol-17p>,
0.050 ± 0.021 j and estriol, 0.051 ± 0.023 JQtgAOO ml and for the U0th week
period: 5.UU ± 2.36; 1.19 x O.hl; and 9.71 ± 5.00
ugA°0
™T->
respectively.
The increase in estrogens during pregnancy also increases the concentration
38
of plasma proteins.
With the above in mind, the results of the present study, which
indicate a fairly constant free estrogen level throughout pregnancy
(except during the
3-Li
week period), can best be explained ir the
following manner.
The phenomenon observed is most likely a combination of factors.
In order to maintain a constant level of an X while the source is pro-
ducing more of X than before, the excess t must be diverted to other
state or places.
In the case of a constant free estrogen level, this
diversion could very well be contributed by a combination of:
1) target- tissue requirements continually and gradually increase, thereby
affecting an increased rate of metabolism of the estrogens;
2)
estrogens,
themselves, are loiown to stimulate an increase in the amount of plasma
proteins which in turn could lead to increased protein binding of the
estrogens; and 3) an increased excretion rate, particularly renal clearance .
The latter
estrogen levels.
ma}'
well be the direct consequence of increased blood
Brown et al. (196U) have studied the renal clearance
of estrogens in the human during late pregnancy and concluded that while
the urinary /plasma ratios for estrone and estradiol-17/2> are essentially
the same, that for estriol is significantly different when compared to
either estrone or estradiol-17/&.
Furthermore, after examining the re-
lationship between plasma concentration and renal clearance, they suggested the possibility of a tubular secretion maximum for estriol.
How-
ever, Brown et al. (1961.0 did not distinguish between the various forms
in which estrogens exist in both plasma and urine, but rather arrived at
39
values for total estrogens.
The work of Brown et al. (196U) may offer an explanation for the
presence of low levels of free estradiol-17<* in plasma while a major
urinary estrogen metabolite in the dog (Siegel et al., 1962).
The
difference between the free plasma level and urinary total estradiol-17<<
level is most likely due to another form of estradiol-17«* (ie., protein-
bound and/or conjugated) and/or an increased conversion of another estro-
gen (eg., estrone) to estradiol-17<* in the liver.
Just the reverse may
be true for estricl, which is in high concentrations in the plasma while
it is not reported to be a major metabolite in the urine (Siegel et al.,
1962).
Although these metabolic conversions and variances in physico-
chemical forms are good possibilities, the possibility of there also
being a selective renal tubular secretion mechanism cannot be excluded.
The estrogen-protein relationship has been investigated by various
workers (Saego & Roberts, 19U6} Bischoff et al., 195U; Antoniades et al.,
1957).
In
a
review by Sandberg et al. (19!>7) on the binding of steroids
to human plasma proteins, it was concluded that steroid-protein binding
involves free and conjugated steroids, that the binding relationship is
weak and reversible, and that of all the steroids examined the estrogens
are the most tightly bound.
Szego and Roberts
(l9ii6)
were able to
progressively dissociate the estrogen-protein complex by dialysis.
Their
results indicate a simple equilibrium between the estrogen and protein
and, therefore, the degree cf binding i3 logically a function of both
the estrogen and protein concentrations.
Since both estrogen and plasma
protein concentrations increase during pregnancy, it would follow that
the amount of estrogen-protein binding would also increase and thereby
Uo
aid in maintaining a constant free estrogen level.
During pregnancy the progressive proliferation of the placenta,
uterus, mammaries and other tissues involved in reproduction is directly-
controlled by a balance of various hormones including estrogens.
It is
well known that hormone ratios change throughout pregnancy, indicating
changes in production rates and/or changes in metabolic rates.
As proposed for the estrous cycle (see page 37)
$
the sex-accessory
tissues require less estrogen for a given response after having been
initially primed by a high dose of estrogen.
If an organ (eg., uterus)
is taken as a whole, its overall estrogen requirement increases through-
out pregnancy since maintenance and proliferation continue.
However,
on the cellular level the amount of estrogen per unit response is lower
than that required initially.
These tendencies would cancel to some
degree, although the former would probably predominate.
The total
estrogen level (free as well as protein-bound and conjugated) may
increase in proportion to the requirement for free (ie., "active")
estrogen.
This would mean a continual drawing off of free estrogen and
a constant readjustment of the equilibrium in order to
free estrogen level.
maintain a constant
This would also imply a shift from combined
estrogens back to free; a subsequent decline in total estsogen if it were
not for the increased rate of estrogen production by the placenta.
In this work the u3# increase above the mean of the dog plasma
estrogens during the 3-U week period can best be attributed to the
initiation of placental secretory activity.
It is during this time that
placental development is completed (Gier, 1965) and its estrogen secretion
combined with that of the ovary without a concomittant increase in tissue
estrogen requirement would result in an increased free estrogen level.
Ill
With the cessation of ovarian activity and re-establishment of a
relationship between tissue requirement and physicochemical equilibrium,
normal mean
the free estrogen level would be expected to decline to its
value of 23.2 jugA0O ml.
Comparison of the present data vith free estrogen levels in whitein
tailed deer plasma (Eleftheriou et al., 1966) indicates a similarity
fairly
that during most of pregnancy free estrogen plasma levels remain
constant.
Eleftheriou et al. (1966) however, report a significant rise
whereas
in free estrogen levels from estrus to 6-8 weeks of pregnancy,
estrus and
in the present study no notable difference was found between
the first week of pregnancy.
This discrepancy need not necessarily be
correspond
true since the 6-8 week period of pregnancy in the deer may
to the 3-h week period of the dog.
On such a basis the dog wouli also
show a significant rise above the estrus level.
Smith (1966) found relatively constant levels of free estrone and
plasma
estriol and slightly increasing levels of estradiol-17^ in human
from 15 weeks before the onset of labor to term.
The present study also
indicates constant free estrogen levels in the dog during the latter
stages of pregnancy, except for estradiol-17(2>, which shows a progressive
decline.
A surprising result was the spectacular rise in free estrogens
during the first week post-partum.
It is generally assumed that in an
animal without a post-partum heat the total estrogen concentration declines
after parturition sin^e the major source of the hormone (ie., placenta) is
expelled.
If this assumption is correct, the results suggest
shift in equilibrium to predominately the free form.
a
sudden
However, this rise
U2
in free estrogen would be expected to be transient since the sex-accessory
tissues show
little evidence of estrogenic activity during anestrus.
If
the phenomenon is not transient, either the tissues are de-sensitized to
estrogen following parturition or the values are incorrect.
appears unlikely*
The latter
Considering the changes in other hormonal concentrations
following parturition, a de-sensitization is quite probable,
CONCLPSIOH
The qualitative and quantitative techniques used in this study appear
to be sound.
Specificity, sensitivity, speed and simplicity are advantages
of TLC plus fluorometry although a drawback is the presence of interferring
fluorescent materials.
The interferring materials, however, can be reduced
to a minimum by using the proper combination of pure reagents.
Addition of
refinements adopted during the course of this study should prove useful.
Final procedural refinements include:
1) use of teflon stopcocks on the
aeparatory funnels in the extraction procedure (overcomes silicone grease
contamination and subsequent difficulties in spotting, distortion of normal
chromatographic migration, and excess residual fluoroescence) and 2) use of
chloroform as the solvent for elution from silica gel rather than methanol
(this decreases the amount of silica gel residue and background fluorescence).
SUMKaRI
Integration of solvent partitioning plus paper and thin-layer
chromatography with sulfuric acid-fluorometry led to the successful
identification and quantitation of estrone, estradiol-17°<, estradiol-17(2>,
estriol and 16-epiestriol as free estrogens in dog plasma.
fluorescence was used to quant itate the estrogens.
Sulfuric acid-
U3
The estrous cycle was divided into early, middle, and late phases
corresponding approximately to early proestrus, late proestrus and
e3trus, respectively.
Mean values (jugA 00 ml plasma) for the early
phase were estrone, 38.1; estradiol-!"**, 22.5; estradiol-17|2>, U9.U;
estriol, 26.1; and 16-epiestriol, 6.6.
Middle phase values in the same
order were 30.3, 7.6, 7.5, lU.8 and 8.9 jugA 00 *1 plasma.
the late phase were:
respectively.
Values for
5.8, 3.2, 5.3, 12.0 and 3.3 JugA 00 ml plasma,
Combined values averaged 1U3.0, 69.2 and 29.6jttgA00ml
plasma for the early, middle and late phases, respectively.
The pro-
gressive decline in total estrogen was attributed to increased tissue
sensitivity to estrogen after a priming effect and to a shift in the
equilibrium from the free form to protein-bound and conjugated states.
Except for estrone and estradiol-17/2> individual estrogen levels
remained fairly constant throughout pregnancy with estradiol-17
the least in concentration and estriol the greatest.
estradiol-17(b decreased after 3-U weeks.
being
Both estrone and
Combined values indicated a
constant level averaging 23.2 jug/1 00 ml plasma throughout pregnancy
except for the
3-ii
week period when the level rose to U0.5.
Mean values
for total estrogen in jug/100 ml plasma during biweekly periods were:
1-2 (2U.U); 3-U (U°.5); 5-6 (26.U); 7-8 (19.6); and 9 (22.U).
A number of hypotheses have been explored in an attempt to explain
the correlations between free estrogen levels and various stages of the
estrous cycle and pregnancy.
hh
ACKNOWLEDGEMENTS
I wish to thank the following people without whose aid this study
would have been exceedingly difficult, if not impossible:
Dr. B. E.
Eleftheriou for his financial and moral support; The Kansas Agriculture
Experiment Station for my research assistantshipj Dr. T. Mellin for
advice on the paper chromatographic and fluorometric procedures; Mr. H.
Minocha for counting the radioactive samples; and Dr. M. Fox for the
plasma samples.
U5
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THE QUALITATIVE AND QUANTITATIVE DETERMINATION OF
FREE ESTROGENS IN DOG PLASMA DURING THE ESTROUS CYCLE AND
PREGNANCY
by
FRED METZLER, JR.
B. A., University of Maine, 1963.
AN ABSTRACT OF A THESIS
submitted in partial fulfillment of the
requirements for the degree
MASTER OF SCIENCE
Department of Zoology
KANSAS STATE UNIVERSITT
Manhattan, Kansas
1966
The direct determination and measurement of biologically-active
molecules, rather than their effects is a major innovation.
With the
means now at hand, it is possible to obtain data on specific hormonal
levels in situ .
The availability of such data greatly contributes to
the understanding of basic physiological processes from the organismic
to the cellular level.
For these reasons,
a
study of the free estrogens
in plasma of cycling and pregnant dogs were undertaken.
The procedure consisted of combining solvent extraction, chroma-
tography (paper and thin-layer), radioisotopes and sulfuric acid
fluorometry.
Prior to extraction estrone-lo-^C and estradiol-rfia-U-^C were
added to each sample for recovery determination purposes.
Extraction consisted of first extracting with diethyl ether,
evaporating the ether fraction and dissolving the residue in toluene.
The toluene solution was then treated with
%
NaCH, the NaOH fraction
neutralised with 6N R^SC^ and extracted with bensene.
The qualitative analysis entailed chromatography of unsubstituted,
acetylated and methylated estrogen derivatives.
Initial chromatography
was on Whatman No. 1 paper (saturated with $0% formamide in methanol)
using a Skellysolve B: benzene (1:1, v/v) system saturated with formamide.
The eluate from the origin (ie., polar estrogens) was re-run in an ethyl
acetate: toluene (l£:8$, v/v) system saturated with
$Of>
methanol.
Separate
portions of these fractions were then acetylated or methylated and run
2-dimensionally on silica gel G thin-layer plates in chloroform :ethyl
acetate (2:1, v/v) for the first direction and ethyl acetate m-hexane (1:1,
v/v) for the second.
Chromatography led to the conclusion that the free
estrogens in female dog plasma include estrone, estradiol- 17*, estradiol-17/2>,
estriol and 16-epiestriol.
Quantitation was carried out by first purifying the sample extracts
with the 2-dimensional thin-layer chromatographic systems mentioned above.
Areas corresponding to standards were eluted with a methanol :chloroform
(111, v/v) mixture.
The eluate wa3 then evaporated, the residue re-
dissolved in 1.0 ml absolute ethanol and a 0.1 ml aliquot taken for
purposes of counting radioactivity.
The remainder was evaporated and
to the residue was added 0.1 ml absolute ethanol and 0.7 ml
acid.
This mixture was heated for 20 minutes at 80°C.
U.3 ml (6% sulfuric acid was added.
sulfuric
The fluorescence of the mixture was
measured with a fluorometer using a primary combination of
a secondary combination of 2A-12 + 105?
filters.
90f>
After cooling,
li7B •
2A and
neutral density Kodak Wratten
After correcting for recovery rate and original sample volume,
values for estrogen in jagAOO »! plasma were obtained.
The estrous cycle was divided into early, middle and late phases
corresponding approximately to early proestrus, late proestrus and
estrus, respectively.
Mean values (ugAOO ml plasma) for the early
phase were estrone, 38.1ij estradiol-17*, 22.5j estradiol-17p>, U9.U;
estriol, 26.1; and 16-epiestriol, 6.6.
Middle phase values in the same
order were 30.3, 7.6, 7.5, 1U.8 and 8.9 jugAoo ml.
phase were: 5.8, 3.2, 5.3, 12.0 and 3.3
AgA^O
bined values averaged 11j3.0, 69.2 and 29.6
middle and late phases, respectively.
Values for the late
ml, respectively.
ugAOO ml for
Com-
the early,
The progressive decline in total
free estrogen was attributed to increased tissue sensitivity to estrogen
after a priming effect and to a shift in the equilibrium from the free
form to protein-bound and conjugated states.
Except for estrone and estradiol-17|2>, individual estrogen levels
remained fairly constant throughout pregnancy with estradiol-17* being
the least in concentration and estriol the greatest.
However, both
estrone and estradiol-17p> decreased after 3-U weeks.
Combined values
indicated a constant level averaging 23.2
ugACO
ml throughout preg-
nancy except for the 3-U week period when the level rose to hO.5.
Mean
values for total free estrogen in ugAoO ml plasma during the biweekly
periods were:
(22.U).
1-2 (2u.h); 3-U (U0.5); 5-6 (26.U); 7-8 (19.6); and 9
It is felt by the author that the following hypotheses best
explain the maintenance of constant free estrogen levels during pregnancy:
for
1) continual and gradual increased sex-accessory tissue requirement
active estrogen; 2) increased protein binding; and 3) increased and
selective excretion.
The 3-h week period increase was considered due to
the initiation of placental secretory activity and an altered free:
combined equilibrium.
The first week post-partum values showed increases
in all free estrogens except estradiol-17*.
p.gA-00 ml.
The combined value was 59.8
This rise suggested a shift in equilibrium to the free form,
concomitant with a decreased tissue sensitivity.
In addition to contributing estrogen data, the present study
stimulated thought on further hypotheses concerning estrogen problems.
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