SPECTRGPHOTOMETREC
mmmm “new 0-? Paocasmaoma

Thesis for tho Dayna—cf M. S.
MICHIGAN STATE COLLEGE

Clyde 1F rank Caiiry
  ' E 945

This is to certify that the

thesis entitled

SpectrOphotonetric Determination
of Progesterone

presented by

Clyde Frank Cairy

has been accepted towards fulfilment
of the requirements for

g, 3, degree in Chemists:

Date December 15. 19g§

 

OQJMW

Major profess r/

_._.~_H_ _ .

 

SPEC TKO PHD ‘i‘ OldEmRIC Difl‘lnn 113A? I OH
OF

PROGE TERO I‘d—E

by

CLYDE FRANK CAIRY

A THESIS
Submitted to the Graduate School oflflichigan
State College of Agriculture and Applied
Science in partial fulfilment of the
_requirements for the degree of
IMASTER OF SCIENCE
Department of Chemistry

1945

- - ~ Q‘HEMISTRX

. . .. .‘ _.
i . l J f

P'T '7'”
it“ ‘0

I

\

i.

1

t‘

 

ACI'CNO WLEDGMEN TS

The author wishes to express appreciation to
Dr. C. A, Hoppert, Professor of Chemistry, for helpful guid-
ance in the course of this work and in the preparation of
this manuscript, to Dr. D. T. Ewing, Professor of Chemistry,
and.Miss Selma Bandemer, Research Assistant in Agricultural
Chemistry, for assistance in mastering the technique of op-
erating the spectrophotometer and for pertinent suggestions
throughout the study, to Dr. C. FR Huffman, Professor of
Dairy, for making available the cows and calves used in the
later phases of this project, and to Dr. E. J. Miller, Re-
search Professor of Agricultural Chemistry for generosity in

permitting the use of the Beckman spectrophotometer.

l?5871

liLLE CE CONTENTS
PAGE
I. INTRODUCTION AND REVIEW OF LITERAI -E . . . . . . l
A. Attempts to find progesterone in the blood and
uril‘le O O O O O O O O O O O O O O O O O O O O O l

B. Difficulty of determining progesterone biolog-

ically . . . . . . . . . . . . . . . . . . . . 2

C. Absorption spectra of progesterone . . . . . . 3

II. EXPER iENTAL PROCED’RES . . . . . . . . . . . . . 4
A. Spectrophotometer operation; graphing proce-

dure . . . . . . . . . . . . . . . . . . . . . 4

B. Preliminary absorption curves . . . . . . . . . 5

l. Hexane . . . . . . . . . . . . . . . . . . . 5

2. Alcohol . . . . . . . . . . . . . . . . . . 6

3. Hater . . . . . . . . . . . . . . . . . . . '7

C. Diluted blood plasma . . . . . . . . . . . . . 9

D. Chromatographic studies . . . . . . . . . . . . lO

Petroleum ether extractions . . . . . . . . . . 11

£21

1. Nith a separatory funnel . . . . . . . . . . ll
a. From aqueous solutions of progesterone . 11
b. From plasma after precipitation of the

proteins . . . . . . . . . . . . . . . . 13

-

lab: or comm-rs (cont-l

c. From plasma without precipitation of
the proteins . . . . . . . . . . . . .
(1) Synthetic mixtures . . . . . . . .
(2) Normal cows and calves . . . . . .
2. Uith a continuous liquid-liquid extractor
a. From aqueous solutions . . . . . . . .
b. From the blood plasma of a cow injected
with progesterone . . . . . . . . . . .
c. From hemolyzed blood of a cow injected
with progesterone . . . . . . . . . . .
III. RESULTS AND DISCUSSION . . . . . . . . . . . . .
IV. SUIEMIRY . . . . . . . . . . . . . . . . . . . .

REF Eljkch ES 0 o o o o o o o o o o o o o o o o o o o o

66
69

INTRODUCTION AND REVIEL‘I 9].: LIT “RAE 11E

 

 

For a number of years attempts have been made, with
some success, to demonstrate the presence of the corpus
luteum hormone, progesterone, in various tissues and fluids
of animals, including the human. The assay for this materi-
al depends upon the production of progestational prolifera-
tion in the endometrium of the rabbit. Corner and Allen in
1929 (1) showed that this effect could be produced by ex-
tracts of the corpus luteum of sow ovaries. Clauberg and
associates in 1953 (2) were able to produce the uterine re-
sponse by using extracts of human ovaries. In the same year
Mazer and Goldstein (3) reported the detection of this mate-
rial in a 6 months human placenta. Other workers have ei-
ther confirmed this (1954-1935) (Fels (4), Ehrhardt (5),
Adler, de Fremery and Tausk (6), Lankeren (7), and Ehrhardt
and Hagena (8)), or failed to do so in earlier attempts
(1931-1953) (Phillips (9), Fels (10), de Fremery, Lucks and
Tausk (ll), Ehrhardt and Weigel (12), and Portman (15)).

Attempts to extract this substance from.urine met
with failure until, in 1935, Loewe and Voss (14) succeeded
in obtaining one Clauberg rabbit unit from 20 liters of hu-
man urine collected during pregnancy or the last half of the

menstrual cycle.

2

In working with blood, Clauberg and his associates (2)
(1955) used up to 555 ml. of human blood and were unable to
show the presence of the hormone. In 1956, Bloch (15) ex-
tracted as much as 500 m1. of human pregnancy blood, obtain-
ing negative results. He also extracted 1.0, 5.0, 4.5, 6.0,
8.0, and 12.0 liters of blood from sows in which the repro-
ductive history was unknown. Of these the smaller amounts
were negative whereas the extracts from.8 liters and 12 1i-
ters showed the presence of 0.5 to 0.7 rabbit unit (one unit
being the amount which suffices, when divided into 5 daily
doses, to change the uterus of a doe weighing 5 to 4 kilo-
grams, to a state representing the 8th day of normal preg-
nancy (1)). V

Since biological assays at best have the variability
of individual animals to contend with, and since the biolog-
ical effect might be due to other substances, the search for
a different method of determining the presence of progester-
one was believed to be wurth while. The use of absorption
spectra has proved usefulixlworking with numerous biologi-
cally important materials, and it was found that progester-
one has an absorption maximum at 240 millimicrons (in abso-
lute alcohol) (16). The curve is single—peaked and the max-
imum falls at a wave-length which is well separated from the
maxima of most other comp01.1nds.

With these ideas in mind, the hope that the presence

of progesterone might be detected in solutions and in blood

by spectrophotometric means, led to this study.

Due to the speculative nature of the problem various

procedures were tried. The following outline will serve to

indicate the order of development of the work. These will

be described in detail under "Experimental Procedures".

A.
B.

SpectrOphotometer Operation; graphing procedure

Preliminary absorption curves

1. Hexane

2.

Alcohol

5, water

Diluted blood plasma

Chromatographic studies

Petroleum ether extractions

1.

With a separatory funnel
a. From.aqueous solutions'of progesterone
b. From.p1asma after precipitation of proteins

0. From.plasma without precipitation of the pro-
teins

(1) Synthetic mixtures

(2) Normal cows and calves
With a continuous liquid-liquid extractor
a. From aqueous solutions of progesterone

b. From the blood plasma of a cow injected with pro-
gesterone

c. from hemolyzed blood of a cow injected with pro-
gesterone ‘

EXPER KEJTAL PROCEDURE“

 

SPECTROPEOTOHETDR TECHNIQUE AID GRAPHING PROCEDURE

 

In all cases the final determinations were made on a
Beckman Quartz Spectrophotometer, Model DU, using the same
pair of 1 cm. square fused silica cells throughout. One of
the cells was always used for the solvent or blank, the
other for the unknown, and during this study these cells
were used exclusively for this purpose. Each cell was
placed in the carrier with the same face toward the light
source on each trial. One of these cells had a light path
length of 1.002 cm. The other was 1.005 cm. long. For the
reason that the cells were always used in the same way, and
since differences in the transmission of the two cells, when
applied to actual data, gave results differing only slightly
from the observed readings, cell corrections have been omit-
ted. Cleaning of the cells was accomplished by immersion in
chromic acid cleaning solution for about 50 seconds, then
rinsing thoroughly with distilled water. Following this,
they were rinsed 5 times with the solvent being used, the
blank was filled with solvent, -nd the second cell was
rinsed twice with the unknown and then filled.

The light source was a hydrogen discharge lamp. As

it was necessary to-change this tube during the work, the

effects of such changes are discussed below.

The sensitivity control was used at the counter-
clockwise limit.

The abs01ptions, in percent transmission, were re-
corded for wave lengths between 208 and 500 millimicrons.
The lower limit was determined by the solvent and the hydro-
gen lamp. In general, determinations were made every 2 to 5

‘

millimicrons except at tie maximum, when readings were taken
for each millimicron. fhese percent transmissions (fiT) were
transformed to log IO/I (or E) by the use of a chart pre-
pared for that purpose. In the above expression, IO repre—
sents the intensity of the light entering the cell, and I
represents the intensity of the light emerging. E is the
xtinction due to the dissolved substances.
In plotting the data, 3 was plotted against wave

lengths in millimicrons in most cases. The concentration
curves were prepared by plotting the E values against the

various concentrations at the wave length where maximum ab—

sorption occurred.
PRELDIINKRY ABSOETIOH CURVES
hexane. Preliminary trials showed that hexane (from

petroleum, practical, Eastman Kodak Co.) absorbed all the

light with wave lengths below 240 millimicrons. It was

therefore discarded in favor of absolute alcohol.

Absolute Alcohol. Crystalline progesterone* to the
amount of 12.5 milligrams was dissolved in 100 milliliters:
of absolute alcohol. This was not used until six months
later when 5 milliliters of this solution was diluted with
95 milliliters of absolute alcohol to make a solution con-
taining 0.00625 mg./ml. Measurements of the absorption of-
t is solution were made with the spectrophotometer 2 days
following the dilution and again on the 4th, 8th, 15th,
21st, and 28th days. The results, presented in Graph.No. l
in the section entitled "Results and Discussion", indicated
that the curves were highly variable as to wave length of
maximum absorption and also as to the degree of absorption.

A fresh stock solution was made up by dissolving 10
milligrams of crystalline progesterone in 100 milliliters of
absolute alcohol. Five milliliters of this was diluted with
95 m1. of alcohol to make a solution containing 0.005 mg./ml.
The absorption of this solution was measured using absolute
alcohol as a blank and was repeated on the 4th, 7th, 15th,
14th, twice on the 19th, and on the 20th day. Graph No. 2
shows the results. The curves are as variable as those in
Graph No. l.

Thirteen days after the above stock solution

(0.1 mg./ml.) was prepared, a new dilution was made by adding

 

*The progesterone used in these studies was obtained
from the Schering Corporation. Melting point z 128-12900.
Optical rotation = +174.0.

95 ml. of absolute alcohol to 5 ml. of the stock solution.
This resulted in an alcoholic solution containing 0.005 mg./
ml. as before. The absorption was measured as previously
described on the same day the dilution was made and also on
the following day to determine if similar results could be
obtained. These curves (Graph Ho. 5) showed that determina-
tions made on successive days using fresh solution were also
variable.

Later in the study, occasion arose to again make up a
stock solution by dissolving 5 mg. of progesterone in 100 ml.
of absolute alcohol (: 0.05 mg./m1.) and to dilute this to
make, among others, a concentration of 0.005 mg./ml. The
results obtained with this solution are presented in Graph
No. 4, and pertinent data from Graphs 1, 2, 5, and 4 are
shown in Graph No. 5. All of these illustrated a lack of
relationship between the extinction, the absorption maximum,
the time since making the solution, and the slit-width.

Distilled Water. Due to the dissimilarity of the re-
sults obtained with alcohol, and because it was desired fi-
nally to work with blood, curves were made using distilled
water as the solvent.

An attempt was made to dissolve 10 mg. of progesterone
in 100 ml. of distilled water. The crystals did not dis-

solve completely. With this solution (0.1 mg./m1.) the ab-

sorption was measured on the 2nd day, twice on the 5rd, once

on the 4th and 8th, and twice on the 9th day following its

preparation. In addition, it was diluted 1:1 to make

0.05 mg./ml. and analyzed on the 1st, 2nd, and 4th days aft-
er dilution. A further dilution of the original stock solu-
tion to 0.005 mg./ml. was made and absorption measured. All
of these results are plotted in Graph No. 6. The curves
showed less variability than the alcoholic curves.

A new aqueous stock solution was made by putting 10
mg. of progesterone into 4 ml. of absolute alcohol (in which
it dissolved almost completely) and then diluting to 250 ml.
with distilled water. The absorption of this solution
(0.04 mg./ml.) was measured once on the next day and twice
on the succeeding day. Further dilutions and determinations
were made, the results of which are given in Graph No. 7.
This graph indicated that dissolving progesterone in a small
amount of alcohol facilitates its solution in water.

The third and a fourth aqueous stock solutions were
made as follows: 10 mg. of progesterone was dissolved in
5 m1. absolute alcohol (all appearing to dissolve). Then
for Stock III distilled water q.s. 500 ml. was added. For
Stock IV the alcoholic solution was ggggg pg sufficient wa-
ter to make 500 ml. Each of these (0.02 mg./ml.) was di-
luted to make concentrations of 0.018, 0.016, 0.014, 0.012,
0.010, 0.008, 0.006, 0.004, and 0.002 mg./In . In addition
Stock IV was diluted further to 0.001 and 0.0005 mg./m1.

The absorption of all of these dilutions was then measured 5

or 4 times at and near the maximum of 248 millimicrons.

From these a concentration curve was constructed (Graph

No. a).
DILUTED BLOOD PLASMA

To determine if progesterone could be detected spec-
trophotometrically in the presence of blood plasma the fol-
lowing anhmals from the emperimental dairy herd were bled
(coagulation was prevented by bleeding into flasks contain-

ing 0.5 gram of sodium citrate crystals per 100 ml. of

blood):
Blood Plasma
11111112 in ml.
0574 (calf) 100 47
A 55 (non—pregnant
cow) , 120 80
A 27 (non—pregnant
cow) 170 105
A 26 (pregnant
covn 190 105

The plasma was obtained by centrifugation.
It was necessary to dilute the plasma in order to al-

low light to pass through it. Dilutions of 5 m1. of plasma

with 95 ml. of distilled water were made using plasma from
C574 and A55. In addition the A55 diluted plasma was dilu-
‘ted.again 5:95 to obtain a 0.25% plasma solution in dis-
‘tilled water. The absorption data from these dilutions, us-
ing; distilled water as a blank,-are shown in Graph ho. 9.
Guarves for 0.25% plasma from.the other animals did not dif—
fer' enough from this one to allow them.to be plotted. Later

in tune work occasion arose to collect blood from a number of

10

animals. The plasma from these was diluted to make 0.4%
plasma solutions. All of these gave curves very similar to
the single example in Graph No. 9.

Progesterone to the amount of 10.75 mg. was dissolved
in 2 ml. of absolute alcohol. Of this, 0.2 ml. was added to
99.8 ml. of A27 plasma which had been diluted 5:95 and this
in turh 5:95 to make 0.25% plasma in distilled water as
above. This made a 0.001075% profiesterone in 0.25% plasma
solution. Graph 30. l0 shows the absorption curve of this
solution as compared with a curve obtained with the 0.25%
A27 plasma without added progesterone. An alteration of the
curve at and near the maximum of 248 millimicrons is evi-
dent.

The results of similarly prepared solutions contain-
ing 0.00055755, 0.001075%, and 0.00215% progesterone in
0.25% of A27 plasma are shown in Graph Ho. 11. The estima-
tions of the progesterone present were found to be in agree-
ment with the amount added to the solutions.

A comparison of the 0.0005575fi and the 0.001075% so-
lutions with 0.25% plasma as a blank is given in Graph No.
12.

CHROMATOGRAPHIC STUDIES

Although very few substances can be adsorbed from an
aqueous solution, it was thought that this should be deter-

mined. It was found that in running water through alumina

11
(grade 7-20, mesh 80-200, Aluminum Ore Co.) a cloudy fil-
trate resulted unless the column (2 cm. x 20 cm.) was first
prepared by running water through it. Hence a series of

steps was devised, as follows:

 

 

 

Amount ,F , Spectro hotometer
in m1 haterial Filtrate samnfe number
50 Distilled water Cloudy
50 n 9! n
50 " II N
50 fl '1 I!
50 " " Less cloudy
50 " " Quite clear I
50 " " Quite clear II
100 0.01 mg./ml.
progesterone in
distilled water Quite clear III
50 Distilled water Quite clear IV
50 Distilled water Clear V

Results in Graph No. 15 proved that alumina did not adsorb

progesterone from aqueous solution.
PETROLEUM ETEER EXTRACTIONS

From.Agueous Solutions. fiiph_g_8eparatory Funnel.
Inasmuch as it appeared that the progesterone in blood would
have to be concentrated, extraction with some organic sol-
vent was indicated. Consequently 100 ml. of the 0.01 mg./ml.
progesterone in water solution, made up 5 weeks earlier, was
extracted with 100 ml. of petroleum.ether (Baker's C. P.
Special, boiling point 200-40O C.) for 5 minutes. The water
layer was saved and the ether layer was distilled off. The
residue was taken up in 100 ml. of distilled water. After

transferring the aqueous solution to another flash, 1 ml. of

l2
absolute alcohol was added to the distilling flask, shaken
and added to 99 ml. of water. This was done to establish
whether there was any residue not taken up by the water
alone. The absorption was determined on the various solu-
tions using water as the blank for the aqueous systems and
petroleum ether for the others. Graph Ho. 14 illustrates
that petroleum ether removed progesterone from aqueous solu-
tions.

In order to determine the character of the extraction
products of water only, a second series was made up as fol-
lows: 100 ml. of petroleum ether was distilled, and the ab-
sorption curve for the distillate determined. The amount of
100 ml. of distilled water was extracted with 100 ml. of pe-
troleum ether in a separatory funnel for 5 minutes. The wa-
ter layer and the ether layer were run on the spectrophotom-
eter. The same procedure was applied to 100 ml. of 0.01 mg./
ml. progesterone solution. The absorption of the water layer
was determined. The ether layer was evaporated to dryness,
the residue being dissolved in 2 ml. of absolute alcohol and
this added to 98 ml. of water. The absorption curves of the
latter and the ether distillate were determined. Graph No.
15 revealed, as did Ho. 14, that petroleum ether extracts
progesterone from aqueous solutions.

To recheck the above procedures, a third series was
developed. The amount of 100 ml. of the progesterone solu-

tion (0.01 mg./ml.) was extracted for 10 minutes with 100 ml.

15
of petroleum ether. The ether layer was evaporated to dry-
ness over a water-bath, 2 ml. of absolute alcohol was added
to the residue, shaken, and this then added to 98 n . of
distilled water. The flask was rinsed with the resulting
solution to take up the residue more completely. In an at-
tempt to dissolve any remaining residue 2 ml. of alcohol was
again shaken in the flask and added to 98 ml. of water. As
a control on this latter procedure a clean flask was subjec-
ted to the same procedure. Absorption curves for these var-
ious products are presented in Graph No. 16, adding further
evidence that petroleum ether extracts progesterone.

From Plasma after Precipitation g£_the Proteins. It

 

was believed that if the proteins could be precipitated from
the plasma without removing the progesterone that this would
allow the latter to show up better. Two experiments were
carried out to see if trichloracetic acid would accomplish
this. In the first, to 100 ml. of mixed plasma (obtained
from cows A26, A27, and A55 about 10 weeks previously) was
added 1 mg. of progesterone dissolved in 0.5 ml. of alcohol.
This plasma was added to 1000 ml. of 10% trichloracetic acid
slowly with stirring. After standing 10 minutes, the mix-
ture was filtered. This yielded 975 ml. of filtrate, which
was diluted to 1000 ml. The amount of 200 ml. was extracted
with 100 ml. of petroleum ether for 10 minutes. The residue
left from evaporating the ether layer to dryness was taken

up in 0.5 ml. of alcohol and 49.5 ml. of water. A control

14
was prepared in the same fashion omitting the added progest-
erone. Graph No. 17 shows the absorption curves.

The second experiment was similar but a proportion-
ately larger amount of progesterone was used. One mg. of
progesterone was dissolved in 0.5 ml. of alcohol and added
to 10 ml. of the mixed plasma used above. .A control 10 ml.
sample of plasma was mixed with 0.5 ml. of alcohol only. To
each of these, 100 ml. of 10% trichloracetic acid was added
as before. After standing 10 minutes and filtering, each
filtrate was diluted to 100 ml. Eighty ml. of each was ex-
tracted with 80 ml. of petroleum ether for 10 minutes. Six-
ty ml. of the ether layer from each was evaporated to dry-
ness, and the residue was taken up in 0.5 ml. of alcohol and
49.5 ml. of water. Graph No. 18 shows the curves for the
filtrates, the water layers, the ether layers, and the re-
dissolved residues. Graphs l7 and 18 indicated that pro-
gesterone could not be detected in the presence of plasma
when the proteins have been precipitated by trichloracetic
acid.

From.Plasma without Precipitation 9: the Proteins.
Synthetic Mixtures. It being possible that petroleum ether
could efficiently extract progesterone from plasma in the
presence of proteins, the following was tried. One mg. of
progesterone was dissolved in 0.5 ml. of alcohol and added
to 10 ml. of the plasma. As a control, 0.5 ml. of alcohol

was added to 10 ml. of the plasma. Each was extracted for 5

15
minutes with 20 ml. of petroleum ether. A sludgy ether lay-
er resulted, but it cleared considerably upon standing 25
minutes. The ether layer was filtered to remove the remain-
ing emulsion. Each was diluted 1 ml. to 19 of petroleum
ether and was then run against petroleum ether. Graph No.
19 shows that progesterone may be removed from plasma by pe-
troleum ether extradtion and establishes the absorption max-
imum at 250 millimicrons.

Hermal Cows and Calves. 0n the possibility that a
difference could be shown between the plasmas of cows in
‘arious stages of reproductive activity, it was decided to
make extracts of the plasma and/or serum of a number of ani-
mals. Chart I indicates the procedure. All samples were
separated into plasma and cells by centrifugation. The ab-
sorption curves of the ether layers (Graph Ho. 20) showed no
significant differences between animals.

E123 g Continuous Extractor. From Aqueous Solutions.
In an attempt to minimize the emulsion formed by the ether,
a continuous liquid-liquid extractor was made. The appacatus
is illustrated in Figure l. The solution to be extracted
(50 to 55 ml.) is introduced into the lower part of the ex-
tractor, the inner tube is inserted, and the condenser
fitted to the top. One hundred ml. of solvent (petroleum
ether) is placed in the flask and warmed to boiling over an
electric coil. when it vaporizes enough to reach the con—

denser, it condenses and falls into the inner tube. As it

16

 

 

 

 

 

 

 

 

 

 

 

 

Hefiao
.eHeo mopeoHeeH o.
.peecwene espeoe N + NN m.Nm H NH mN Hm + mm Na:
.pcecmene enscoe N + NN o.m: H NH mN He + OHH NNH
.peeemone ensues N + NN m.Hs mN.H NH mN a: - NoH NNH.
.eHee oHo espeoe 3H + MN o.Ha m.H NH mN mm + mHH HNmo
.oHee eHo enscoe :H + NN 0.0: m m mN mm + oOH meme
.otoeen ease mH wean + mN o.ma m m mN mm + OOH NN«
.enoaon mane NH eons + MN 0.:: m m mN o.mm + QQHANV
.enoaen osee NH oenm + mN o.s: HN m mN m.mm . OOHAH. NN<
.weEpom con
(anew mansheeHmcoo ,
"enoeen ease m oenm 4 mN o.ms NN m mN m.ms + mN Nm«
. . meHSOS hmflpm .HE Efihmm HOG .
use as as .m: .c s x a no no as .0:
mapssmm v p .pozsa .pomea H H On an .eoan c

IHHm hops: hogan (ensue .eEHB cmpowpp mammam copay .p54 stdc<
r eeHa -xo .pe« .oHeH» -pHo

 

58 I'll.

 

 

 

 

 

 

m.
Continuous Liquid-
liquid Extractor
1° -0
whim
1
120 III.
250 \7/ 15 an. tubing
m.
f 5 In.
tuna; '
100 I1. (Ink
_(_ - '

 

 

 

figure 1
30-10: 1 II. I 10 u.

17

18
piles up there its weight forces solution down and out the
holes in the bulb at the lower end of the tube. The sol-
vent, being lighter than the aqueous layer, rises to the top
and accumulates there. Uhen it surpasses the level of the
sloping side-arm it gravitates bacx into the flask. Thus
the solvent circulates through the solution and has an ep-
portunity to dissolve any soluble material.

The extractor was first used to remove some progest-
erone from 50 ml. of the 0.01 mg./ml. aqueous stock solu-
tion made up 5 1/2 months before. The extraction proceeded
for 2 1/2 hours, when the ether in the extractor was added
to the flask ether and the absorption curve determined using
petroleum ether as a blank. Graph No. 21 shows that this
extraction was at least partly successful.

It was necessary to start using a new bottle of pe—
troleum.ether. This was a.Merck product, boiling point 500-
6000. This was found to transmit no light between 250 and
260 millimicrons;so it was discarded.

A bottle of petroleum ether similar to the first was
used with interesting results. The amount of 50 ml. of the
0.01 mg./ml. aqueous solution of progesterone was extracted
with 100 ml. of this ether (hereafter called petroleum
ether II) for 1 hour and 40 minutes. An equal amount of
distilled water was used as a control. The extractor ether

in both cases showed per cent transmissions above that of

the pure solvent. The curves for the flask other are shown

19
in Graph No. 22, along with a plotting of the slit-widths.
The latter, being highly erratic, indicated that the petro-

‘

leum ether diff e1 ec from the first let used (called petro-

q

leum.e her I). Petroleum ether II was therefore discai ded
in favor of another lot, petroleum ether III. The labels of

1

these th ree lots of Baker's C. P. Special Ietnaleum LtLer,

 

 

 

h. P. 200-4000. gave the following information:

Lot I Lo t II Lot III
Sp. ”r. at 150/1500. 0.050 abt. 0. 03 abt. 0.52
Initial Boiling Point 51.50C. 51.50 C. 50.000.
Haximum boiling Point 55.OOC.. 15. 400 , 55. 200.
:onvolotile mutt 01 0.0002fi 0.000éfl O .OOOBfl
Sulfur canpounds (as S) 0.000% 0.000% 0.000g
heavy oils _949005 0.000fi_ 0.QQQ&_

 

 

As some difficulty was encountered when working with
petroleum ether in the spec crophotometer cells, due to rapid
evaporation of the more vols- tile fractions, it was decided to
evaporate the petroleum other off and redissolve the residue
in alcohol or water before determining the absorption
curves. F 1fty ml. of the old 0.01 mg./ml. stock solution

0 4.1.,

was extracted 1100 petroleum ether II for 1 hour. The resi-

due, after evaporating the mixed flask and extractor ether,

.15-;

was dissolved in 0.5 ml. of absolute alcohol, and 49.5 ml.
of water was added to it. A cloudy solution resulted, but
the cu1 ves showed the extraction, when compa1 red with the ex-
traction of plain vater, wa nevertheless successful (Graph
No. 25).

A new aqueous stock solution of progesterone was pre-

pared as before by dissolving 10 mg. in 5 ml. of absolute

20
alcohol and adding this to distilled water to make a liter.
A 50 m1. portion of this and a 50 m1. portion of plain water
were extracted exactly as described in the preceding para-
graph, xcept that the flask ether and the extractor ether
were kept separate, and the alcoholic residue was added :9_
the 49.5 ml. of water. This resulted in a very nearly
clear solution. In making the absorption curves, the flask
extract of progesterone solution was used against the flask
extract of water as a blalk, and the extractor other ex-
tracts in a similar way. Graph No. 24 offered further proof
of the ability of petroleum ether to extract progesterone.

From the Blood Plasma of a Cow Injected with Progest—
grgpg, It was decided at this stage to attempt to recover
some injected progesterone from.the blood stream of a cow;

It was estimated that a 1000 pound COW'would require 40 mg.
of progesterone in order to have about 0.25 mg. in 200 m1. of
blood, a quantity with which it was convenient to work.
Therefore, 40 mg. was dissolved in 5 ml. of absolute alcohol
and injected in that form as follows:

Blood was collected in 125 m1. flasks each containing

0.5 gm. of sodium citrate crystals.

21

Approximate S

 

 

 

 

Thne amount bled ample Remarks

m1. no.
4:24 100 1 From left jugular vein
4:25 100 2 From.left jugular vein

 

 

4:29 40 mg. progesterone in

- 5 m1. absolute alcohol
injected into right
jugular vein

 

 

4:51 100 5 From left jugular vein
4:52 100 4 " " " "
4:55 100 5 " " " "
4:3 57 100 6 II '1 fl "

4:40 Finished bleeding_

Samples 5, 4, 5, and 6 represent continuous bleeding
interrupted only by changing needles between samples 4 and
5. All samples were stored in a refrigerator over night.

The following day the plasma and the cells of each

sample were separated by centrifugation with results as fol-

 

lows:
Total Plasma
Sample volume, volume, Remarks
no . ml 0 ml 0
1 108 71.0
2 115 72.0
5 110 72.5
4 105 70.0 Red tinge, Due to hemol-
ysis when needle was
changed.
5 104 70.0
6 _._._.lo___9 .2415...

 

The plasma samples were stored in a refrigerator when
not being used.

Two days later, the extracting of these samples was
begun. Samples 1 and 2 were extracted with the same 100 m1.

portion of petroleum ether II as were samples 5 and 4, and

22

also 5 and 6. The table shows the procedure:

Amt. of Amt. of
ether

Sample plasma Extraction
no. nfld time, hrs. nu,

 

Remark 5

 

 

1 50 1.5 100

 

 

 

 

 

Large bubbles in
ether layer. Let
stand 2 hours 20
min.
1 l6} Heated.more slowe
2 50 1'7 100 1y. Emulsifica-
tion not so ex-
tensive.
2 55 1.5 100 Little emulsifi-
cation
5 50 1.5 100 Little emulsifi-
_ cation
5 17} Little emulsifi-
4 32 1-5 10° cation
4 52 1.5 100 Little emulsifi-
cation
5 50 1.5 100 Some emulsifica-
tion
16} r Some emulsifica-
6 25 1-0 100 tion
6 42 1.5 100 Some emulsifica-

tion

After evaporating the ether samples to dryness in a water-

bath, each residue was dissolved in 0.5 ml. of absolute a1-

cohol, and added to 24.5 ml. of distilled water. The first

sample, A, resulted in a slightly cloudy solution; B was

less cloudy than A; C was less cloudy than A but more cloudy

than B. Using distilled water as a blank, the absorption

curves (Graph No. 25) were found to be influenced consider-

ably by the cloudiness and to indicate that progesterone was

25
not recovered.

The cloudiness described above might have been due to
some ether reacting with the rubber stopper in the distill-
ing flask; so an experiment was run as above, using 100 m1.
of petroleum.ether II and 58 ml. of a.mixture of the aqueous
solutions B and C above. The ether layer was evaporated to
dryness without the use of a rubber stopper. When redis-
solved in 0.5 m1. of alcohol and 24.5 ml. of distilled wa—
ter, a cloudy solution resulted, indicating that some non-
volatile materials in the ether were forming an emulsion
with water.

From.Hemolyzed Blood ping Cow Injected with Progest-

 

 

erone. Bloch (15), in attempting to detect the presence of

progesterone by biological means, used hemolyzed blood. To

determine if hemolyzed blood could be used in the extractor,
the following experiment was set up. All blood collections
were made into 125 m1. Erlenmeyer flasks containing 0.5
gram of sodium citrate.

Approximate Sample

{‘10
lime amt. bled; ml. no . Remarks

4:50 100 1 FlaSk contained 2 mg. of
progesterone (and citrate).
From left jugular vein.

 

 

 

 

 

 

4:51 100 2 From left jugular vein.
4:55 100 5 From leftpjugular vein.
4:55 75 mg. of progesterone in
10 ml. of absolute alcohol
into right jugular vein
4:58 100 4 ‘From.left jugular vein
4:59 100 5 From left jugular vein
4:40 100 6 From.1eft jugular vein
4:42 100 7 From left jugular vein
4:44 Finished bleedingp

 

 

 

24

Samples 1, 2, and 5 represent continuous bleeding, as do 4,

5, 6, and 7. The blood was stored in the refrigerator.

The hemolyzing of this blood was accomplished by add-
ing 200 ml. of distilled water to 100 ml. of blood. Then
each resulting solution was extracted in the continuous ex-

. tractor as outlined below.

Amount of “xtraction Amt. of Ether
hemolyzed time, ether lot
no. bloodJ m1. hours usch m1. no.
50
51
56
55
55
55
5O
5O
5O
5O
5O
50
55
55

Ether
sample

Sample

 

 

 

100 III 1A

 

 

100 II B

 

 

100 III C

 

 

200Eli II

 

I
"’" 200* 1'11" " ' E

 

 

 

 

100 III F

mmmomommeemmmmmmmmmmmmwpwwww
HHHHHmmmmmHHHHHHHHHHHHHHHHHH
D O O O O O O O O O 0 . O C C O O O O O O O O C 0 O O

cfi<1mtnUHDCDOcfiCDmCflUHmcnUHPCRUHmcnmcnUHmcnpfi>

50
*A new, larger extractor was made and used for J
samples 4 and 5 but discarded for sample 6 as the results
were not satisfactory.

 

 

 

All ether samples were evaporated to dryness over

steam. A 100 m1. portion of petroleum ether II and a like
amount of petroleum ether III were evaporated to dryness
over steam. The residues from A, B, C, petroleum ether II,
and petroleum ether III were taken up in 25 ml. of absolute
alcohol. Those from D and B were taken up in 15 ml. of ab-
solute alcohol, and that from F in 20 ml. The absorption
curves were determined using absolute alcohol in the solvent
cell. Graph he. 26 showed that progesterone was partially
recovered from hemolyzed blood when it had been added to the
whole blood and that it was not recovered from the blood of
a cow following intravenous injection.

To get an estimate of the amount of progesterone re—
covered from the blood to which it had been added, a concen-
tration curve for progester ne in absolute alcohol was con-
structed. Five mg. of progesterone was dissolved in 100 m1.
of absolute alcohol (= 0.05 mg./m1.). This was diluted with
alcohol to make 0.02 mg./ml., 0.01 mg./m1., 0.005 mg./ml.,
0.0025 mg./ml., and 0.001 mg./m1. The absorption curves
were determined on the same day using absolute alcohol as a
blank. These curves were used in Graphs No. 27 and he. 28,
which showed that progesterone in alcohol follows Beer's law
in the concentration range used.

Absorption curves on the same alcoholic samples were

made again 6 days later and showed the variations noted in

Graph No. 5.

RESUQUS AND DISCUSSION

 

PRELIMIIARL ABSORPTION CURVES

—_

 

Absolute Alcohol. Graphs l, 2, 5, and 4 indicate
that alcoholic solutions of progesterone shOW'spcctrophotom-
etric curves which are highly variable, both as to wave
length of the maximum absorption and also as to the amount
of absorption. It was noted that the results obtained on
the day the solutions were prepared, were quite consistent,
but after that there was no correlation between the curves
and the time elapsed. It was apparent that the compound
changed on standing, or that temperature or some other fac-
tor had objectionable effects. A.combination of these ef-
fects is possible, and in spite of explanations, for the
purpose of this problem, such variability was not to be de-
sired. The variations in the slit-widths at the maximum are
explained by the necessity of adjusting the hydrogen lamp in
order to get maximum intensity from it. This change alone
would affect the curves but other factors were obviously op-
erating, as a change in slit-width did not always produce
the same effect upon the curve. (See Graph he. 5.)

Graph Ho. 5 assembles pertinent data from the preced-
ing graphs and illustrates the lack of definite correlation

between the extinction, the wave length of maximum,absorp-

 

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52
tion, the time since making the solution, and the slit-
width. However, there seems to be a general tendency for:
the extinction (E) to increase as the slit-width increases;
the maximum to be lowered when the slit-width increases; the
maximum to be lowered as the extinction is increased. None
of these holds true in all cases.

Distilled Water. Graph 50. 6 shows less variation
between distilled water curves than between the alcoholic
curves, and these variations may be explained in part by a
factor which did not influence the alcohol curves. Progest-
erone is much less soluble in water than in alcohol and the
upper curves in Graph 6 are from saturated solutions. The
lower curves represent a half-saturated solution and might
therefore be more constant, as they appear to be from the
curves.

Graph 7 illustrates that preliminary dissolving of
the progesterone in alcohol facilitates its solution in wa-
ter. The greater E values indicate more progesterone in so-
lution. There is a good correlation between the height of
the curves and the concentration, with the exception of the
curve for the 0.04 mg./ml. solution run on the second day.
If all are run the same length of time after they are made
up, satisfactory curves are obtained. This is shown in
Graph Ho. 8 where E values are plotted against concentra-
tion. These determinations were made within 24 hours after

the solutions were made up, and the results show a nearly

 

 

 

 

 

 

 

 

 

 

 

 

 

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straight line. This was taken to mean that if progesterone
could be obtained in an aqueous solution, an estimate of the

concentration could be made.

DILU‘l‘liD BLOOD pm... .3

 

The protein present in blood plasma shows a maximum
absorption at 280 millimicrons. Considerable dilution
(1:400) is necessary to bring the extinction down to a suit-
able level. Ko difference was found in comparing the curves

from different animals (Graph No. 9). A difference could
hardly be expected in the pr sence of such dilution.

Progesterone added to this highly diluted plasma
caused an alteration in the curve as shown in Graph No. 10.
If the absorptions at wave length 248 millimicrons (the maxi-
mum.for progesterone in water) are compared, the following
is revealed. The extinction (E) of the progesterone-contain-
ing sample is 0.6200. From the concentration curve (Graph
No. 8) this would represent 0.0127 mg./ml. if it were not for
the fact that the diluted plasma alone shows some absorption
at this wave length. r“his latter shows an extinction of
0.1000 which would subtract 0.0016 mg./ml. The difference
is 0.0111 mg./ml. or 0.00111fi progesterone. The actual
amount added was in a percentage of 0.001075. While the
above method of calculating may be open to criticism, it ap-
pears that the analysis of the mixture gave quantitative re-

sults for the progesterone added. (See Graph Ho. 12 for re-

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sults of running the solutions against a plasma blank.)
An extension of the work seen in Graph No. 10 is
shown in Graph No. ll. Interesting data from this graph are

summarized below:

 

 

 

 

Per cent
Per cent Observed E progesterone Calculated
progesterone at 248 my. *renresented per cent
in sample by23 value progesterone
None 0.1045 0.00014
0.0005575 0.3778 0.00074 0.000600
0.001075 0.5751 0.001175 0.001055
0.00215 1.0810 0.002200 0.002060

 

 

 

 

The per cent progesterone represented by the E value is ob-
tained from.the concentration curve (Graph No. 8). The cal-
culated per cent progesterone is determined by subtracting
the percent progesterone represented by the extinction of
the sample containing no hormone (0.00014) from.those for
the various concentrations. Comparing the first column in
the table with the last, the weakest solution is the only
one noticeably out of line. This may be explained by noting
that, in Graph No. 8, E values below 0.2 and above 0.7 give
points which fall to one side of the straight line. In
other words, very low or very high absorptions result in
readings which are inconsistent with those in the optimum
°ange. These deviations indicate that progesterone, like
many other substances, does not adhere to beer's law at all
concentrations.

Graph No. 18, which compares these plasma solutions

with diluted plasma in the blank cell, shows maxima at 248

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42

millimicrons as expected, and no other peaks are encoun-
tered. However, the calculation of the amount dfpmogester—
one present, using the water concentration curve, gives re-

sults which are too low (55-36% below the actual values).

 

 

 

 

Per cent progest- Per cent -
Per cent Observed E erone represented deviation from
progesterone at 248 mr, by E value (from. progesterone
1n sample concentration curve) in sample
0.0005575 0.1959 0.00054 -56
0.001075 0.5595 0.00070 ’ ~55

 

 

 

 

This indicates that a new concentration curve, using
0.25% plasma as a blank and dissolving the progesterone in
the same, would be necessary if this type of study were to

be extended.

CHROHAEOGEAEBIC STUDBES

 

It is shown clearly in Graph he. 15 that progesterone
is not adsorbed from aqueous solution by passing it through
alumina. All is accounted for by estimating the amount
which came through and comparing this with the amount in the

solution at the start (1.0 mg.).

 

 

 

 

Progesterone cal- 1 Calculated
E at culated from con- Volume Of progesterone
Sample 248 my. centration curve, sample, in sample,
a mg ./ml. m1 ' ma.
III 0.5545 0.00685 100 0.6850
IV 0.5448 0.00667 50 0.5555

Total 1.0185

 

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Graph Ho. 14 shows that petroleum e ohor re 1.oves pro3ecste1 one
from aqueous solutions. The stock solution (0. 0l m,./ml.)
was checked and showed a concentra ' on, according to Graph
No. 8, of 0.0095 n3./ml. The ether extract, after drying
and dissolving in water (Curve II) contained 0.0065 m3./ml.
or 68% of the original amount. The water layer (Curve I)
accounted for 0. 0005 m3./ml. or 5% of the original. The
loss is quite large, but this work suggested that more con-
plete ex traction mi3ht us more satisfactory. It will also
be not ed that the distilled ether gives a characterrstic
type of curve. This will be considered nore fully when com-

.iffe rent lots of petroleum ether (see Graphs 22

’31

paring the
and 86).
Graph No. 15 likewise shows incoriplete e: {traction of
the prog3esterone, probably from too short an extraction
thne. The dissolved residue from the proresto one solution
(Curve IV) has a calculated pr03esterone content of
0.00775 ng./ml. or esp of the original amount. The water
layer (Curve II) still contained 0.00125 m3./ml. or 15%.
This accounts for 95% of the pro3esterone used. Curve I
also indicates that water dissolves enough petroleum ether
to alter its absorption slightly.
Recheciinr the above gave the da a for Graph No. 16.

The stock solution was checked and was found to contain a

 

  

 

   

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

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calculated progesterone level of 0.00955 n ./ml. (from con-
centration curve, Graph 8). The ether extraction residue
(Curve II) when run a3s in.t distilled water was calculated
to contain 0.0089 13./ml. or 95p of the original quantity.
When compared with the control solution (alcohol and water
from a clean.Ilas1:) Curve V), 0.00785 mg./ml. were found.
This represents 82% of the total. The water layer after ex-
traction (Curve I), the second Lattcmpt to the up the resi-
due (Curve III), and the control consisting of 2 ml. of al-
cohol and 98 ml. of water rinsed in a clean flask (Curve IV),
show no absorption maxima at the 248 millimicron level.

Thus it is assumed that the extraction we 3 quite complete

.1.

and that the residue was quite co: 1pletcly taken up. however,

"1

it appears tha' 2p ethyl alcohol in water diifers sufficient-

ly from pure water to warrant setting up a concentration

curve using this solution as a blank, although for compara-

c)

tive purpo es raph No. 8 will be used in this thesis.
... -- 9 ..

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'rom.Plas 11a after Precipitation 01 the lroteins. In
Graph No. 17 and Graph ho. 18 is shovm.that progesterone was
not detected by the spectrophotometer in the presence of
blood plasma in which the proteins had been precipitated by
trichloracetic acid. This may have been due to adsorption
of the hormone on the surface of the protein particles. The
great extinction in Curves I, II, and III of Graph 18 is
presumably due to absorption of li3ht by the trichloracet ic

acid.

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51

From Plasma without Precipitation f the Proteins.

 

Graph he. 19 shows that progesterone may be removed from
plasma by petroleum ether, and it also establishes the max-
imum.for progesterone in petroleum ether at a wave length of
230 millimicrons. The maximum in alcohol is at 240 millimi-
crons, and in water it is at 248 millimicrons. From these
data it appears most efficient to work with the hormone in
aqueous solution as the maximum is not so near the lower
limit of the Spectrophotometer.

Normal Cows and Calves. These curves are petroleum

 

ether curves, and the maximum for progesterone should there-
fore be at 230 millimicrons.

Graph No. 20 shows that there is no significant rela-
tion between the state of the reproductive cycle and the
character of the absorption spectra. While many of the
curves show a tendency for a peak at or near.250 millimi—
crons, this is a general characteristic of many of the ether
extracts made in this study and should likely be discounted.

With a Continuous Ligpid-Liquid Extractor. The first

 

attempt to use this extractor gave results which are plotted
in Graph No. 21, in which the absorption of the extraction
product was determined, using petroleum ether as a blank.
The definite peak at 250 millimicrons is taken to represent
the progesterone which was extracted.

The data plotted in Graph he. 22 illustrate an inter-

esting point in regard to the use of similar but not identi—

 

 

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56

cal samples of petroleum ether. fetroleum ether II required
unusual adjustments of the slit~width as seen in Curve III.
All other solvents, including distilled water, absolute al-
cohol, and other lots of petroleum.ether, required progres-
sively decreasing slit-widths as the wave length increased.
The effect of the erratic slit-widths when using petroleum
ether II is observed in both Curves I and II of Graph 22.
The extinction (E) tends to increase when the slit-width
must be increased. However, the great absorption of light
at 230 millimicrons in Curve I indicates that extraction of
the progesterone was successful.

Comparison of the labels on the petroleum ether
bottles indicates a difference not only in the boiling point
range but in the nonvolatile matter (see page 19). Petrole-
um ether II contains twice as much non-volatile matter as
either petroleum ether I or III. This probably accounts for
the differences in the spectra of these solvents.

Graph No. 23 illustrates a further effect of this
nonvolatile material in the ether. On evaporation and re-
dissolving in alcohol and water, a cloudy solution resulted.
This was expected to increase the extinction of light and ac-
counts for the higher E value (Curve II) when compared with
the stock solution (Curve I). Running the extract against
the control (III) resulted in bringing the extinction (Curve
IV) closer to that of the stock solution (I). This was due

to the fact that the control (extract of distilled water

 

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alone) was also cloudy.

Clearer solutions resulted when the alcoholic solu-
tion was added tg water. Graph No. 24 shows that a small
amount of the extracted progesterone is found in the ether
left in the extractor (Curve II). The ether in the flask
was calculated (from Graph 8) to contain 0.00 25 mg./ml. or
72.5% of the total. The extractor ether was calculated to
represent 0.0002 mg./ml. or 2%. It appears from these data
that the extraction was not complete, but it must be borne
in mind that the calculations were made on the basis of a
concentration curve set up using distilled water as a blank.
At any rate it appears that considerable extraction of pro-
gesterone from aqueous solutions by the use of petroleum
ether is possible. It is also likely that extraction for a
longer time would tend to make the recovery more complete
and at the same time to cause the progesterone in the ex-
tractor ether to pass over into the flask.

From the Blood Plasma.g§_§_Cow Injected with Progest-
erone. In this study, the influence of cloudiness is seen
again. There appear to be no significant differences in the
curves (Graph No. 25) at the 248 millimicron level where
progesterone would be expected to ShOW'up. Variations in
the curves are most likely due to the cloudiness (due to pe-
troleum ether II) as the most cloudy one (A) shows the
greatest extinction and the least cloudy sample (B) shows

the least extinction. It appears that the progesterone in-

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61
jected is not recoverable by this technique.

From.Hemolyzed Blood 9:,a Cow Injected with Progest-

 

 

erone. This graph (No. 26) illustrates many related points.
Curve A shows that the technique will recover progesterone
from hemolyzed blood when the hormone is added as the blood
is drawn. The concentration (see Graphs 27 and 28 for a con-
centration curve for progesterone in absolute alcohol) cal-
culated from the extinction (1.409) at the maximum, was
0.0278 mg./ml. As there were 25 m1. of solution, this rep-
resents a total of 0.695 mg. This is only 54.75% of the orig-
2 mg. This may be accounted for on the basis that all of the
crystals may not have dissolved. Earlier studies demonstra-
ted the difficulty of dissolving the hormone in aqueous solu-
tions (see Graph No. 6).

In addition, Curves B and C show the difference be-
tween petroleum ether II and petroleum ether III. The former
was used in preparing Curve B only and shows an absorption
characteristic of an ether sample with a high nonvolatile
matter content (see Curve II). In contrast, Curve C, repre-
senting an extract of blood prepared as in B except that pe-
troleum ether III was used, reveals an entirely different
curve which compares favorably with the pure solvent, dried
and redissolved in alcohol (III).

Curves D and E show that the use of larger quantities
of solvent (200 ml. in place of 100 ml.L causes an accumula-

tion of nonvolatile material. This results in final curves

 

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65
similar to those from petroleum ether II. It is therefore
objectionable to use the larger amounts.

Curve F is difficult to GXplain. It was expected to
approximate Curve C, indicating tne absence of progesterone
or to be similar to Curve A with a maximum at 240 millimi-
crons, meaning that the injected hormone had been success—
fully extracted. While the result did not coincide with the
first possibility, certainly there is no evidence to suggest
that the blood as drawn contained any of the injected pro-
gesterone. It is concluded that the technique used was in-
sufficiently effective or that progesterone is quickly re-

ibilities

(I)

moved from the circulation in some fashion. Pos
which ar« suggested to explain the latter are: the hormone
might be metabolized, for example, by the uterus to form
pregnandiol (the latter may be conjugated with glucuronic
acid in the liver); or it could simply be selectively ab-
sorbed by some tissue and thus removed from the circulation.
hany encouraging facts were learned from this study,
but it is not possible to say at this time that the hormone
progesterone can be demonstrated in the circulating blood by
spectrophotometric means. It seems likely, however, that
when a suitable extraction procedure is worked out, absorp-

tion spectra curves will be useful in measuring the progest-

erone quantitatively.

iflflEQMKY

l. Alcoholic solutions of progesterone resulted in
speetrophotometric curves which varied considerably from day
to day in regard to the wave length at which maximum absorp-
tion occurred and also in regard to the degree of absorption.
These variations may be due to changes in the solutions,
changes in the functioning of he apparatus, or both.

2. Fresh alcoholic solutions showed maximum absorp-

tion at a wave length of 259 to 240 millimicrons. This is -n
agreement with previously reported curves (l6).

5. Aqueous solutions of progesterone were found to
be more suitable for spectrophotometric measurements than
alcoholic solutions.

4. The preparation of an aqueous solution was great-
ly facilitated by first dissolving the crystals in a small
quantity of absolute alcohol.

5. The maximum absorption in aqueous solution was
found to be at a wave length of 248 millimicrons.

6. Concentration curves for aqueous solutions of
progesterone showed that this substance follows Beer's law
at least between 0.004 mg./ml. and 0.018 mg./ml., that is,
that absorption is closely proportional to the number of

molecules in the light path. On either end of the above

67
range, he deviation may be caused by limitations of the
spectrophotometer rather than by failure of the hormone so-
lutions to follow Beer's law.

7. Ho significant difference could be detected be-
tween the curves obtained from the diluted blood plasma of
different cows and calves. This is probably due largely to
the high dilutions of plasma used for the determinations.

8. Cristalline progesterone added to diluted plasma
showed characteristic absorption at 248 millimicrons. The
amount calculated to be present, using concentration curves,
coincided nicely with the amount added.

9. Progesterone was not adsorbed from aqueous solu-
tion by passage through alumina.

10. Progesterone was extracted from aqueous solu-
tions, from blood plasma solutions, and from hemoljzed blood
solutions, by the use of petroleum ether. The amount recov-
ered ranged from 68 to 93%.

11. The absorption maximum of progesterone in petro-
leum ether was found to be 230 millimicrons.

l2. Progesterone was not detected in the presence of
blood plasma in which the proteins had been precipitated by
trichloracetic acid.

13. The absorption curves of petroleum ether extracts
of the plasma of normal cows and calves did not show signif-
icant differences at the absorption maximum for progester-

one.

68

14. Different lots of petroleum ether from the same
sourde showed large variations in their absorption curves.
This was apparently associated with the nonvolatile matter
as the satisfactory samples contained 0.0002fi while the un-
satisfactory lot contained 0.0004%. This nonvolatile resi-
due tended, in addition, to form an emulsion with water.
This latter was minimized by taking the residue up in a
small amount of absolute alcohol and adding this to water.
Adding the water to the alcoholic solution of the residue
resulted in an emulsion.

l5. Progesterone injected into cows could not be de-
tected spectrophotometrically in the petroleum ether ex-
tracts of the blood plasma or of the whole blood following

hemolysis.

REF ERIE-"f C ES

 

8.

9.
10.
ll.

12.

l5.

14.

70

 

Corner, G. W., and -. Ii. Allen, "PhysiOIO{:y of the Cor-
pus Luteum II. Production of a Special Uterine Re-
action (Progestational Proliferation) by Extracts of
the Corpus Luteum.,' Am. J. ths 101., LXYTVIII (2)
(1929), pages 526- 559.

Cleuberg, C., H. W.“1hiel and R. Ziecker, Arch. 3.
Gvnd£., 152 (1955), page 61.

Hazer, C., and L. Goldstein, Clinical Endocrinology g:
the Female H. B. Saunders 00., Philadelphia, 195

 

Eels, E., Arch. f, Gypdk., 158 (1954), ma :e 564.

Ehrhardt, K., Mfinchner med. Wchnschr., 81 (1954), page
869.

 

Adler, A. A“, P. de Fremery, and.h. Tausk, Nature, 155
(1954), page 295.

Lankeren, Cornelius van, Arch. 3, Gvndk., 160 (1955),
page 150.

Ehrhardt, K., and A“ Hagena, Endokrinologie, 16 (1955),
page 51.

Phillip, 3., Zentralbl. :, Gynflx., 54 (1950), page 2754.

 

 

Fels, E., Zentralbl. i. ngflk., 55 (1951), page 514.

 

de Fremery, P., A.. Luchs, and’i Tausk, Arch. 3. Q. ggg.
thsiol., 251 (1952), page 541.

 

Ehrhardt, K., and W. weigel, Endokrinologie, 15 (1955),
page 225.

Portman, K., "Studies and Investigations into the Corpus
Luteum.hormone," Acta. path. et microbiol. Scandinav.
Suppl. 25 (1955), pages 1— 124.

 

Loewe, S. and H. E. Voss, Schweiz. med. Nehnschr., 64
(1954), page 1049.

71

15. Bloch, P. W., "Progestin Content of Blood," Endocri-
nology, 20 (1956), pages 507-510.

16. Slotta, K. H., H. Ruschig and E. Fels, "Reindarstellung
der Hormone aus dem Corpus luteum," Berichte der
Deutschen Chemischen Gesellschaft, 67B (1954),
pages 1270-1275.

 

      
    
   

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