an improved procedure for the extraction of the … · an improved procedure for the extraction of...

AN IMPROVED PROCEDURE FOR THE EXTRACTION OF THE OVARIAN HORMONE AND SOME CHEMICAL

PROPERTIES OF THE PRODUCT.*

J. 0. RALLS, C. N. JORDAN, AND EDWARD A. DOISY.

(From the Laboratories of Biological Chemistry, St. Louis University School of Medicine, St. Louis.)

(Received for publication, June 1, 1926.)

Since our first publication in this field, a number of investigators have recently turned their attention to the ovarian hormone. No attempt will be made to discuss all of the recent literature, but attention should be called to a few of the papers in which the extraction of the hormone is considered. Zondek and his co- workers (1925), E. Laqueur and his collaborators (1926), Glimm and Wadehn (1925), Dickens, Dodds, and Wright (1925), and Loewe (1925) have published their experiences with the preparation of the hormone. Some of these investigators have used placenta, while others have used ovaries as the source material; but all (except Glimm and Wadehn who used immature rabbits) have obtained extracts which produce artificial estrus in ovariec- tomized rats or mice.

Zondek and Brahm (1925), using either placenta or liquor folliculi, obtained the hormone in a water-soluble condition by a procedure which is not fully described. Laqueur, Hart, deJongh, and Wijsenbeek (1926) remove the proteins of the liquor folliculi with colloidal iron and concentrate the resulting aqueous solution in vacua. The subsequent treatment is not given but the hormone is finally obtained in a highly purified water-soluble form which contains neither phosphorus, amino nitrogen, hydroxyl, nor pro- tein, but is dialyzable through collodion and parchment and gives less color than that produced by one-twentieth of its weight of cholesterol by the Liebermann-Burchard or Salkowski reactions.

* This investigation has been made with the assistance of a grant from the Committee on Therapeutic Research, Council on Pharmacy and Chem- istry, American Medical Association. The authors take this opportunity to express their appreciation of this assistance.

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358 Extraction of Ovarian Hormone

Although Laqueur’s investigations appear to be published in preliminary form, they are very important in the study of the ovarian hormone. Though we have had preparations (0.01 to 0.02 mg. per rat unit) which seem to approach more closely the pure hormone than his, we have not found that the hormone forms a true aqueous solution; of course crude preparations may be held in colloidal solution but Laqueur’s product is too well purified to fall in this category. The nature of the contaminating substances could conceivably account for this difference in solubility of the

TABLE I.

Yield and Purity of Hormone from Hog Liquor Folliculi by Improved Methods of Preparation.

2 cc.

5000 800

5 2-6 2200 1390

6a 14% 375 860

6b 14; 375 615

7a 0 62 835 7b 0 62 610 8 15 650 830

10 5 892 950 12 16 1050 1060 16 12 1279 1000 R 1 440 750 18 0 550 835

Time of xeserva-

tion.

mos.

V0hIlle 3f liquor folliculi xtracted.

-

I I

.-

Yield. iat unitl per liter.

ml.

1.39

0.030

0.125)

I 0.124

0.11 0.031 0.03

0.055 0.022

-

_

Remarks.

Not purified, prepared by old method.

Partial petroleum ether extraction and digitonin treatment to remove cholesterol.

Purification was not com- pleted; conducted to test yield by two different pro- cedures.

Modified procedure.

products obtained by Laqueur and by ourselves. The claims of Zondek and Brahm and of Glimm and Wadehn of a water-soluble ovarian hormone need not be considered further until more data are published by them.

Both Laqueur and Dodds and Dickens failed to obtain yields as large as we reported in our first paper. We are unable to offer any explanation; when the procedure is followed exactly as published,


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J. 0. Ralls, C. N. Jordan, and E. A. Doisy 359

we have invariably obtained from 600 to 1400 units per liter of liquor folliculi in our later work. In Table I we present figures on the yield by the new modifications of our technique.

Improved Method of Preparation of the Hormone from Hog Liquor Folliculi.

In a preliminary report before the 1925 meeting of the American Society of Biological Chemists, we (Ralls, Jordan, Heusinkveld, and Doisy, 1926) outlined an improved method of extracting the ovarian hormone from liquor folliculi which was based upon the principles used in the method described in the present paper. As our subsequent investigations, tested step by step, have shown it to be possible, we have gradually broken away from the procedure which we described in 1924 (Doisy, Ralls, Allen, and Johns- ton) and have introduced many simplifications and improvements in the preparation.

The important points in the preparation of the ovarian hormone are : (1) the separation from proteins, and (2) the separation from lipids, including cholesterol. The first separation is effected by precipitating the proteins of fresh liquor folliculi with two volumes of 95 per cent alcohol and by subsequent solution of the crude and partially purified hormone in acetone and ether which do not dissolve proteins.

The removal of the lipids is much more complicated and difficult. As we pointed out in our earlier paper, the phospholipids may be precipitated from an alcoholic solution by acetone, and the triglycerides may be almost completely frozen out of 60 or 70 per cent alcohol with very little loss of the hormone in the precipitates. However, the final product produced by this procedure contains soap which probably is due to the fact that the liquor folliculi contains about as much bicarbonate as blood plasma and, during distillation of the filtrate from the proteins, this bicarbonate loses COZ to form carbonate which in the warm alcoholic solution hydro- lyzes some of the lipid material. This then adds the removal of the soaps to our problem. As illustrative of the additional com- plications produced by the soap, we may cite our figures in Table II on the distribution ratio of cholesterol between petroleum ether and 70 per cent alcohol in the presence and absence of soap, which again brings out the old point that certain solutes may affect the solubility of other solutes in a common solvent.


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Since the publication of our earlier observations upon the preparation of the ovarian hormone, a great deal of time has been spent in a detailed study of the various steps of purification. It soon became apparent that a considerable part of the material precipitable by acetone from alcoholic solution which we formerly called phospholipid is soap. Our attention being directed to the soap, consideration of the distribution ratios showed it to be desirable to remove the soap in one of the earlier steps of purification. We have partially accomplished this by extraction of the residue from the distillation of the alcoholic solution with 85 to 90 per cent acetone in which both soap and phospholipid are poorly soluble.

TABLE II.

Distribution of Cholesterol between YO Per Cent Alcohol and Petroleum Ether.

70 per cant alcohol. Petroleum ether.

I Cholesterol: Cholesterol:

“bufme Total sample. solids. s0aps. YY .zg, Soaps. In

,2k In 1 cc. sample. sam- In

ple. pie. 1 CD.

__~~__ ~-~-- cc. ml. ml. mg. mg, cc. mg. mg. mg. ml.

5.0 53.6 48.0 5.61.12 2.93 29.7 00.0 29,710.14 5.0 53.1 47.4 5.71.14 2.93 31.3 00.0 31.310.44 5.0 94.4 88.7 5.71.14 3.00 32.9 00.0 32.910.96

25.03 11.3 00.0 11.30.45 24.89 324.8 00.0324.813.05 25.03 11.5 00.0 11.50.46 24.89 325.0 00.0325.013.06 24.89 10.9 00.0 10.90.43+ 24.89 326.1 00.0326.113.10

Ratio cholesterol in petroleum

ether: cholesterol in alcohol.

9.1 9.2 9.3

Average.. . . . . . . . . . . 0.44+* I I

Average . . . . . . . . . . . . 13.07* <29.7* >29.0* I I

*Value for last three figures only.

The acetone was distilled off and the remaining soaps were completely removed by distribution between water and ether which, if properly executed, caused practically no loss of hormone in the aqueous soap medium. When it was found that this procedure entailed very little loss of the hormone, the process was shortened still more by leaving out the acetone precipitation and going di- rectly to the extraction of the hormone from the aqueous suspen- sion with ether.

This ethereal extract contained a large proportion of cholesterol whose separation from the hormone has caused considerable


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J. 0. Ralls, C. IT. Jordan, and E. A. Doisy 361

trouble. To be sure, this cholesterol can be almost completely removed by digitonin but the excess digitonin added in the precipitation is another source of annoyance. The digitonin can be a contaminating substance just as much as cholesterol; and because of the solubilities of the hormone and digitonin, we found this a troublesome mixture to handle. Our earlier experiences with cholesterol, combined with our later studies of the solubilities of the hormone, showed clearly the way to separate the two. Cho- lesterol is roughly twenty-nine times as soluble in petroleum ether as in 70 per cent alcohol and therefore can be removed rather well by repeated extraction of the alcohol with petroleum ether, while the solubilities of the hormone are such that this produces very little loss of potency of the alcoholic solution. Calculation showed that six extractions of a solution of the hormone and cholesterol from 1 liter of liquor folliculi in 100 cc. of 70 per cent alcohol with 25 cc. portions of petroleum ether would be sufficient to remove all but about 0.001 mg. of the cholesterol. Slaving no exact quantitative data on the distribution of the hormone, we tested our deductions experimentally. The cholesterol was removed as anticipated and, to our satisfaction, less than 5 per cent of the hormone was lost to the combined petroleum ether extracts (Table Ivb).

Extraction and PuriJication of the Hormone.

Concisely stated, the preparation is carried out upon liquor folliculi from which the proteins have been precipitated by the addi- tion of two volumes of 95 per cent alcohol. (For stability of hormone under these conditions, see Table I.) The proteins are filtered off and extracted with alcohol for 6 to 8 hours in a Soxhlet apparatus and the combined extract and filtrate distilled to dryness in vacua. In this step the foaming which may be troublesome can be avoided by allowing the vacuum to draw the alcoholic liquid into the practically empty distilling flask at the same rate as the liquid is removed by volatilization. At the conclusion of the distillation, some water or alcohol should be added and the liquid gently heated for a short time to complete the hydrolysis of the lipids. (Subsequent directions are based upon the assumption that 1 liter of liquor folliculi is being used in the preparation.) The residue is dissolved in 150 cc. of water, centrifuged to remove some insoluble material, and transferred to separatory funnels.


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Extraction of Ovarian Hormone

Since we prefer to use centrifugation to accelerate the breaking of the emulsions which may form, and the capacity of our centri- fuge is limited, we divide the aqueous solution between two separatory funnels. The aqueous solution is made alkaline with NaOH so that a few drops added to an equal volume of alcohol will redden phenolphthalein; 30 cc. of ether are added to each funnel, the mixture shaken well, centrifuged, and the aqueous solution run into another funnel. The extraction with ether is repeated three or four times (see Table III), the combined extracts washed with water and the ether distilled off. The residue is dissolved in petroleum ether and alcohol alternately, keeping record of the volumes used, and transferred to a separatory funnel. Enough alcohol and water are added to give a volume of 100 cc. of 70 per cent alcohol and the quantity of petroleum ether is made up to 25 cc. The alcoholic solution is extracted six times with 25 cc. portions of petroleum ether and then distilled to dryness in ~~cuo. The residue is leached with redistilled dry ether and the ethereal extract centrifuged to remove any insoluble solids. This ethereal solution which seems to retain its potency remarkably well is our stock solution with which we expect to start in our futuxe work on purification of the hormone. As a general rule, preparations made in the described way or by processes embodying these principles yield a product of which 0.03 to 0.04 mg. (see Table I) is 1 rat unit. By additional fractionations the potency may be doubled, the weight of the rat unit decreasing to 0.01 to 0.02 mg.

Discussion of Method of Preparation.

The most important points to be gleaned from Table I are: (1) the stability of the hormone of liquor folliculi preserved with two volumes of 95 per cent alcohol; (2) the yield expressed in rat units of the hormone from liquor folliculi of hog ovaries; (3) the weight of a rat unit by our improved method of preparation.

Preparation 12 which had been preserved with alcohol for the longest period of time, i.e. 16 months, gave 1060 rat units per liter of liquor folliculi, which is only slightly less than the maximum reported in this paper of 1390 for No. 5. The smallest number of rat units, 610 in Ko. 7b, was obtained from freshly collected liquor. Our source of supply yields from 30 to 90 cc. of liquor folliculi per day and as the volumes listed in the table generally run into the hundreds, it is evident that each sample is a composite of many


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days collection. This would tend to diminish any gross variations in the individual preparations and, therefore, permits us to conclude that the means of preservation used seems to be efficient.

As we recounted earlier in this paper, Laqueur, Hart, deJongh, and Wijsenbeek (1926) have obtained a water-soluble product which far exceeds in potency the preparation described in our earlier paper. Though we are not certain that we understand their statements correctly, it seems that they have prepared an extract containing 0.01 mg. of solids or even less per mouse unit; or, since the mouse unit is equal to about one-sixth of a rat unit, a rat unit of their product would weigh 0.06 mg. or less. From a reading of their paper, it seems probable that they may have been handi- capped by either lack of material or lack of a balance suitable for weighing the minute quantities at their disposal and, therefore, did not determine the weight of the mouse unit of their product with certainty. From our Table I, it is clear that they may have had a product whose potency was greater than they felt justified in claiming. In three preparations, Nos. 5, 10, and 12, made by our new simplified method, we obtained a product of which approximately 0.03 mg. equalled 1 rat unit; in one, No. 18, made by a slightly modified technique, the unit was 0.022 mg. In our subsequent fractionations of these products we have reduced the unit in one case to less than 0.01 mg.

Table II requires little additional comment but the importance of its data may be emphasized by the following considerations.

Whereas formerly we had been effecting the removal of cholesterol by the use of digitonin for its precipitation, we now use the favorable differences in the solubilities of cholesterol and the hormone in 70 per cent alcohol and petroleum ether. As has been stated before, the hormone is more soluble in 70 per cent alcohol than in petroleum ether, while the cholesterol is twenty-nine times as soluble in the latter solvent as in the former. Knowing the distribution ratio of the substance to be removed and the total amount present, and being able to fix the volumes of the two solvents, the number of extractions to effect a certain degree of purification can be calculated from the formula developed here.

I f A = total quantity of substance present, 5 = quantity of that substance remaining in one solvent after one

extraction,


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A-x= quantity present in the other solvent after one extraction, Va = volume of the one solvent used, VP = volume of the other solvent used, Ca = concentration of the substance in the one solvent, Cp = concentration in the other solvent,

then

Va - = b, the ratio of the volume of the one solvent to the volume VP

of the other, and

x - = Ca;

A-s

Vu - = cp.

VP

But

CP - = K, or the distribution ratio of the substance in question Ca

between the solvents concerned, or Cp = KCa;

A -x Kx hence - = --. Vu

And sincyg = b, or Vu = bVp, we have A%x = g;

b A-x=K$;Ab-xb=Kx; Kxfxb=Ab;x=A-. K-i-b

I f now we wished to calculate the value of 22 or the amount of cholesterol, or other substance whose distribution ratio is K, left in the one solvent after two extractions with a volume Vp of the other, we have:

b - where the previous x is 6he A or the total amount

xz=xK+b present at the start of the second extraction. But

b x=A-,

K+b

hence

@I b x~=A(K+~)~ or m=A


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In the same way

2 b -------=A K+b

and in like manner

x,, = A

Having this formula, and by taking the logarithm of both sides of the equation, the number of extractions necessary to effect the removal of all but an x amount of substance can be calculated, thus

b = log A + n log -

K+b

b Log zn - log A = n log -J

K+b

log Cr* - log A log x, - log A n=

b log -

Or log b - log (K + 6)

K+b

To illustrate the use of this equation, take a case where

b = 4, K = 29, A = 200 mg., $1 = 0.1 mg., and x2 = 0.01 mg., then

log 0.1 - log 200 - 3.3010 n* = log log 33

= ~ = 3.6 - 0.9160

and log 0.01 - log 200 - 4.3010

n2 = = ~ = 4.7. log 4 - log 33 - 0.9160

It seems clear, then, that four extractions under the conditions outlined above will remove all but a little less than 0.1 mg., and five extractions will remove all but a little less than 0.01 mg. of the material distributed between the solvents. If it is desired to approach the question of removal of cholesterol in a simpler way, the cholesterol left in the 70 per cent alcohol after

b each extraction is -

Ktb X 100 per cent of the cholesterol present in that


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medium just before extraction. Here where K = 29, and b = 4, this is g

or 12.1 per cent and the second extraction will remove 87.9 per cent of the 12.1 per cent remaining after the first extraction, and so on with each successive extraction.

At this point the effect of the presence of soap can be well brought out. When soap is present to the extent of 10 to 18 mg. per cc. in the 70 per cent alcohol, the distribution ratio of cholesterol between the alcohol and petroleum ether is 9.2 instead of 29. Hence the per cent of the original amount of cholesterol left in 70 per cent alcohol after one extraction with petroleum

400 ether is now - or 30.3. From the formula for the number of extractions

13.2 necessary to effect a certain degree of purification, it may be shown that approximately twice as many extractions must be made in the presence of soap as in the absence of soap in order to effect the same removal of cholesterol. Thus in the example cited, seven and nine extractions would be necessary to remove all but 0.1 and0.01 mg. of cholesterol in the presence of soap.

We were curious to learn the value of the distribution ratio for the cholesterol in 70 per cent alcohol and in petroleum ether under the conditions prevailing in some of our preparations without sacrificing some of the alcoholic solution containing the valuable hormone. By the use of the formula developed below one can determine this ratio if determinations of cholesterol in successive petroleum ether extracts are made and a record of the volumes of alcohol and petroleum ether in these manipulations is known.

Thus if x=

CPl =

cp? =

then

and

amount of cholesterol in volumes Vu2 of 70 per cent alcohol after one extraction with volume Vp, of petroleum ether is made, concentration of cholesterol in the first petroleum extract of VUI cc. of 70 per cent alcohol, concentration of cholesterol in Vpz cc. of petroleum ether of second extract when Vu2 cc. of 70 per cent alcohol was extracted,

X -= Val

concentration of cholesterol in 70 per cent alcohol after

one extraction,

Cpl=KorC2?e3=K X 2

from which Cpl Val x=--’

K


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But since

CP2 VP2 = amount of cholesterol removed from 2 quantity of cholesterol in 70 per cent alcohol by second extraction

x - cp2 VP2 = Va2

concentrat,ion of cholesterol in 70 per cent alcohol after

second extraction.

Then

CP2 = K or Cp2 Va2

=K 5 - CP2 VP2 x - cp2 VP2

Va2

and since, from above,

Cpl Bar xc- K

we have Cp2 Va2

= K; CPZ Vaz K

Cpl Val = K;

___ - cp2 VP2 Cpl Val - Cp2 Vp2 K

K

Cp2 Va2 Cpl Val - Cp2 Vp, K

=l; CpzVat=CplVal-CpzVp2K;

Cp2 Vpz K = Cpl Val - Cp2 Va2 ; K = Cpl Val - Cp2 Va2

CPZ VP2 *

Or since in our cases Val = Va2 or Va,

K = Va (Cpl - Cp2)

CPP VP2

Given the following figures for Preparation 12:

VP1 CPl Val 23.0 cc. 0.345 100

VP2 CP2 Va2

21.5 0.17 100

VP3 CP3 Var 22.0 0.035 100

K = 100 (0.345 - 0.17) = 21. 21.5 X 0.17


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K _ 100 (0.17 - 0.035) = 18

22.0 x 0.035 *

The differences in the two figures and their deviations from the determined value for pure solutions may well be due to errors in the determination of such small amounts of cholesterol and pos- sibly to incomplete equilibrium between the two phases, and to the effect of the hormone or other substances upon the solubility of the cholesterol in 70 per cent alcohol. Regardless of this difference, it is seen that the previous removal of soaps has been of value in keeping the distribution ratio of the cholesterol between 70 per cent alcohol and petroleum ether high.

TABLE III.

Number of Ether Extractions Required to Remove Hormone from Soaps.

No. of ether extract.

Total.. . . . . . . . . . . . . . . . . . . . . .

Left in aqueous solution.. . . . .

-

0

_

-

Preparation 12. Preparation 16.

Solids, Rat units rganic. total.

-___

mg. per cc.

14.2 840 1.9 156 0.4 78 0.17 36 0.05 9

--

Solids, Ra;okunits Irganic.

-I-

mg. per cc.

. . . . . 1119

--

31.8 >15<4E

21.1 1000 1.0 170 0.7 80 0.08 41 0.06 9

--

1300

-I- 43.9 1 >s<3:

-

Solids. rganic 1

.-

mQ. per cc.

5.4 0.56 0.18 0.13

- -

Rat units. total.

444 125 27

<15

I >596 <611

--

27.1 10

Since it appears desirable to remove the soaps before attempting the distribution between 70 per cent alcohol and petroleum ether, we have studied the question of the number of extractions of the soap solution necessary to remove the hormone. As we have found that soap increases the solubility of cholesterol in water, and since many investigators seem inclined to the view that the hormone is a lipid related to cholesterol, it is probable that soap would increase the solubility of the hormone in water. Though this may be true, our results in Table III indicate that the hormone may be rather well removed by ether extractions. It is evident


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that about 80 per cent of the hormone is removed by the first extraction and that four extractions are adequate to remove over 96 per cent of the active principle. As we generally find a little soap in the ether extracts, it is advisable, in view of the subsequent step, to wash these ether extracts carefully with dilute alkali and then water. The ether extract is then distilled to dryness and is ready for the petroleum ether-alcohol procedure.

In Table IVa and IVb, data are given bearing upon the value of the distribution of the hormone, cholesterol, and solids between 70 per cent alcohol and petroleum ether. In Preparations 8, 10, 11, and 12 the solids from the preliminary ether extraction were

TABLE IVa.

Number of Extractions of Alcoholic (70 Per Cent) Solution with Petroleum Ether Necessary.

E%z:

8 10 11 12 18

Extraction No. Solids per 1 CD. in alcohol.

1 2 3

I I

4

---

Tl. ml. ml.

5 6

ml. ml.

1.7 0.8 0.23 0.28 3.5 1.25 0.27 0.12 0.26 2.3 0.40 0.25 0.13 0.43 1.8 0.4 0.12 0.07 0.27 1.3 0.4 0.14 0.235*

*Alcohol distilled off without determining solids; value is calculated from ethereal solution of solids.

mg.

19 45.8 56.6 48.2 24.4

mg. 0.36

0.17 0.08 0.07

- Solids per 1 cc. of petroleum ether.

- I

suspended in approximately 100 cc. of 70 per cent alcohol which was then extracted with six successive portions of 25 cc. of petroleum ether. The values of solids per cc. for these successive extractions appear in Table IIIa; from these data it can be seen that the purification by this process becomes rather slow and ineffec- tive after the fourth extraction. For instance, in No. 12, both the fifth and sixth extractions removed less than 2 mg.

Table IIIb illustrates the advantages of the petroleum ether extraction. For example, in No. 12 over 96 per cent of the solids and nearly 100 per cent of the cholesterol, but less than 2 per cent of the hormone, were removed from the alcoholic solution by


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the petroleum ether. This is indeed an advantageous method of purification; by a simple easy extraction process the weight of a rat unit is decreased from 0.82 to 0.03 mg. with practically no loss of hormone. Jt will be noted that under the column headed “Petroleum ether. Rat units,” each figure is preceded by a < symbol; this merely means that in no case have we used enough of the petroleum ether extract to obtain a positive test.

It seems rather clear from the data and discussion that the procedure described yields a product of which about 0.03 mg. is 1

TABLE IVb.

Distribution of Solids, Cholesterol, and Rat Units between YO Per Cent Alcohol and Petroleum Ether (B.P. 40-60”).

Prepa- ration

No.

5* 6a 6b 8*

10 11 12 R 18

-

Alcohol 70 per cent.

Vol- ume.

cc.

100 50 50

100 90 81

100 40 50

Total solids left.

T?.

704 54 28.5

980 26 33 27 18 10

Choles- nat terol. units.

m7.

<12 <l <I

0.01t 0.01t 0.01t 0.01t 0.01t

3060 432 230 540 800

900 338 457

T No. of

extractions.

-

1

-

Petroleum ether. -

VolunM xed in

each extrao- ticn.

_ cc.

25 25 25 25 25 25 25 10 15

-

Total solids extrao-

ted.

“0.

2205 292 198 285 585 963 717 340 208

m0.

615 94 68

248 309 326 162 85.f

Rat units.

<I87 <34 <27 <22 <34 <20 <21 <15 <lO

* Distribution of solids differs from other data of table, due to the fact that soaps had not been completely removed.

t Not determined; maximum value calculated from distribution ratio.

rat unit and that the losses incurred by the two important steps in the technique do not total more than 5 or 6 per cent. We have not determined the loss during the distillation of the alcoholic extract of the liquor folliculi, but are not disposed to think that it can be very extensive. The distillation is conducted at a low temperature in vacua which limits possible odixation, and our yields are much larger than those reported by Laqueur and his collaborators or by Dickens, Dodds, and Wright.


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J. 0. Rails, C. N. Jordan, and E. A. Doisy 371

Attempts da Purify the Hormone.

As our work increases the potency of the preparations, the supply of material (in mg.) for chemical investigation decreases. Though this is common knowledge to those working with hor- mones, vitamins, or other substances of this type, it may be worth while to give a concrete example derived from the data of this paper. We have just stated that the rat unit might be reduced by proper fractionation to 0.02 mg. or less per rat unit. Upon the basis of 0.02 mg. and 1000 units per liter of liquor folliculi, we

TABLE V.

Solvent I”ractionation.

Solvent fractionation of 5.4 mg. or 174 units: 1 unit = 0,031 mg.

Dry residue extracted with

petroleum ether.

I

Solutlion concentrated and ooo!ed,

e-7 SoIution : Residue :

3.0 mg. 1.75 mg. 0.15mg. = 1 unit D.06 mg. =

30 units 30 units

Units recovered, 117.

Residue extracted with ether.

Resiciue : so1utron:

1.04 mg. 0.80 mg. 1 unit O-26 mg. = 1 unit D-D15 mg. =

4 units 53 units

can expect 20 mg. of this refined product per liter providing none is lost in the process of fractionation. Since we obtain about 1000 cc. of liquor from 10 pounds of hog ovaries and the present price of ovaries from the packing house is about $2.00 per pound, the cost of this semipure product exclusive of work and reagents is about $1.00 per mg. Should Lipschiitz’s (1926) preliminary report be confirmed by later work (0.5 mg. from 500 cc. of liquor folliculi, and therefore about 1 mg. from $20;00 worth of ovaries), the price of isolation of the pure hormone and its chemical study assume staggering proportions.


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With the above figures in mind it may be easiIy understood that we have attempted to work on an ultramicro scale. Pregl’s proce- dures for ultimate analysis and other methods devised by ourselves which require only minute quantities of material have been used. Fractionations of small amounts by means of solvents and by distillation in uucuo have been attempted.

;In exampIe of solvent fractionation is given in Table V. Of the total units, approximately two-thirds were recovered; this discrep- ancy is probably not due to mechanical loss but more likely is due to cumulative errors in the biological testing or to loss by oxidation, since we have sometimes found that purified preparations lose their potency quite rapidly. It will be noted from the table that this preparation was separated into two fractions, one soluble in petroIsum ether and a residue which was partially soluble in ethyl ether. The latter was approximately twice as potent as the original preparation, but contained only one-third of the hormone. Obviously such a method of purification introduces too much loss of material to be of any practical value in the purification of the minute quantities at our disposal.

Because of the numerous reports (Herrmann, 1915; Frank, 1917; Fraenkel and Fonda, 1923) on thermostability of the hormone, we have turned our attention to the possibility of purification by fractional distillation in a high vacuum. In our best recoveries we have rarely obtained more than half of the rat units with which we started and in some cases much less. It has seemed to us that our better recoveries occurred when preparations of a high degree of potency were used for distillation, and though this might be explained upon the shorter time the hormone was exposed to the higher temperatures, it is quite probable that the chemical activity of the impurities may play an important rale in the loss of activity of the hormone. We are inclined to use thermostability as a relative rat,her than an.absolute term. Depending upon the conditions prevailing during the distillation, the amount of loss of potency varies a great deal. In the experiments given in Table VI the loss was unusually small due probably to the brief time occupied in the distillation.

Another point with respect to thermostability is worthy of mention; i.e., the criterion of thermostability. In work prior to 1923 the method of t.esting extracts for potency was not adapted to


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J. 0. Rails, C. N. Jordan, and E. A. Doisy 373

quantitative accuracy. Though results of a rough quantitative character could be obtained (Giesy, 1920) it may have been some- what difficult to detect losses in activity of 20 to 50 per cent. With the improved method of standardization of extracts (Allen

TABLE VI.

Vacuum Distillation.

Preparation Glah.-Fractional distillation of 4.45 mg. at 0.03 mm. pressure. > 90 units.

Solids. Units.

Distillate up to 184”. 2.7 mg. 1 unit 0.04 to 0.06 mg. > 45 - < 67 Heating begun at 10.36. 178-184” from 10.45 to

10.53.

Residue above 184”. . . .1.96 mg. 1 unit 0.08 mg. 24 Recovered . . . . . . . . . . . .4.66 mg. > 69 < 91

Preparation Iold.-Fractional distillation at 0.1 mm. pressure of 3.9 mg. 125 units.

Distillate up to 172’. 1.19 mg. Heating begun at 12.43. 170” at 12.51; stopped

1.01. Cooled rapidly.

Distillate 172-195”. 0.48 mg. Heating begun at 1.34. 182” at 1.41. 195” at 1.51; cooled

rapidly.

Residue above 195”. . . . .1.27 mg. Recovered . . . . . . . . . .2.94 mg.

1 unit 0.016 t,o 0.026 mg. 46 - 74

1 unit 0.05 to 0.075 mg. 6 - 10

1 unit 0.098 mg. 7 - 13 > 59 < 97

Distillation was conducted in a flask blown from a piece of 10 mm. Pyrex tubing; the side tube came off at the top of the flask. The entire flask up to the side tube was immersed in the oil bath.

and Doisy, 1923), the loss of potency becomes easier and more certain of detection. According to Loewe (1925) the mouse smear method has an accuracy of 20 per cent. Though in some cases our losses do not exceed 20 per cent, in others the loss is as much


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as 80 per cent and invariably we do find loss. Admittedly our results on thermostability are not clear cut, but they have impressed us so much that we have discontinued our attempts to purify the hormone by vacuum distillation.

It is clear from Table VI that with Preparation 1Old we have lost about 1 mg. of solids. This loss is inexplicable unless it was due to lower boiling substances in the distillate which were not condensed in our receiving tube. In the distillation of No. 61ah the decrease of activity was approximately the same as with No. 1Old but the recovery of solids (104 per cent) was complete.

Chemical Characterization of the Purified Product.

In attempting to learn something of the chemical nature of the hormone which might aid us in its actual isolation, we have per- formed a number of reactions which are given in detail below. Herrmann (1915)) Fraenkel and Fonda (1923), Giesy’ (1920), Frank and Gustavson (1925), and others have utilized some of these and still other reactions with the same object in mind. As none of us has worked with the pure hormone, a great deal of cau- tion should be exercised in the interpretation of results. It is easily conceivable and indeed probable that the pure substance will differ in some of its reactions from crude or semipure products, but since we believe that our preparations are considerably more potent than others which have been studied and that our test reaction is better adapted to the detection of loss of activity, we present our results for what they may be worth.

Due to the excessive cost of material and the limitation of its supply, we have not attempted in this preliminary work to prepare and crystallize derivatives but have contented ourselves with a study of the effect of various reactions upon the potency. While this is not an entirely satisfactory procedure, it may give us use- ful information which may lead to the isolation of a derivative of the hormone.

1 Attention should be directed to P. M. Giesy’s Dissertation, A chemical study of the placental hormone, (1920) which due to the kindness of Dr. Giesy the authors have had the privilege of consulting. This thesis is an extensive painstaking piece of work which appears to be a distinct advance over the earlier investigations of others interested in the placental hormone.


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Prep

aratio

n No

.

5il

5sl

6ah

dist

illate

.

161

TABL

E VI

I.

Chem

ical

Beha

vior

of

the

Purij

ied

Prod

uct.

Origi

nal.

mg.

0.03

8-

0.04

8+

0.02

2f

0.03

0+

0.22

++

0.00

67-t

0.00

59-

Acety

l- ati

on.

w.

0.12

6-

Benz

oyl-

ation

.

%a.

0.04

5-

0.12

f 0.

18+

Weig

ht of

1 un

it aft

er

treatm

ent.

-Nsp

hthy

iaocy

a- na

te.

ml.

0.08-

0.14

+

Brom

i- na

tion.

ml.

0.20

-

Ffyd

roge

n-

ation

.

WI.

~.03

3f

~.04

5$$

Sapo

nifica

tion:

In air

.

WJ.

0.05

-

0.00

98$

0.00

84-

In hy

dro-

gen.

WI.

0.05

+

0.00

98&

0.00

84-

- I 1

- _

-

Phen

yl-

xydra

zine.

mg.

0.03

6+

KMnO

4

ml.

0.48

-


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Oxidation.-As indicated in Table VII certain reagents will destroy the activity of the hormone, some slowly, others rapidly. Apparent’ly any oxidation reaction where free oxygen is involved causes a loss. The ease with which activity is lost in some preparations that stand in air or come in contact with air in heating has astonished us. That the same preparation standing in an inert gas or heated in such a gas is more stable points to oxygen or air as the offending agent. We have found that due to the fact that the access of air to such preparations is reduced to a minimum, our preparations in solvents with high vapor pressures are quite stable. It is an interesting point that solutions of the hormone in oil (Mazola) seem to be quite stable in air, which indicates that some mechanism for oxidation is absent, as oxygen can certainly pene- trate such solutions. We are inclined to believe also that oxidation (by HOBr) and not bromination accounts for inactivation when carried out in dilute bromine solutions at 0°C.

That inactivation can be produced under non-oxidizing conditions by some reagents is certain. Such reagents as acetyl chloride, benzoyl chloride, and cY-naphthyl isocyanate are examples. This is interpreted as meaning that some distinctive group in the molecule of the substance is affected. It is not clear yet that some derivatives produced by such chemical reactions are them- selves totally inactive. Much work must be done to clear up such obscure points.

Reactions of the Hydroxyl or Amino Group.

a. Benxoyl Chloride.-When benzoylation was carried out using pyridine as solvent (Einhorn, 1898), there was an almost total loss of activity. The manner of carrying out the procedure was such that we believe the loss of potency to be due to a chemical action on some group in the molecule of the active substance. Either a hydroxyl group or an amino group could be involved. From the ultimate analysis about 8 per cent oxygen may be present. It would appear to be most reasonable to assume from this inactivation by benzoylation that part at least of the oxygen is there as the hydroxyl group.

b. Ace&l Chloride.-Acylation in pyridine carried out by the above method gave a product which was also inactive. This would seem to be additional evidence that the action was due to the presence of the hydroxyl group.


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c. Naphthyl Isocyanate.-It was desirable to study other types of reactions that might further throw light on the presence of the hydroxyl group which seemed to be indicated by benzoylation and acylation. Bickel and French (1926) have shown that cl-naphthyl isocyanate reacts well with primary and secondary alcohols to produce urethanes. This led us to try this reagent on our preparations with the result again that most of the activity was lost. Although we feel that these reactions and the probable presence of oxygen in the hormone establish with a fair degree of certainty the presence of a hydroxyl, we recognize also that an amino group could also be acted upon by these reagents. For the present, however, we have disregarded the possibility of an amino group. In later work we expect to establish more definitely the presence or absence of these groups.

Reactions of CarbonyL-Attempts to prepare a hydrazone with p-nitrophenylhydrazine were unsuccessful. With phenylhy-

TABLE VIII.

Ultimate Analysis.

C H N Molecular weight.

per cent per cent per cent 5il 80.5 10.7 0.93 418

81.0 10.3 458

drazine the reaction was conducted as usual for the preparation of a hydrazone. If such a product forms, it is clear from Table VII that the potency of the hydrazone must be of the same order as that of the hormone. We are inclined to doubt this possibility and would rather tentatively conclude that the carbonyl group is absent.

HydrolysQs.-Though we have lost part of the hormone in saponification reactions, we are inclined to believe that this may have been due to causes other than hydrolysis. Active lipase preparations did not produce much loss of potency and saponification with 0.01 N sodium ethylate in 95 per cent alcohol caused very little loss of activity.


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Reactions of the Double Bond: Hydrogenation and Bromination.

The behavior of Preparations 5il and 5sl toward hydrogenation and bromination which is typical of other preparations seems significant. The ease with which all preparations are quickly inactivated by dilute bromine at 0°C. suggests that the active substance is unsaturated. From the instability of the hormone in air and toward oxidizing agents, workers in this field have generally believed it to be unsaturated. Hydrogenation of samples for 1 hour using platinum black and a pressure of 2 atmospheres did not materially reduce the activity. Under the same conditions oleic acid was hydrogenated in 5 minutes. This could mean that either the substance is difficult to hydrogenate or that saturation with hydrogen does not appreciably destroy its activity, or pos- sibly both.

Ultimate Analysis.-Analysis of the 0.04 mg. product gave C 80.8 per cent; H 10.4 per cent; N 0.9 per cent; P. 0.00. These percentages total only 92 per cent but further analyses were not made due to lack of material. It seems probable from some reactions that oxygen is present as hydroxyl which might make up the difference between 92 and 100 per cent. We have been much impressed by the similarity in certain properties between our preparations and the very active preparations of vitamin A of Takahashi, Nakamiya, Kawakami, and Kitasato (1925), particu- larly the ultimate analysis, molecular weights, and boiling point under reduced pressure. Although their molecular weights are a little less than ours, their boiling points are correspondingly lower. We realize, however, that any conclusions drawn from our preparations with an activity of only 0.04 mg. per rat unit are highly speculative.

Does the Active PrincQple Contain Nitrogen?-Frankly, we have been much concerned over the fact that two of our preparations (Nos. 5il and 5k) have shown a nitrogen content by the micro- Dumas method (0.9 per cent) much in excess of any blank which we obtain on known nitrogen-free compounds. We cannot account for the presence of this amount of nitrogen as an impurity, but hesitate to say positively that it is associated with the active principle of our preparation. Realizing the impossibility of settling such a question until the limit of potency of our preparation has been reached, our attention has been turned more to the


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J. 0. Ralls, C. E. Jordan, and E. A. Doisy 379

purification of the substance. The amount of material involved (3 to 5 mg. for each analysis) has also been a factor in deferring other analyses. We hope that we may be able to settle definitely the question in the near future.

Molecular Weight.-.By the freezing point depression method, the molecular weight using two different solvents turns out to be 418 and 458.. Based upon the assumption that nitrogen is a com- ponent of the hormone, and that there is 1 atom of N per molecule, the molecular weight is a little over 1500. This lack of agreement is impressive evidence that we have not obtained the hormone as a pure compound as yet.

Chemical Methods Used.

Acetyl ChZoride.--0.18 mg. of Preparation 5sl in 0.5 cc. of pyridine was treated with 1 drop of acetyl chloride; tube became warm. 1 drop more added which produced a large precipitate. Evaporated off ether and let stand 8 hours. Treated with 0.5 cc. of alcohol to destroy excess acetyl chloride; heated slowly to 95%. and evaporated to dryness with vacuum. Oily residue dissolved in ether and transferred to bottle with 2 cc. of Mazola. Warmed to dissolve. 0.126 mg. -.

Benzoyl Chloride.--+.18 mg. of Preparation 5~1 dissolved in 0.5 cc of pyridine. To this mixture 1 drop of benzoyl chloride was added. Heat developed and amount of precipitate increased when additional drop of CsHsCOCl was added. Tube heated to remove ether; precipitate dissolved but appeared again on cooling. After standing 8 hours 0.5 cc. of alcohol added to destroy excess GHsCOCl. Heated on water bath and evaporated to dryness with vacuum. Oily residue taken up in ether and transferred to bottle with 2 cc. of Mazola. Warmed to dissolve. 0.045 mg. -; 0.12 f; 0.18 +.

or-Naphthyl Isocyanate.-O.25 mg. of Preparation 5~1 in ether was evaporated to dryness in a stream of CO,. To the residue was added 0.4 mg. of a-naphthyl isocyanate in 2 cc. of ligroin. Tube was heated in water bath for 10 minutes. Distilled off solvent under reduced pressure and dissolved residue in 2.6 cc. of Mazola. 0.08 mg. - ; 0.14 mg. +.

Hydrogenation.-In our tests on hydrogenation which we have carried out on several of our preparations we used PtOz prepared by the method of Adams and Shriner (1923). From 0.1 to 1.0 mg. of the hormone and about an equal quantity of PtOz in ether or alcohol, preferably the latter, were treated with a slow stream of hydrogen from a tank at about 2 atmospheres pressure. Time varied from 15 minutes to 2 hours, but in all cases the efficiency of the catlyst was checked against oleic acid. A typical experi- ment follows.

0.86 mg. of Preparation 5sl in ether was evaporated to dryness in a stream of hydrogen. To the residue 1 cc. of alcohol and 1.0 mg. of PtOz were added and the mixture treated with a slow stream of hydrogen at 1.5 atmospheres


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Extraction of Ovarian Hormone

pressure for 1 hour. Catalyst turned black in 4 minutes. Solution centrifuged and catalyst thoroughly washed with ether. Total ether-soluble solids recovered 0.80 mg. Solution had lost its yellow color. Hydrogenated 0.033mg. f; 0.044mg. ++.

Bromination.-In all the bromination experiments the hormone was dissolved in a 0.5 to 1 per cent solution of bromine in chloroform at 0°C. and maintained at that temperature in the dark from 5 to 30 minutes. Excess bromine was removed with a stream of CO, and the chloroform by using reduced pressures.

PhenyZhydrazine.-O.27 mg. of Preparation 5sl was treated with 0.3 mg. of phenylhydrazine in 0.3 cc. of alcohol and a trace of acetic acid added (Kamm, 1923). Stood 20 minutes; evaporated to dryness with vacuum and mixture dissolved in Mazola for testing. 0.036 mg. +.

SUMMARY.

1. An improved method for the preparation of the hormone from hog liquor folliculi is described. The procedure advocated yields a product virtually free from cholesterol whose potency is from 25 to 40 rat units per mg. of solids.

2. Some chemical reactions of the product having an activity

of 0.04 mg. per rat unit are described.

BIBLIOGRAPHY.

Adams, R., and Shriner, R. L., J. Am. Chem. Xoc., 1923, xiv, 2171. Allen, E., and Doisy, E. A., J. Am. Med. Assn., 1923, lxxxi, 819. Bickel, V. T., and French, H. E., J. Am. Chem. Sot., 1926, xlviii, 747. Dickens, F., Dodds, E. C., and Wright, S., Biochem. J., 1925, xix, 853. Doisy, E. A., Rails, J. O., Allen, E., and Johnston, C. G., J. Biol. Chem.,

1924, Ixi, 711. Einhorn, A., Ann. Chem., 1898, ccci, 95. Fraenkel, S., and Fonda, M., Biochem. Z., 1923, cxli, 379. Frank, R. T., Xurg. Gynec. and Obst., 1917, xxv, 329. Frank, R. T., and Gustavson, R. G., J. Am. Med. Assn., 1925, Ixxxiv, 1715. Giesy, P. M., Dissertation, Columbia University, New York, 1920. Glimm, E., and Wadehn, F., Biochem. Z., 1925, clxvi, 155. Herrmann, E., Monatschr. Geburtsh. u. Gym%., 1915, xli, 1. Kamm, O., Qualitative organic analysis, New York, 1923. Laqueur, E., Hart, P. C., deJongh, S. E., and Wijsenbeek, J. A., Deutsch.

med. Woch., 1926, lii, 2,52. Lipschiitz, A., Vesnjakov, S., Tuisk, R., and Adamberg, L., Compt. rend.

Sot. biol., 1926, xciv, 738. Loewe, S., Klin. Woch., 1925, iv, 1407. Pregl, F., Die quantitative organische Mikroanalyse, Berlin, 1923. Rails, J. O., Jordan, C. N., Heusinkveld, C. T., and Doisy, E. A., J. Biol.

Chem., 1926, lxvii, p. v. Takahashi, K., Nakamiya, Z., Kawakami, K., andKitasato, T., SC. Papers

Inst. Physic. Chem. Research., 1925, iii, 81. Zondek, B., and Brahm, B., Klin. Woch., 1925, iv, 2445.


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J. O. Ralls, C. N. Jordan and Edward A. DoisyPROPERTIES OF THE PRODUCT

HORMONE AND SOME CHEMICALEXTRACTION OF THE OVARIAN

AN IMPROVED PROCEDURE FOR THE

1926, 69:357-380.J. Biol. Chem.

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