Estrogen storage in fat

Estrogen storage in fat G.

H.

M.

BASSETT,

TWOMBLY,

D.

MEISEL

M.

LE\‘ITZ,

New

York,

M.D. B.S.

PH.D.* New

York

When ‘AC-labeled estradiol is injected intravenously into obese women, unconjugated labeled estrogens will be found in their body fat, the amount roughly $ro@ortional to the weight of the fat. The half-life of these stored hormones is about 10 hours, Rabbits implanted with pellets of estradiol occasionally develop carcinoma of the endometrium (4 of 20 in 219 to 597 days), but this continuous type of stimulation seems no more effective than intermittent injections as used by Meissner, Sommers, and Sherman.” CBA mice made obese by injection of gold thioglucose go into constant estrus and show hypertrophied uteri. They do not stay in estrus any longer, however, than do normal controls following castration. Obese Vob mice show no evidence of chronic estrogenic stimulation. Pregnant women at term have no evidence of stored estrone, estradiol, or estriol in their body fat. The level of these hormones appear to be somewhat less gram for gram than those found in their serum. Our observations suggest that obesity is a concomitant phenomenon and not a cause of cancer of the endometrium.

N u M E R o u s writers have remarked upon the tendency of women with carcinoma of the corpus to be overweight. A study of all patients with endometrial cancer seen at Bellevue Hospital in the 10 years from 19511961 showed their average weight to be 169 pounds. It has also been pointed out that certain women who appear to have had chronic estrogen stimulation have a higher incidence of carcinoma of the corpus than would be expected. Diddle l found 6.1 per cent of women with granulosa cell or theta cell tuFrom the Department of Obstetrics Gynecology, New York University School of Medicine.

mors to have concomitant endometrial cancers. Larsen2 puts the figure at 10.3 per cent for postmenopausal patients. Many other observations have tended partially to support this thesis, such as the previous appearance of endometrial hyperplasia, the tendency for a later menopause, and menstrual irregularities at the time of the menopause perhaps associated with anovulatory cycles, the increased incidence of carcinoma of the corpus in cases of Stein-Leventhal disease, and occasional case reports in which estrogen therapy appears to be related directly with the appearance of such cancers. Parenthetically, it may be mentioned that if one is looking for an estrogen-induced etiology of cancer in women, the time to look is 10 to 15 years before the onset of the neoplasm if we can reason from analogy with other known human carcinogens. In trying to explain the association of obesity with chronic estrogenic stimulation, most authors have postulated a common cause in a lesion or congenital abnormality in the hypothalamus. When Davis and Plot? reported on their studies of the fate of l*C-

and

This investigation was supported in part by a United States Public Health Service Research Grant CA-02071-14 from the National Cancer Institute and from American Cancer Society Grant P-206H. Presented at the Ninetieth Meeting of the American Society, Phoenix, Arizona, 1967. *Research Career K3-HD-18,422-05, Institute of Child Development.

Annual Gynecological May 4-6,

Development Awardee, of the National Health and Human

785

786

Twombly

et ul.

labeled progesterone in women and pointc$ out the storage of this tnaterial in the hod\ fat, it occurred to us that a similar storage, of natural estrogens might result in lotigcontinued low levels of stimulation in the obese, and thus a direct cause-and-rfft%ct relationship with the following sequencct : obesity + estrogenic stimulation + endotnetrial cancer. At peak production rates. natural estrogens might go into body fat: particularly in obese women, to slowly ltaak back into the circulation during parts of the monthly cycle when the ovaries were less active. Obesity would be like a reservoir, flattening out the peaks but filling in the Lfalleys of blood estrogen levels. In a previous paper from this laborator),’ observations have been recorded on the cxcrttion rate in the urine of carbon-14 in fat and thin women who had been injected intramuscularly with labeled estradiol- 17/3. Fifty-two women were so treated and the radioactivity of their urine determined fol the ensuing 72 hours. Twenty-two women weighing 150 pounds or less excreted an a\.erage of 68 per cent of the injected “(:. Thirty women weighing 151 to 240 pounds t,xcreted an average of 56 per cent. This observation then tended to confirm our thesis. Two preliminary experiments showed storagp of radioactivity in the body fat 15 and 18 hours after injection of l”C-labeled t-stradiol. In one of these cases, it was estimated that 10 per cent of the injected radioactive material was so stored. The present paper is an account of out subsequent laboratory studies along thest* lines. First. WC have determined in much treater detail the amount and chemical nature of labeled estrogens found in fat aftrr injection. We have attempted to determinr how long such material remains. Carrying the idea that cancer of the endometrium results from long-continued steady estrogenic stimulation into the animal laboratory, we have produced four carcinomas in 20 rabbits implanted with pellets of estradiol. We have produced obesity in CBA mice by injecting them with gold thioglucose and have observed that these preposterously fat

Finally, if fat storage of natural rndogenol+ estro,qens t&s place in wo~n~~~. it ought to I,(, possible to drrrronstrate large quantities in fat from pregnant patients .iust before term. This has proved ;t difficult trchnical procedurr. but we are convked from the few studies WV have done that no such storage takes plart,. The levels of unconjugated estrone. rstradiol, and estriol sct’u1 to hc lower, if anything, tharl one finds in the blood drawn at the same tinit, that the sample of fat \~as removed. Storage of ‘T-labeled in women

estrogens

in fat

Estradiol labeled with “C at the 16 position was synthesized with a specific activity of 27,000,OOO counts per minute per milligram. This material was dissolved in propylene glyco1 and injected in the amount of 1 mg. 01 2.5 ~I,C intravenously into women about to undergo Lgynccologic abdominal surgery. At varying: titnes after injection, hlood sanlplrs were drawn and pieces of fat were taken from the edgt,? of their abdominal womids, or ilr some cases, from their omentums. To removr rstro,qerts from the fat. it was extracttad onct’ with 100 prr cent ethanol. once with 80 pt‘r cent ethanol. and twice with a one-to-one mixture of aceton? and 100 1je-r ccmt ethanol. To make this as thorough as possihle, the fat and solvents tvere mixed for 2 minutes in a Waring Mendor. By t.his procedure virtuall) all the lahekd estronr or estradiol was cstracted by thr ethanol and the fat could he discarded.
Volume Number

99 IS

Estrogen

Table I. Radioactivity ‘C-estradiol to human

in fat and blood subjects*

,- _I* Time 120 min. 57

ii 150 60 60 60 60 55 60 60 10 hr. 9.5 12.5 7.5 10.5 48 hr. 49

Pounds of fat?

Weight

$The

of fat Chicago, fat

was

were Vol. extracted,

Fat (c.p.m./Gm.)

1,115 840 777 540 713 273 487 917 357 443 267 236 202 388 -

627 450 295 454 207 139 181 91 232 100 193 176 106 61 166 107 14 14

5 23 33 54 42 71 89 101 130 249 23 17 59 54 111 54 39

22

*One milligram (25 JLC) of W-&radio1 taken, ex-iracted, and assayed for 1%. ?Pounds Actuaries

calculated 1: 17, purified,

by 1959. and

the intravenous

Blood (c.p.m./ml.)

7

91 9.5 12,4 13:3 150 151 162 189 198 245 350 106 114 14ii 150 209 145 152

following

in using

propylene the

assayed

glycol

height-weight-age for

radioactive

The results of these studies are shown in Table I and in Fig. 1. In general, it can be seen that the amount of ‘“C found in the fat is roug.hly proportional to the total amount of body fat present. At one hour, it varied from 3 to 89 per cent of the injected dose. A calculated value for a normal woman was about 12 per cent. After 10 hours the radioactivity recovered from the fat had fallen to 3 to 17 per cent. Again the amount found was proportional to the total fat as calculated. The normal radioactivity was about 6 per cent. Two patients were studied after 48 to 49 hours and they were slightly overweight. They showed radioactivity in their fat of 1 per cent of the injected dose. We may conclude, then, that the half value period for injectecl radioactive estrogens in fat is in the neighborhood of 10 hours. It is obvious that what we have measured so far i;s only radioactivity extracted from fat by ethanol-acetone mixtures and has been characterized in no further fashion. In order

was

estrone

administration

Injected dose in total fat (%I 7 3 10 23 15

prior for

and

to

American estradiol

surgery.

in fat

787

of

14C in fat identified Estradiol$ (%) 60

E&one (%) 27

2; 17 29 23 89 6 3 6 12 17 1 1

given tables

storage

At

4 10

92 32

2 20

95 74

9 55

81 42

surgery,

women

as published

as described

in

the

fat

and by

the

blood Society

were of

text.

to determine just what these radioactive materials were, we carried out the following procedures in seven of the extracts. The extract was submitted to column chromatography on silica gel as described by Goldzieher, Baker, and Riha.5 This procedure is recommended for fat extracts. The eluted radioactive material was diluted with 200 pg each of estrone, estradiol, and estriol carriers. The mixture was chromatographed on celite using the gradient elution method described by Engel and associates.6 The solvent system is 90 per cent methanol-trimethylpentane and the gradient is started by the addition of trimethylpentane-ethylene dichloride (1 : 1) . Zones of radioactivity corresponding to estrone and estradiol were obtained, but the estriol zone was devoid of radioactivity. The elution pattern was in agreement with that previously described.” Twenty milligrams of carrier estrone and estradiol was added to the corresponding eluates. The estrogens were crystallized three times from ethanol. After the third crystallization, the crystals were

788

Twombly

et al

LOCALIZATION OF “C IN FAT KXLOWING INJECTION OF ‘“C-ESTRADIW i

120 Per cent of standard

INTRAVtNOlJS

160

200

240

weight

Fig. 1.

acetylated and the acetates were crystallized twice. In none of the 7 cases was radioactivity dissociated from the carrier. In fact, Table I indicates that in 6 of the 7 cases virtually all the radioactive material extracted from the fat was identified as estrone and estradiol. Animal

experiments

Greene and Saxton, in 1938, reported from the Rockefeller Institute in Princeton that they had observed the development of carcinoma of the endometrium in Dutch rabbits in their colony. This followed an epizootic which they interpreted as a pregnancy toxemia followed by cirrhosis of the liver. The carcinomas occurred in animals which not only showed cirrhosis at autopsy but also evidences of low-grade chronic estrogenic stimulation. They felt that their animals were unable to detoxify and excrete normally their own endogenous estrogens. ‘The observations of Greene and Saxton

brought to our attention the fact that carcinoma of the endometrium occurred in the rabbit, an observation that had been made by several investigator?, ‘* I0 and suggested that this tumor might be the result of chronic estrogenic stimulation. In 1957, Meissner, Sommers, and ShermanI] reported the production of six carcinomas of the endumetrium in 18 rabbits injected repeatedly, every 3 weeks, with stilbestrol and made diabetic with alloxan. The tumors were found after 315 to 616 days. It was our hope that continuous stimulation would be even more effective than the intermittent injections of the above experiment. Consequently, we obtained female Dutch rabbits 2 or 3 months old and implanted 25 mg. pellets of estradiol which, being relatively insoluble, exert a constant effect for many months. Long-continued experiments in laboratory animals always encounter the obstacle of epizootic disease. Finally, however, a group of 20 animals was kept alive

Volum8c Number

Estrogen storage

99 6

in fat

789

ESTRUS SMEARS IN CBA MICE TREATED WITH GOLDTHIOGLUCOSE FOLLOWED BY CASTRATION

Fed

Starved

Control

Fig. 2.

219 or more days. Of these 20, 4 showed endometrial carcinoma, the slowest appearing after 597 days. Two of these tumors metastasized, one to a regional lymph node and one to the peritoneum of the abdominal wall, the intestine, and the omentum. We concluded that long-continued estrogenie. stimulation did indeed lead to the production of adenocarcinoma of the endomettium, but that in our hands at least longcontinued uniform stimulation by pellet was no more effective than the intermittent stimulation used by Meissner, Sommers, and Sherman.ll At the suggestion of R. A. Liebelt and associates,12 who supplied us with breeding stocks of his CBA mice, we undertook an investigation of the relationship of obesity to constant estrus. Injection of mature females intraperitoneally with 50 mg. of gold thioglucose resulted in a 4-O per cent mortality, but of the survivors, approximately 50 per cent became obese, some excessively so, i.e.,

double their normal adult weight. These animals showed constant estrus by vaginal smear. If, as we had theorized, this result had been brought about by storage of endogenous estrogens in the body fat and slow release, then castration should be followed by continued estrus for a number of days while this stored estrogen was released. It was thought that starvation would release even more hormone and prolong estrus. Fig. 2 shows some of the results of these experiments. The smears in the upper part of the diagram show the animals going into constant estrus one to 3 weeks after injection. When all were in full estrus, they were spayed. In this figure, the upper 3 animals were fed and the lower 5 were starved. It will be noted that all animals stayed in estrus one to 4 days after being spayed, the fed ones a day longer than the starved ones. This might seem to have some significance, were it not for the controls shown in the lower part of the figure. These animas had smears taken

790

Twombly

et al

regularly. They were spayed an the first da! of an estrus cyck. It is seen at once tllat these animals, even though untreated and not obese, stayed in estrus 3 to 4 days aftc%l castration also. We conclude that gold thioglucose obesity is associated with constant estrus but that storage of estrogens in fat is not suggested by our castration experiments. Autopsies cm these fat mice showed great uterine hypertrophy and diffuse stromal luteinization in the ovaries. Recently, Liebelt and associates” havr published a similar study on BALB/c/Ki mice. They note that animals which attained a body weight of 40 grams (10 gram weight gain) in 90 days showed persistent vaginal cornification. This started in his animals approximately 15 days after gold thioglucose injection, before the obesity was apparent. The constant estrus and the obesity were correlated with the amount of damage found on histologic examination in the ventral medial region of the hypothalamus. The) conclude that altered ovarian function in gold thioglucose- treated mice is not a function of

Fig. 3. An obese mouse of the Vob strain.

In the COIII‘S(~ of these experiments w(’ learned that ;i stock oi Vob mice was be& maintained hy 1Dr. W. E. Heston at the National Institutes of Health in Bethesda. These mice are obese: not becaustr of an) treatment, but because of their genetic composition. Dr. Heston supplied us with somt and they are really quite striking in their appearance (Fig. 3 i . Some are so fat that the) are almost unable to walk. At autopsy, however, these mice. in contrast to the gold thioglucose--treated ones. show atrophy of thek uterine horns :’Fiq. -4J A third lint, of obese mice is known as “yellow” or 3r*BR/Wi. They are represented by two genotypes which differ as far as is known in regard to one gene. Individuals possessing the Y’ gene are yellow coated and become obese when fed enriched diets. Individuals not possessing the Y gene are gra) coated and do not become obese. These aniulals have t,ccn studied very carefully by M. and K. Silberberg’,’ who noted that many of

Volume 99 Number 6

Estrogen storage

the females showed marked changes of the endometrium. The endometrium was hyperplastic and hypertrophic. The glands were closely packed and often cystically dilated. Adenocarcinoma was seen in one mouse 18 months old and 2 animals developed myosarcoma at 18 months and 20 months. The really critical observation from our point of view was that these changes were entirely independent of whether the animals were obese or not. The Silberbergs record 46 gray nonobese females, mean age 19.7

Fig. 4. Autopsy

of an obese Vob

mouse

showing

in fat

791

months, with endometrial hyperplasia in 36 (78 per cent) and 55 yellow obese females, mean age 18.7 months, with endometrial hyperplasia in 39 (71 per cent). Storage the fat

of endogenous estrogens of pregnant patients

in

It is obvious that if obesity plays an etiologic role in cancer of the endometrium according to our storage hypothesis, it must be possible to show that such storage is operating not only with injected estrogens but

minute

unstimulated

uterine

horns.

792

Twombly

et al,

also with host-produced endogenous steroids. One can study the estrogen content of fat in patients with functioning estrogen-producinp ovarian tumors, but these are rare and often not diagnosed until they have been removed. Pregnant women, on the other hand, are easily available and we know that near term they excrete very large quantities of estrogens in their urine. Consequently, it seemed reasonable to test our hypothesis by determining the estrogen content of fat removed from patients undergoing elective cesarean section and compare the amounts found with that in blood drawn at the same time. Methods for such determinations, however, are far from standard. Even though estrogens may he comparatively plentiful, they are still present only in microgram amounts. This means that one must process 15 to 20 Gm. of fat to find 0.2 pg of estrone, estradiol, 01’ estriol if these are present at about the same

PRINCIPLE

OF THE

Added estrone

0

Endogenous estrone

0

DOUBLE

concentratiou lound in plasma. I LI the www of such extraction procedures, all activity ma\ be lost at a single step or there ma! he VU!ing losses which vary from sample to samply. In order to obtain what we f4 are real]) reliable figures, we ha.vc employed the foilowing procedure: Patients who were near term but not in labor were used as experimental subjects at thcx time of electivt cesarean section. Fifteen to 20 Gm. of fat was cut from the edges of their abdominal incisions and 20 ml, of venous Mood withdrawn by vcnipuncturtl. The fat was cut in small pieces; 4 parts of ethanol was added, and the mixture was homogenized briefly in a Waring Blendor. Tn order to determine possible losses in subsequent procedures, 10,000 counts per minutfx each of pure tritiated estrone, estradiol, and estriol of high specific activity were added and the homogenization continued, This in

ISOTOPE

DILUTION

METHOD

“H

1

‘1 Purification

+

0 ‘) H3C--‘4C ’ 0 HzC ---14C < 0 & 3, Plrification

Acetic anhydride

\

14po

H3C’

“0

3H

Estrone acetate Fig. 5. By adding tritium-labeled estrone, losses occurring during purification procedures are accurately detected by subsequent measurements of tritium radioactivity. After purification, the amount of endogenous estrone present in the original sample can be determined by measuring 14C and tritium radioactivity. If only added tritium-labeled estrone were in the sample, 2 atoms of tritium would be present for each 14C atom. Endogenous estrone increases the ratio of 14C to tritium.

Volume Number

!Ki 6

effect labeled these three estrogens with tritium (Fig. 5). The mixture was centrifuged and extracted three times more with 80 per cent ethanol. The fat was discarded, the ethanol was evaporated under vacuum, and the residue dissolved in 70 per cent methanol. Storage of this mixture at -17* C. for 18 hours resulted in precipitation of the fat remaining in the alcohol. Followng centrifugation at -15’ C., the supernatant was removed and evaporated to dryness. The residue was dissolved in toluene. The toluene solution was extracted with 1N sodium hydroxide to isolate the estrogen!<. After neutralization, the aqueous solution was extracted with ether. The ether was evaporated to dryness and the residue was ta.ken up in benzene : N-hexane ( 1 : 1) and extracted with an equal volume of water. The benzene-hexane contained estrone and estradiol, the water, the estriol. The estriol was extracted from the water with ether. The estrogens were purified by chromatography on two paper systems. First estrone and estradiol were separated in toluene : trimethylpentane : methanol : water (75:25:80:20). Then each estrogen was purified further by chromatography in benzene : Skelly solve C : methanol : water (40:60:70:30). The estriol was purified by paper chromatography in toluene : ethyl acetate : methanol : water (85: 15:50:50) and benzene : methanol : water ( 100:5.5:45). The estrogens were located on the paper by scanning for radioactivity in a paper strip counter. The estrogens were t&ted with methanol and assayed by a modification of the double isotope dilution technique of Kliman and PetersonI as follows: the estrogen was dissolved in pyridine and acetylated with 14C-acetic anhydride. By this procedure, estrone, estradiol, and estriol were converted to the monacetate, diacetate, and triacetate, respectively. :Each acetate was dissolved in methylene chloride and extracted four times with water to remove acetic acid. The solvent was removed in a gentle stream of nitrogen and the residue was submitted to thin-layer chromatography in cyclohexane : ethyl acetate (3 : 1) The area corresponding to the ace-

Estrogen

storage

in fat

793

tate was eluted, diluted with 30 mg. of carrier acetate and the mixture was crystallized to constant 14C/3H ratio. The counts per minute per microgram of the estrogen with respect to 3H was calculated from equation I.15 c.p.m. 3H M c.p.m. ‘%I y -!!103 x SA,, x -N

c.p.m. = L@ where

c.p.m. 3H = c.p.m. 14C

the isotope crystals

n=

number

ratio

in the

of acetate groups

specific activity of cortisol acetate synthesized from cortisol and the 14Cacetic anhydride

SA, =

M=

molecular tisol

N=

molecular weight of the estrogen analyzed.

The micrograms were calculated a

=

weight

of cor-

of estrogen in the sample from Equation II.

c.p.m. (added at the start) c.p.m./pg (from equation I)’

Finally, conversions were made to micrograms per 100 Gm. of fat. The procedure for the analysis of plasma was somewhat simpler. The tritiated standards were added to the plasma which was then extracted with ether. The ether was evaporated to dryness and the residue was submitted to the phenolic separation and then the rest of the procedure as described for the fat. The results shown in Table II indicate that the concentrations of the three classical estrogens, estrone, estradiol, and estriol were slightly higher in the plasma than in the fat. At least in these experiments no evidence was obtained for storage in fat.

Table II. Comparison

of the concentrations of endogenous estrogens in fat and plasma in human pregnancy at term* Plasma ml.) 0.4-3.0 1.3-2.4 0.3-1.4

(pg/lOO

Estrone Estradiol Estriol *Four

subjects

were

studied.

(pg/lf% Cm.) 0.1-4.0 0.6-1.6 0.1-0.27

794

Twombly

et al

Comment

In the early stages of our studies, it was shown that following the injection of labeled estradiol, fat women excreted radioactivity in their urine at a slower rate than thin ones. This was confirmed in part by Brown and Strong *” in studies with unlabeled hormones, In the present study, we found that radioactive material could be isolated from the fat of women injected with labeled estradiol. However, the half-life for such storage was unexpectedly short, about 10 hours, Perhaps the most significant finding in this part of the study was that the radioactive material found in the fat was mainly estradiol, physiologically the most active estrogen, accom panied by smaller amounts of estrone. ‘Thf> exception was a single 10.5 hour experiment in which estrone predominated. The inferc’nce is that adipose tissue does not retain estrogens for lengthy time intervals and that there is no evidence of extensive rnetabolisrrl of estradiol by fat. The conclusion that the fat is not a significant site of localization of estrogens was confirmed by the results obtained on analysis of

thr fat and plasma 01 pregnant women. 111 tht: subjects studied, thy 1~~1~ (of uncorr.jugated estrone, estradiol. and cstriol fomci irk fat were not too different from those iri plasma. The animal experiments shed little light cm the etiology of endometrial carcinoma. Obesr mice gave no evidence of estrogen storage in their fat. 111 the rabbit. 4 of 20 rabbits developed endometrial carcinoma following the implantation of estradiol but this rate of incidence is not sianiflcantly different from that observed following intermittent injections of the cstropen. It would appear that if estrogen is the promoting agent in endometrial carcinoma, it cannot he linked PVPI~ indirectly to the fat. Perhaps carcinoma of the endometrium has its etiology ill a hypothalamic peculiarity involving estrogen while obesity and, at times, diabetes arp by-products of the same abnormality. We are determining anhydridr.

indebted to Dr. Hortense Gandy fo! the specific activity of the L4C--acetic.

REFERENCES

1. Diddle, A. W.: Cancer 5: 215, 1951. 2. Larson, J. A.: Obst. & Gynec. 3: 551, 1954. 3. Davis, M. E., and Plotz, E. J.: AM. J. OBST. & GYNEC. 76: 939, 1958. 4. Twombly, G. H., Scheiner, S., and Levitz, M.: AM. J. OBST. & GYNEC. 82: 424, 1961. 5. Goldzieher, J. W., Baker, R. A., and Riha, E. C.: J. Clin. Endocrinol. 21: 62, 1961. 6. Engel, L. L., Cameron, C. B., Stoffyn, A., Alexander, J. A., Klein, O., and Trofimow. N. D.: Anal. Biochem. 2: 114, 1961. 7. Greene, H. S. N., and Saxton, J. A., Jr.: J Exper. Med. 67: 691, 1938. 8. Burrows, H.: J. Path. & Bact. 51: 385, 1940, 9. Polson, C.: J. Path. & Bact. 30: 603, 1927.

10. I I. 12.

13. 14. 15. 16.

Rusk, G. Y., and Epstein, N.: .4m. J. Path. 3: 235, 1927. Meissner, W. A., Sommers, S. C.. and Sherman, G.: Cancer. 10: 500, 1957. Liebelt. R. A.. Sekiba. K.. Ichinoe. S.. and Liebelt: A. C.:’ Endochnol& 78: 8bi5, ‘1966. Silberberg, R., and Silberberg, M.: Yale J. Biol. & Med. 29: 525, 1957. K&man, B., and Peterson, R. E.: J. Biol. Chem. 235: 1639. 1960. Hobkirk, R., and Nitsen, M.: J. Clin. Endocrinol. 26: 625, 1966. Brown, J. H., and Strong, J. A.: J. Endocrinol. 32: 107: 1965.

Discussion DR. M. EDWARD by Dr. H. Close fate of Cl’-labeled

DAVIS, Chicago, Illinois (read Hesseltine). Our studies of the progesterone in women and

tion, and endometrial body

fat

would

serve

cancer. Thus, as a reservoir

the excessive providing a

constant source of estrogens.

its storage in body

fat suggested to the authorg that similar storage of endogenous estrogens might result in long-continued low levels of stimulation

This is an excellent of injected estrogens depots. It confirms

in the obese and thus a direct cause-and-effect relationship connecting obesity, estrogenic stimula-

vious studies on the fate of injected

cholesterol,

progesterone,

a common

and

study of the metabolic fate in relation to the body fat and elaborates on our preestradiol,

wherein

Volume 99 Number 15

Estrogen storage

pattern emerges: ( 1 ) 10 to 12 per cent of the radioactive material is stored in body fat, (2) the storage of the hormone is in a physiologically active form, and (3 :I the radioactive material has an unusually short life. Lipids function primarily in the cellular transport of hormones, and their role as a storage site for these steroids is of relatively minor importance. With the exception of estrogen-producing tumors, a mechanism of prolonged maintenance of estrogen levels has been hard to postulate. A storage and slow release mechanism would seem logical. Employing the concept of the storage of Cl”-labeled estrogens, the authors have come to the conclusion that endogenous estrogen is not stored in body fat for subsequent slow release. They adroitly offer another explanation for their findings by saying that obesity and endometrial carcinoma are not causally related but exist instead ;as only concomitant phenomena. But they then tie it all together and suggest a hypothalamic dysfunction common to both. It is known, and shown by the complexities of the authors’ technique, that analysis of small amounts of estrogen is difficult. An equally likely conclusion to be drawn from their work is that endometrial carcinoma and obesity are indeed related by a common hypothalamic dysfunction but that the amounts of estrogen stored in the body Fat are so small as to escape analysis. We have r:hown that exogenously labeled estrogen is stored in body fat as have the present authors. Why then should we conclude that endogenous estroglln is not? The complexities of neuroendocrine relationships and the difficulties of present assay techniques. present difficult obstacles, but further studies should be carried out. Claude Bernard’s words of insight are encouraging: “A discovery is usually an unforeseen relation not confirmed in theory, for otherwise it would have been foreseen.” REFERENCES

1. Davis, M. E., Plotz, R. G., and Werbin, 72: 740, 1956. 2. Davis, M. E., and & GYNEC. 76: 939,

E. J., LeRoy, G. V., Gould, H.: AM. J. OBST. & GYNEC. Plotz, E. J.: AM. 1958.

J. OBST.

in fat

7%

DR. SAUL B. GUSBERG, New York, New York. Dr. Twombly has expressed very elegantly the frustration of workers in this field seeking animal models to study the possible endocrinopathy in some tumors. I quite agree with his speculation that there may be a common origin for both these abnormalities-that is, the obesity with possible derangement of the carbohydrate metabolism and the tumor in the uterus. Some persons have tried to examine these problems in natural experiments in humans by studying patients with functioning ovarian tumors and patients with Stein-Leventhal disease. The fact in these studies must be stressed that the abnormality in these individuals may occur perhaps 20 or more years before the cancer appears. From some unpublished studies in our own laboratory we found that if one selects obese women beyond the menopause and compares those with and without cancer of the endometrium, there is no detectable difference in the estrogen metabolism. DR. TWOMBLY (Closing). I am a little surprised that no one has asked me more about the method of determining estrogens in fat in pregnant individuals. This is an extremely complicated sort of procedure because it is like trying to find something about the size of a codeine tablet dissolved in an oil tank full of fluid. The radioactive tracing techniques are of great sensitivity, and, as I said, Dr. Levitz and I think that our determinations on the fat in pregnant individuals are reliable. It takes months to study a single case. It is disappointing that the techniques are so difficult because one would like to study, for instance, the estrogenic content of fat in patients with SteinLeventhal disease. It has only been in patients who presumably have such high levels as are present in the last few weeks of pregnancy that we can get sufficient quantities of these hormones to make our determinations really satisfying to us. I hope that this is only an interim report and that at some time in the future I may be able to bring you some more results of these rather prolonged labors.