A . W H E R E A R E ESTROGENS FORMED?
Modification in sexual function or in the secondary sex characteristics of the organism may be correlated with the extirpation of certain endo-crine organs or with changes in the morphology of these tissues. Simi-larly, the rate of excretion of estrogens under normal and pathological conditions may give evidence of an indirect nature as to the ultimate source of estrogen elaboration. For example, estrogen excretion rises markedly during the course of pregnancy and drops precipitously with the termination of pregnancy but the removal of the ovaries of pregnant women and of mares does not result in disappearance of estrogens from the urine. The placenta is therefore implicated as a source of estrogen.
Studies of this sort have been reviewed previously (49,139) and will not
T A B L E I I I
I S O L A T I O N OF C R Y S T A L L I N E E S T R O G E N S FROM L I K E L Y S I T E S OF S Y N T H E S I S
Estrogen Organ Investigator
«-Estradiol Ovaries (sow) MacCorquodale et al. (108) Estrone0 Ovaries (sow) Westerfeld et al. (191) Estrone Placenta (human) Westerfeld et al. (190)
«-Estradiol Placenta (human) Huffman et al. (87) Estriol Placenta (human) Browne (19) Estrone Adrenals (beef) Beall (8) Estrone Testes (stallion) Beall (9) α-Estradiol Testes (stallion) Beall (9)
° Demonstrated but not isolated.
X . THE CHEMISTRY AND METABOLISM OF THE ESTROGENS 381 be discussed further. Such indirect evidence complements the more direct evidence furnished by the actual isolation of crystalline estrogenic compounds from extracts of these organs. Table III lists the estrogen and the endocrine organ from which it was isolated. The ovaries and placenta are generally regarded as the chief sources of estrogen in the organism. The testes and adrenals appear to produce much smaller quantities although, in the stallion, testis tissue appears to be a pro-digious producer of estrogen. ThusJ Levin (101) finds that certain specimens of stallion urine are the richest sources of α-estradiol to date and Beall (9) reports that the estrogenic content of horse testis is higher than that of any other endocrine organ.
B. ISOLATION OF ESTROGENS FROM SOURCES OTHER T H A N THOSE OF ELABORATION
Pregnancy urine is a very rich source of estrogenic material. The estrogens are present, for the most part, in the form of conjugates such as estrone sulfate5 or estriol glucuronide; brief acid hydrolysis suffices to liberate the estrogen (Marrian in 37). The urine of nonpregnant women is a poor source as is also the urine of males with the notable exception of stallion urine (Zondek, 200; Levin, 101). The estrogen and its urinary source is indicated in Table IV. Not all species elaborate estrogens of identical structure although estrone and estradiol are common to those species which have been studied. Estriol appears to be characteristic of the human species; estrogens in which ring Β is aromatic or partially saturated are found only in the mare.
C . INTERMEDIARY METABOLISM OF ESTROGENS
A comparison of the chemical structure of the various estrogens which have been obtained in exhaustive isolation studies (Tables III and IV) suggest metabolic interrelationships, some of which have been sub-stantiated by experiment. It has been fairly well established that the following reactions occur in the mammalian organism:
α-estradiol ^ ± estrone —> estriol
^-estradiol
This scheme is based on the isolation or detection of metabolites of the estrogen under study following its administration in massive doses to an experimental subject. The results of such experiments are summarized in Table V. There are differences among the species with respect to the course of estrogen metabolism. For example, the formation of
/3-estra-6 The preparation of estrone sulfate from estrone has been described b y Butenandt and Hof steter (27).
382 W I L L I A M H . P E A R L M A N T A B L E I V
I S O L A T I O N OF E S T R O G E N S FROM U R I N A R Y S O U R C E S0
Estrogen Source Investigator
Ketonic estrogens
Estrone Pregnancy urine (human) Doisy et al. (50) Butenandt (21) Pregnancy urine (mare) DeJongh et al. (42) Male urine (human) Dingemanse et al. (44) Male urine (stallion) Haussler (69)
Deulofeu and Ferrari (43) Male urine (bull) Marker (110)
Castrate male urine (steer) Marker (110)
Estrone sulfate Pregnancy urine (mare) Schachter and Marrian (154)
• Butenandt and Hofsteter (27) Equilin Pregnancy urine (mare) Girard et al. (62)
Hippulin Pregnancy urine (mare) Girard et al. (62) Equilenin Pregnancy urine (mare) Girard et al. (61)
Nonketonic estrogens
«-Estradiol Pregnancy urine (human) Smith et al. (169) Pregnancy urine (mare) Wintersteiner et al. (199)
«-Estradiol Male urine (stallion) Levin (101)
ß-Estradiol Pregnancy urine (mare) Hirschmann and Wintersteiner (80)
Estriol Pregnancy urine (human) Marrian (119) Doisy et al. (48) Estriol glucuronide.. Pregnancy urine (human) Cohen and Marrian (36) /3-Dihydroequilenin
(" δ-follicular
h o r m o n e " ) Pregnancy urine (mare) Wintersteiner et al. (198)
° Exclusive of metabolism experiments.
diol rather than a- estradiol is favored in the rabbit when estrone (Stroud, 175; Pearlman and Pearlman, 132) or α-estradiol (Heard et al., 71; Fish and Dorf man, 59) is injected, whereas little or no ß-estradiol can be detect-ed in the urine of men following estrone administration (Pearlman and Pin-cus, 134). In human pregnancy urine little or no ß-estradiol can be detected (Pearlman and Pearlman, 132), although ^-estradiol (Smith etal,
169) has been isolated from this source. Estriol has been isolated only from human source material but this substance (or one that closely re-sembles it in its physical properties and/orits biological action) may be formed in the guinea pig (Fish and Dorf man, 58), rabbit (Pincus and Zahl, 140; Pearlman and Pearlman, 132), monkey (Doisy et al,, 49), dog (Long-well and McKee, 103; Pearlman et al., 131), and rat (Schiller and Pincus,
X . THE CHEMISTRY AND METABOLISM OF THE ESTROGENS 383 T A B L E V
E S T R O G E N M E T A B O L I S M E X P E R I M E N T S
Estrogen metabolites0
Subject Investigator Isolated Indicated6
Subject Investigator
β Excreted in urine except where otherwise noted.
b Indicated indirectly b y comparison of the chemical and physical properties of the substances responsible for the estrogenic activity of postinjection urine extracts with those of pure crystalline hormones.
384 WILLIAM H. PEARLMAN T A B L E V (Continued) Estrogen metabolites0
Subject Investigator Isolated Indicated6
Nonketonic estrogens M o n k e y s : female o v a - Westerfeld and Doisy riect.; ovariect. and ( 1 8 9 )
hysterect.
Estriol R a b b i t : female (func- Pincus and Zahl ( 1 4 0 ) tional uterus
essen-tial to conversion)
Estradiol W o m e n (simultaneous Smith and Smith Estriol progesterone admin- ( 1 6 8 )
istration)
Nonketonic estrogens D o g s : both sexes Dingemanse and Tyzlowitz ( 4 5 ) Nonketonic estrogens D o g s : sex? Longwell and M c K e e
( 1 0 3 )
In bile: " w e a k " and
" s t r o n g " phenolic estrogens
Estradiol Men and women Pincus and Pearlman
Estriol ( 1 3 8 )
«-Estradiol R a t and rabbit: liver, Heller ( 7 6 ) uterus, etc., slices
/3-Estradiol Rabbits: female Pearlman and
Pearl-Estriol man ( 1 3 2 )
Estradiol R a t s : partially hepa- Schiller and Pincus Estriol tectomized ( 1 5 6 )
α-Estradiol R a b b i t (pregnant): Szego and Samuels endometrium ( 1 8 0 )
Administered estriol
Estriol; no estrone, Monkey : female ; nor- Doisy et al. ( 4 9 ) nor estradiol mal intact; ovariect.
and hysterect.
Rabbits: female Pincus and Zahl ( 1 4 0 ) Estriol; no estrone, Men Schiller and Pincus
nor estradiol ( 1 5 5 )
Administered /3-estradiol
Estrone, α-estradiol?, M o n k e y : female ; o v a - Doisy et al. ( 4 9 ) estriol riect. and hysterect.
X . THE CHEMISTRY AND METABOLISM OF THE ESTROGENS 385
1 5 5 , 1 5 6 ) ; identification of this estrogen metabolite by isolation of a crys-talline product would be desirable. Marrian ( 1 2 0 ) suggests that estriol may have some specific but unknown function during pregnancy.
Studies of the metabolism of equilin or equilenin have not been reported and there is only conjecture as to the relative position of these substances in the scheme of estrogen metabolism. Fieser ( 5 6 ) suggests that compounds of the equilenin group may represent successive stages in the dehydrogenation of estrone. In the laboratory, estrone can be dehydrogenated to give a stereoisomer of equilenin (see Section I, D , 6 ) . Equilin readily undergoes dehydrogenation to yield equilenin (see Sec-tion I, D , 3 ) , a process which may also occur in vivo.
D . Is ESTROGEN METABOLISM CONFINED TO ORGANS OF S E X HORMONE PRODUCTION AND STIMULATION?
As far as is known, very little or no estrogenic material is produced in organs other than the ovaries, testes, placenta, and adrenals; possibly the pituitary gland produces some estrogen but this has not been estab-lished. It is quite logical to assume that the end organs of estrogen stimulation, such as the uterus, transform the estrogens into other prod-ucts in a process intimately linked with the biological utilization of these hormones. Indeed, in vitro experiments indicate that uterine tissue will modify the structure of the estrogen molecule. Thus, Heller ( 7 6 ) incu-bated estrone with rat and rabbit uterine tissue and observed an increase in estrogenic potency. Szego and Samuels ( 1 8 0 ) demonstrated an almost complete conversion of estrone into α-estradiol following incubation of estrone with the endometrium of the pregnant rabbit; however, this change could not be effected by the endometrium obtained from non-pregnant bovine or from a non-pregnant woman. Such experiments do not necessarily mean that the intermediary metabolism of estrone is confined to the uterus. As a matter of fact, the bulk of the evidence indicates that the uterus (and ovaries) (see Table V) are not essential to those chemical transformations which the estrogens are known to undergo;
but then it must be borne in mind that our knowledge of estrogen metab-olism is meager. Marker ( 1 0 9 ) made the interesting suggestion that the regulation of the menstrual cycle is controlled by estrogenic substances by a process involving the reduction of these substances to the biologically inactive estranediols ( L X X ) , which he had isolated from nonpregnancy urine but not from pregnancy urine. It has not been proved that the biological effect is dependent on this chemical change nor has it even been established that the estrogens are reduced in the organism to estranediol.
It seems that our knowledge of the biochemical mechanisms whereby estrogens exert their biological effects is practically nil.
386 W I L L I A M H. P E A R L M A N
There is considerable evidence that the liver plays a major role in the transformation of the estrogens into products lacking biological activity (see Section II, F). The liver and certain other organs can also convert the estrogens into other products possessing more or less estrogenic potency. Heller (76) incubated estrone with liver slices poisoned with cyanide and noted the formation of a product of increased estrogenic potency, presumably α-estradiol. The addition of cyanide inhibits the inactivating system of the liver; the in vitro conversion of estrone into a substance possessing greater biological activity cannot be demonstrated otherwise. In the perfusion experiments of Schiller and Pincus (155), rat liver appears to convert α-estradiol into estrone and estriol or into estro-genic substances which are very similar in physical and chemical prop-erties. If the estrogenic substances found in bile are assumed to be formed by metabolic processes occurring in the liver, estrogen metabolism studies on bile fistula dogs may be of special interest in this regard.
Longwell and McKee (103) found that, if dogs were injected with estrone, a nonketonic estrogen appeared in bile. This estrogen is extractable from benzene with sodium carbonate solution. Pearlman et al. (131) detected a similar estrogen (estriol?) in the bile following the intravenous injection of α-estradiol; a ketonic estrogen, presumably estrone, was the major estrogenic metabolite detected. According to Szego and Roberts (147,178), the liver is essential for estrogenic activity as measured by the uterine water response in the adult, partially-hepatectomized or eviscer-ated rat. Two mechanisms are envisaged which may explain this phe-nomenon: (1) the liver inactivates estrogens and is also essential for the chemical "activation" of estrogens and (2) the liver may furnish some metabolite essential for the uterine response.
E . ISOLATION OF NONPHENOLIC STEROIDS STRUCTURALLY RELATED TO ESTROGENS: A R E THESE METABOLICALLY R E L A T E D ?
The isolation of certain nonphenolic steroids from urinary sources (see Table VI) has provoked speculation as to whether these substances may not be derived in vivo from the estrogens since there is a close structural relationship. In the laboratory, the conversion of equilenin into A5»7'9-estratrienol-3-one-17 has been realized (see Section I, D, 2);
the latter substance was isolated by Heard and Hoffman (72) from mare pregnancy urine. Whether the organism can effect a similar conversion is not known. The estranediols isolated by Marker et al. (114,117) from human nonpregnancy urine may have arisen from estrone in vivo since catalytic hydrogénation of estrone yields substances of this type. Doisy et al. (49) have pointed out that dehydrogenation of some saturated ring
X. THE CHEMISTRY AND METABOLISM OF THE ESTROGENS 387 T A B L E V I
I S O L A T I O N OF N O N P H E N O L I C S T E R O I D S0
Steroid Urinary source Investigator Estranediol A and Β ( L X X ) . .
A5'7-9-Estratrienol-3-one-17 (see L X X I I )
3-Desoxyequilenin ( L X X I X a ) 1 l-Keto-3-desoxy-equilenin
(after C r 03 oxidation) ( L X X I X b )
Human pregnancy Pregnant mare Pregnant mare
Pregnant mare
Marker et al. (116,117) Heard and Hoffman (72) Prelog and Führer (141)
Marker and Rohrmann (114,115b)
° Of close structural relationship to estrogens.
structures is effected by mammals but that the reduction of aromatic rings is of less frequent occurrence. It was suggested that these reduc-tion products of estrogens may be anabolites rather than catabolites of the estrogens. In this connection the experiments of Bernhard and Caflisch-Weill (10) are pertinent. They found that hexahydrobenzoic acid labeled with deuterium is dehydrogenated in the dog to benzoic acid;
the aromatization of the cyclohexane ring readily occurs in vivo. It may be that aromatization of the steroids occurs in the organism.
The recent isolation by Prelog and Führer (141) of 3-desoxyequilenin (LXXIXa) from pregnant mares' urine is exceedingly interesting.
Marker and Rohrmann (115b) had previously suspected the presence of this substance in pregnant mares' urine since ll-keto-3-desoxyequilenin ( L X X I X b ) was isolated from the neutral fraction after chromic acid oxidation; similar oxidation of equilenin acetate will result in the intro-duction of a keto group at Cn (see Section I, D, 4, b). There is no basis of chemical analogy to support the hypothesis that desoxyequilenin arises directly from equilenin in the organism. It is true that catalytic reduction of the estrogens in an acid medium will result in the elimination of the hydroxyl group at position 3 but saturation of ring A occurs con-comitantly. On the other hand, it is conceivable that 3-desoxyequilenin arises in vivo from A5'7,9-estratrienol-3-one-17 (LXXII) by a process involving dehydration in ring A followed by aromatization since, on chemical grounds at least, this process is easily visualized. (By virtue of the same reasoning, 3-desoxyequilenin may possibly have arisen as an artifact in the course of isolation; the formation of artifacts has been a sore point with the student of steroid metabolism in the past—for examples, see "Artifacts in the study of the intermediary metabolism of andro-gens" in 139.) It is not unlikely that 3-desoxyequilenin is converted to equilenin in view of the demonstrated hydroxylation in vivo of certain
388 WILLIAM H. PEARLMAN
naturally occurring aromatic substances, e.g., the conversion of phenyl-alanine (labeled with deuterium) to tyrosine (Moss and Schoenheimer, 124); the conversion of stilbene to 4,4'-dihydroxystilbene in the rabbit is another example (Stroud, 176).
H Y P O T H E T I C A L C O N V E R S I O N in vivo OF T H E E S T R O G E N S0 TO C A R C I N O G E N S6
Ο C O O H
V
+I C H3
Pyruvic acid
/ \ / \ /
?I U I '
A A V
0 CHI
201
Cholanthrene 3-Desoxyequilenin
( L X X I X a ) (formula inverted)
JF?
equilenin ( X I V )
Jh
Estrone ( X I X )
β L X X I X a , X I V , X I X have actually been isolated from pregnancy urine,
* Adapted from Fieser (55).
The fact that a highly aromatic steroid such as 3-desoxyequilenin has been isolated from natural sources is highly significant in connection with that theory of carcinogenesis which postulates the formation of poly-nuclear substances akin to synthetic substances of demonstrated carcino-genic activity, e.g., 20-methylcholanthrene, by a "faulty" metabolism of the steroids (see Fieser, 56); 20-methylcholanthrene can be prepared synthetically from the innocuous bile acids and also from cholesterol.
More recently, Fieser (in 55) suggested that equilenin (or estrone) might undergo condensation with pyruvic acid in vivo, resulting eventually in the formation of 3-hydroxycholanthrene. (The latter substance has not been prepared synthetically.) In view of the fact that 3-hydroxy-20-methylcholanthrene, which has been prepared synthetically, is lacking in potency as a carcinogen (probably due to the presence of the 3-hydroxyl group), Fieser does not feel too sanguine with regard to the hypothetical formation of cholanthrene or its derivatives; it was difficult to visualize how the phenolic hydroxyl group could be eliminated in vivo. However, the recent isolation of 3-desoxyequilenin clearly removes, at any rate, this objection, although to be sure it is not known whether 3-desoxy-equilenin actually arises from 3-desoxy-equilenin or from some other substance.
3-Desoxyequilenin may therefore conceivably give rise to cholanthrene, a substance which has been synthesized and which is known to be very
X . THE CHEMISTRY AND METABOLISM OF THE ESTROGENS 389
potent as a carcinogen. The fact that only 3.6 ßg. of 3-desoxyequilenin is present per liter of pregnant mares' urine (calculated from the isolation data) is in itself interesting, for it indicates that significant steroid metabolites, e.g., carcinogens or procarcinogens, may be present in biologic material in amounts that would ordinarily escape detection.
F . R O L E OF LIVER IN ESTROGEN INACTIVATION
1. In Vitro Studies
a. Incubation Experiments. Zondek (201) first demonstrated a loss of estrogenic potency on incubation of estrogens with liver pulp; acid hydrolysis did not restore the estrogenic activity. Since the inactivating property of the liver is destroyed by heating and since cell-free extracts retain their inactivating capacity, Zondek concluded that the estrogen inactivation is probably an enzymic process; the enzyme responsible was designated an "estrinase." The observations of Zondek have been con-firmed by Engel and Rosenberg (54); the latter authors succeeded in obtaining aqueous extracts from beef liver which are capable of rapidly inactivating the native and synthetic estrogens. Zondek and Sklow (203) demonstrated that the reticuloendothelial cells of the liver play no part in the process of inactivation; the liver cell is believed to contain
"estrinase."
In the experiments by Heller et al. (76-78), α-estradiol was incubated with liver slices from the rat and rabbit; complete inactivation of the estrogen was observed and the estrogenic activity was not restored by acid hydrolysis. Partial inactivation was effected by kidney slices;
incubation with heart, lung, spleen, uterine, or placenta tissue did not decrease the estrogenic potency. Estrone was completely inactivated by rabbit liver slices and partially by rabbit kidney and rat liver. Estriol was only partially inactivated by rat liver and kidney and also by rabbit liver. It appears then that there are differences in the rate of inacti-vation of the various estrogens and that there are also differences among species in the rate of inactivation of the same estrogen. In vitro experi-ments by other workers further substantiate the latter conclusion.
Twombly and Taylor (183) showed that human liver tissue inactivates α-estradiol less rapidly than does liver tissue from mice and rats. In the experiments of Samuels and McCauley (153) the rate of estrogen inactivation was lowest when human liver mince was employed; inacti-vation was most rapid following incubation with liver mince obtained from the rat and mouse; other species studied were the rabbit and dog.
b. Perfusion Experiments. Israel et al. (94) perfused the heart-lung system of the dog with estrone; no inactivation was observed. On the
390 W I L L I A M H . P E A R L M A N
other hand, when a heart-liver-lung preparation was substituted, rapid inactivation ensued; histologic examination of the liver at the termina-tion of the experiment appeared to indicate that the functermina-tional state of the liver had been maintained. Schiller and Pincus (155) perfused rat liver with α-estradiol. They observed some inactivation which they suggested might be accounted for by conversion of α-estradiol to the less potent estrogens, estrone and estriol. If small amounts of α-estradiol were perfused, complete inactivation resulted in the course of five hours;
acid hydrolysis of the perfusate did not restore the activity. On the other hand, perfusion of rat heart with α-estradiol resulted in no loss of activity.
2. In Vivo Studies
a. Estrogen Experimentally Diverted into Hepatic Portal Circulation.
Golden and Sevringhaus (65) transplanted the ovaries of the rat to the mesentery and to the axillae; estrus did not occur in animals with the ovaries in the portal circulation but estrus did occur in those animals with transplants in the axillae. Biskind and Mark (13,14) implanted pellets of estrogen in the spleen of castrated rats. They observed no estrogenic effect as long as the spleen remained connected with the hepatic portal circulation. When the spleen was transplanted so that its venous blood flowed directly into the systemic circulation, the estrogenic effect of the implanted hormone became apparent. Biskind and Meyer (15) implanted estrone pellets in the spleen of male rabbits; degeneration of the testicles did not result as is the case when estrone is implanted sub-cutaneously. These observations were confirmed by Segaloff et al.
(160,162), who carried out experiments of a similar nature.
b. Liver Damage. In animals with liver damage induced by hepato-toxic agents or by dietary factors, there is an increased sensitivity of the
b. Liver Damage. In animals with liver damage induced by hepato-toxic agents or by dietary factors, there is an increased sensitivity of the