Janice M. Larner

The naturally occurring C-17 fatty acid esters of estradiol are long-acting estrogens.

C-17 fatty acid esters of estradiol are naturally occurring biosynthetic metabolites of estradiol. A representative component of this family of esters, estradiol-17-stearate, was studied in order to determine the estrogenic properties of these unusual hydrophobic steroids. Following the classical estrogen bioassay, a solution of this ester in oil was injected subcutaneously into immature rats once a day for 3 days. There was little effect on the uterus on the first day after the third injection. However, on subsequent days a large stimulation of uterine growth occurred. The course of this estrogenic effect was exactly opposite to that obtained with estradiol. In order to eliminate the possibility that this effect on the time course of estrogenic stimulation was caused by increased solubility of the hydrophobic esters in the carrier oil, the steroids were administered to adult ovariectomized animals in aqueous medium via a single intravenous injection. The uterotrophic response to estradiol was maximal at 12 h and was completely dissipated in 48-60 h. Estradiol-17-stearate produced a uterotrophic effect of twice the duration of estradiol. In the immature rat, aqueous intravenous injections of estradiol-17-stearate produced a greater uterotrophic effect than estradiol and this effect was still maximal 96 h later. In addition, this single injection of estradiol-17-stearate advanced the time of vaginal opening, a marker for puberty in the female rat. The mechanism of the prolonged estrogenic stimulation was investigated by studying the steroidal content of the uterus after injecting [3H]estradiol and [3H]estradiol-17 -stearate i.v. into immature rats. At 1 and 4 h there was significantly more radioactivity in the uteri of the [3H]estradiol treated animals. At later times (8 h and onwards) the total radioactivity in the uterus did not differ appreciably between the two groups. However at these later times, the amount of [3H]estradiol was far greater in the uteri of animals receiving [3H]estradiol-17-stearate. Consequently, the prolonged estrogenic effects of the endogenous C-17 fatty acid esters of estradiol are caused by the increased duration of the estrogenic signal. It is hypothesized that one of the roles of the fatty acid is to protect the steroid nucleus from metabolism and thereby prolong the life of the parent C18 steroid. Thus, the results of these experiments are consistent with the family of endogenous alkyl esters of estradiol having a physiological role as long-acting estrogens.

Steroidal fatty acid esters

Several years ago we discovered an unexpected family of steroidal metabolites, steroidal fatty acid esters. We found that fatty acid esters of 5-ene-3 beta-hydroxysteroids, pregnenolone and dehydroisoandrosterone are present in the adrenal. Subsequently, others have shown the existence of these non-polar 5-ene-3 beta-hydroxysteroidal esters in blood, brain and ovaries. Currently, almost every family of steroid hormone is known to occur in esterified form. We have studied the esters of the estrogens and glucocorticoids in some detail, and have found that these two steroidal families are esterified by separate enzymes. In a biosynthetic experiment performed simultaneously with estradiol and corticosterone, we established that the fatty acid composition of the steroidal esters is quite different. The corticoid is composed predominantly of one fatty acid, oleate, while the estradiol esters are extremely heterogeneous. Our studies have demonstrated that the estrogens are extremely long-lived hormones, that they are protected by the fatty acid from metabolism. They are extremely potent estrogens, with prolonged activity. Esterification appears to be the only form of metabolism that does not deactivate the biological effects of estradiol. We have demonstrated the biosynthesis of fatty acid esters of estriol, monoesters at both C-16 alpha and C-17 beta. They too are very potent estrogens. These fatty acid esters of the estrogens are the endogenous analogs of estrogen esters, like benzoate, cypionate, etc., which have been used for decades, pharmacologically because of their prolonged therapeutic potency. We have found that the estradiol esters are located predominantly in hydrophobic tissues, such as fat. Sequestered in these tissues, they are an obvious reservoir of estrogenic reserve, requiring only an esterase for activation. To the contrary the biological activity of the fatty acid esters of the glucocorticoid, corticosterone, is not different from that of its free parent steroid. We have shown that the rapid kinetics of its induction of gluconeogenic responses is caused by its labile C-21 ester which is rapidly hydrolyzed by esterase enzymes. While it appears that the physiological role of the estrogen esters may be related to their long-lived hormonal activity, the role of the other families of steroidal esters is not yet apparent. They, and perhaps the estrogen esters as well, must serve other purposes. Indeed they may serve important biological functions beyond those which we ordinarily associate with steroid hormones.

Synthesis of estradiol fatty acid esters by human breast tumors: fatty acid composition and comparison to estrogen and progesterone receptor content

The estradiol-17 beta-fatty acid esters are non-polar metabolites of estradiol, formed in many tissues, including human breast tumors. It has been shown in the rat that the synthesis of these esters is greatest in those tissues that respond to estrogen stimulation. Thus the possibility was explored that the biosynthesis of the estradiol esters in human breast tumors occurs mainly in those tumors that are estrogen sensitive; and thus that the synthesis of this family of non-polar metabolites of estradiol could be used as an additional marker for the identification of hormonally dependent tumors. However, the conversion of estradiol to the esters did not correlate with other indicators of estrogen responsiveness, the progesterone or estrogen receptors. Interestingly, the composition of the fatty acids in the estradiol-17-esters synthesized in the human tumors was markedly different from those originally identified in the bovine uterus. In the bovine uterus, the esters were predominantly unsaturated, 85%, while in this study the saturated esters were the major component. Since after systemic administration the saturated estradiol-17-esters have been found to be much longer-lived than the unsaturated esters, the biosynthesis of the relatively high proportion of saturated esters by human breast tumors may indicate a significantly prolonged duration for the estrogenic signal produced by endogenously formed estradiol esters. These esters formed and sequestered within the tumor cell, may serve as a preformed store of estradiol, which after enzymatic hydrolysis, can locally stimulate growth of tumors that are estrogen responsive.

Estrogenic action of estriol fatty acid esters

Recent studies suggest that, estriol, like estradiol, is biosynthetically esterified with fatty acids. We have synthesized the stearate estriol, at C-16 alpha, C-17 beta and the diester, C-16 alpha,17 beta and tested these D-ring esters for their estrogenic action both in vivo and in vitro, comparing them to estradiol, estriol and estradiol-17-stearate. None of the estriol esters bind to the estrogen receptor. They are only weakly estrogenic in a microtiter plate estrogen bioassay: stimulation of alkaline phosphatase in the Ishikawa endometrial cells. However, both estriol monoesters are extremely potent estrogens when injected subcutaneously (in aqueous alcohol) into ovariectomized mice. Compared to the free steroids, they produced a dramatically increased uterine weight with a greatly prolonged duration of stimulation. The 16 alpha,17 beta-diester also induced a protracted uterotrophic response, but the stimulation of uterine weight was comparatively low. Since the esters of estradiol and estriol do not bind to the estrogen receptor, their estrogenic signal must be generated through the action of esterase enzymes. These naturally occurring esters have the potential of being extremely useful pharmacological agents for long-lived estrogenic stimulation.