Proc. Natl. Acad. Sci. USA Vol. 92, pp. 1545-1549, February 1995 Biochemistry Long-lived testosterone esters in the rat WALTER BORG*, CEDRIC H. L. SHACKLETONt, SHAM L. PAHUJA*, AND RICHARD B. HOCHBERG*4 *Yale University School of Medicine, Department of Obstetrics and Gynecology, and the Comprehensive Cancer Center, New Haven, CT 06510; and tChildren's Hospital-Oakland Research Institute, Oakland, CA 94609 Communicated by Seymour Lieberman, St. Luke's-Roosevelt Institute for Health Sciences, New YorkA NY, November 7, 1994 ABSTRACT Over the past decade it has become increas- There are now numerous studies showing that fatty acid ingly clear that steroid hormones are enzymatically esterified esters of almost all of the families of steroid hormones are with fatty acids. These steroidal esters are the natural analogs synthesized in vitro (12, 13). However, there are only a few of synthetic esters that are used therapeutically. One such which demonstrate that steroid esters exist endogenously. family ofpharmacological steroids is the synthetic alkyl esters After the esters of the A5-33-hydroxysteroids were discovered of testosterone, androgens with great hormonal potency. We in the adrenal gland (1, 2), esters of dehydroisoandrosterone have investigated whether testosterone esters exist naturally and pregnenolone were found in blood (14) and pregnenolone by using the rat as a model. Most tissues of male rats, includ- esters were found in human ovarian follicular fluid (15). Fatty ing blood, have very little if any ester (quantified by immu- acid esters of reduced metabolites of progesterone and tes- noassay as a nonpolar saponifiable metabolite), but fat and tosterone have been isolated and characterized by MS, reveal- testes have sizable quantities, -3 ng of testosterone equiva- ing androsterone esters in human breast cyst fluid (16) and lents per g of tissue. Testosterone in fat averages 9 ng/g. The allopregnanolone (as well as pregnenolone) in bovine corpora fat from female rats and long-term (>2 weeks) castrated luteum (17). There are very few studies of endogenous esters males has no detectable testosterone ester. The presence of of biologically active steroids. Esters of E2 are present in testosterone esters was confirmed by GC/MS, which clearly limited concentration in blood (17), with much greater showed the presence of testosterone in the hydrolyzed ester amounts in fat (18). Relatively large amounts of E2 fatty acid fraction of fat from intact males but not long-term castrates. esters have been found in human ovarian follicular fluid, Upon castration, testosterone levels in the fat completely enabling their complete characterization (18). Other reports of disappear within 6 hr. To the contrary, it is not until 48 hr esters of active steroids are more tenuous. Esters of testoster- after castration that a measurable fall in the testosterone ester one (19,20) and the corticoids, cortisol and corticosterone (21, fraction was observed; even after 10 days a small amount of 22), have been reported to circulate in sizable amounts in ester is still present in the fat. These experiments demonstrate human blood. Although the report of corticoid esters in blood the existence of a previously unknown androgen with a po- was first published in 1960, there has been no independent tentially important physiological impact; testosterone esters, confirmation. The evidence for testosterone esters in blood is natural analogs of potent therapeutic agents, occur in the fat uncertain since neither long-term inhibition of steroidogenesis where they can serve as a reservoir of preformed androgen to nor castration reduced the concentration of the putative com- stimulate neighboring target tissues. pound (20). The existence of natural esters of androgens is of impor- Although fatty acid esters of sterols, such as cholesterol, have tance because, like estrogen esters, synthetic alkyl esters of been known for decades, the existence of naturally occurring androgens have been used therapeutically for decades due to fatty acid esters of steroids is a much more recent discovery. their high potency and prolonged action (23). Thus, biological In 1979, putative steroidal esters of the A5-3(3-hydroxysteroids, esterification of androgens, such as testosterone, would be pregnenolone (1), dehydroisoandrosterone, and 17a-hy- expected to have a dramatic effect on both the potency and the in life of the male hormone. This paper reports a study in which droxypregnenolone (2), were discovered the adrenal gland. the existence of fatty acid esters of testosterone (TL) in tissues They were named lipoidal derivatives to convey their nonpolar ofthe male ratwas examined and the hypothesis was tested that nature and their ability to be converted back to the parent steroid TL is long-lived when compared to testosterone. as a result ofmild hydrolytic procedures. They were subsequently identified as fatty acid esters (3). These findings raised the pos- sibility of the existence of similar esters of biologicaLly active MATERIALS AND METHODS steroids. Such compounds would be the natural analogs of syn- [1,2,6,7,16,17-3H]Testosterone (100 Ci/mmol; 1 Ci = 37 GBq) thetic steroidal esters that have been used pharmacologically as purchased from New England Nuclear was refluxed with alkali extremely potent and long-lived hormones. The well known ther- to remove labile 3H (24) and then purified by HPLC (25); final apeutic use of one such family of pharmacological steroid hor- specific activity was 92 Ci/mmol. Testosterone stearate and mone esters, the estrogens (4), led to experiments which showed [3H]testosterone stearate were synthesized by esterification that estradiol (E2) is biosynthetically converted into a lipoidal with stearyl chloride and purified exactly as described (25). derivative, LE2 (5), a nonpolar metabolite, which was identified Sprague-Dawley rats (Charles River Breeding Laboratories) as a family of C-17 fatty acid esters of E2 (6). Although LE2 is not between 3 and 5 months old were castrated under methoxyflu- estrogenic when esterified (7)-i.e., it does not bind to the es- rane (Metofane; Pitman-Moore, Washington Crossing, NJ) an- trogen receptor directly (8)-it is converted to E2 by esterase esthesia (day 0). At the stated times, animals were decapitated action. The fatty acid esters comprising LE2 are extremely long- while under Metofane anesthesia and the tissues were removed lived (9) and, thus, act as a reservoir of E2. They represent the and immediately frozen. Blood was obtained by cardiac puncture, most potent of the naturally occurring steroidal estrogens (10, placed on ice, and centrifuged at 4°C, and the serum was re- 11). moved. Fat from various areas was combinded and mixed. Ap- The publication costs of this article were defrayed in part by page charge Abbreviations: TL, testosterone ester; E2, estradiol; LE2, E2 lipoidal payment. This article must therefore be hereby marked "advertisement" in derivative; THF, tetrahydrofuran. accordance with 18 U.S.C. §1734 solely to indicate this fact. VTo whom reprint requests should be addressed. 1545 Downloaded by guest on October 1, 2021 1546 Biochemistry: Borg et al. Proc. Natl. Acad. Sci USA 92 (1995) proximately 250 mg of each tissue was weighed, transferred to a displaces '20% of the bound tracer. The blank (no tissue) test tube (16 x 125 mm) containing 2 ml of methanol and carried through the entire procedure was generally 7-10 pg. homogenized with two 10-sec bursts of a Polytron homogenizer The results of the RIA were corrected for the blank and for (Brinkmann). To correct for experimental losses, a representative recovery of the internal standard and normalized for the size fatty acid ester, 5000 cpm of [3H]testosterone stearate (13 pg; of the aliquot and the weight of the tissue. They are reported testosterone molar equivalent), was added as an internal standard as pg, molar equivalents, of testosterone (pg T equiv) per g of in 50 ,ul of ethanol. When testosterone was measured, an internal tissue. standard of [3H]testosterone, 5000 cpm, was also added. The To verify that the alumina chromatography eliminates tes- suspension was mixed, 4 ml of chloroform was added, and it was tosterone from the nonpolar TL fraction, two experiments Vortex mixed again. Two milliliters of water was added and, after were performed. In one, 100,000 cpm of [3H]testosterone was thorough mixing, the suspension was clarified by centrifugation at added to 250 mg of fat and the amount of radioactivity con- 1600 x g and the bottom organic layer was removed with a taminating the TL fraction was measured: it was - 100 cpm, or Pasteur pipette. The aqueous layer with the tissue residue was 0.1%. In another experiment, 10 ng of testosterone was added extracted again with 4 ml of the organic layer obtained by par- to 250 mg of female fat and the TL was analyzed. There was titioning chloroform/methanol/water in the ratio 2:1:1. The or- no measurable testosterone in the TL fraction (see Table 1). ganic extracts were combined, evaporated under N2, and then put When the fat samples from female rats or long-term castrated under vacuum at 50°C for 10 min to remove all traces of alcohol. males (>2 weeks) were analyzed by this protocol, the amount With serum, a slightly different extraction procedure was of TL measured was not significantly different than the blank used to increase the extraction yield. Four milliliters of freshly (see Fig. 2) and in most experiments it was the same as the distilled tetrahydrofuran (THF) was added to 1 ml of serum, followed by the 3H internal standards and then 1 ml of brine. blank. When TL in fat samples from male rats was analyzed, The solution was vigorously mixed and then centrifuged. The significant amounts were found (see Table 1 and Fig. 2). If the THF was removed and the aqueous residue was extracted TL fraction was not saponified (mock procedure) no testos- again with an additional 2 ml of THF.