Abstract
The estradiol-fatty acid esters are highly potent and long-lived estrogens that were first isolated and identified from in vitro biosynthetic experiments (Mellon-Nussbaum S., Ponticorvo, L., Schatz, F., and Hochberg, R. B. (1982) J. Biol. Chem. 257, 5678-5684). Other studies have indicated that these esters exist endogenously, but the evidence is indirect, resting solely on the presence of a nonpolar saponifiable metabolite. Although there are similar reports of other naturally occurring fatty acid esters of biologically active steroid hormones, likewise, none has been isolated and characterized. In this study we have found that follicular fluid from the ovaries of women stimulated with gonadotrophins contains relatively large amounts of a nonpolar saponifiable derivative of estradiol (approximately 10(-7)M), which we presumed to be fatty acid esters. Using a combination of chromatographic techniques we isolated these estradiol metabolites and identified them by mass spectral analysis. They are a mixture of five different estradiol-17 fatty acid esters. The amount of each present was determined by separating the esters by reversed phase high performance liquid chromatography and then quantifying each peak by radioimmunoassay and UV absorption. The esters of estradiol are predominantly unsaturated, with linoleate the most abundant comprising 43% of the total. The other esters are: palmitate (20%), arachidonate (19%), oleate (14%), and stearate (4%). Thus these studies conclusively demonstrate the existence of these unusually powerful estrogens.
Highlights
From the $Department of Obstetricsand Gynecology, Mass Spectroscopy Facility, and Comprehensive Cancer Center, Yale Uniuersitv School of Medicine, New Hauen, Connecticut 065510 and $Children’sHospital Oakland Research Institute, Oakland, kalifornia’94609
Other studies have indicated that these esters exist endogenously, butthe evidence isindirect,resting solely on the presence of a nonpolar saponifiable metabolite
In this study we have found that follicular fluid from the ovarieosf women stimulatedwithgonadotrophinscontainsrelatively large amounts of a nonpolar saponifiable derivativoef estradiol, which we presumed to be fatty acid esters
Summary
The LE, fractions were analyzed by FAB-MS on a ZAB-SE mass spectrometer (VG Analytical, Manchester, United Kingdom) model, using a cesium ion gun with amatrix of m-nitrobenzyl alcohol. The columns were washed with 5 ml of benzene, and thLe E, fraction was eluted with 12 mlof benzene/ethyl acetate (94:6) Under these conditions neither E, nor any of its conjugates is eluted from the column [16].The solvent was removedunder vacuum and theresidues saponified by dissolving them in 900 pl of methanol, followed bythe addition of 100pl of 10%aqueous KzC03. The ethyl acetate phases were combined, dried under vacuum, and theresidues dissolved in ethanol.An aliquot was counted for recovery of the internalstandard. To 500 pl of follicular fluid, 800 cpm of [3H]estradiol (3 pg, 10 fmol) was added in 25 p1 of ethanol to serve as an internal standard.Each of them was extracted twice with 2.5 volumes of diethyl ether, and the ether phases were combined and dried under vacuum. E, measured directly in the serum by RIA was corrected for recovery of the internalstandard
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