MEASUREMENTS OF BIOACTIVE ESTROGENS IN THE RAT BRAIN AND SERUM AFTER PREMARIN TREATMENT

Purpose: To investigate estrogen uptake into brain tissues after the administration of conjugated equine estrogens (Premarin®) using an animal model. Methods: Ovariectomized Spague-Dawley rats were treated with a single dose of Premarin® (1 mg/kg body weight, i.v.). The blood was collected by cardiac puncture, clotted on ice and centrifuged in order to obtain serum samples. Tissue homogenates were prepared in pH 7.4 phosphate buffer. Samples were spiked with internal standards, and estrogens were extracted with ethyl acetate. The organic layer collected was dried under a nitrogen stream, and the residue was derivatized with dansyl chloride. Quantification of bioactive estrogens was performed by isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: To assess biodistribution of selected bioactive estrogens formed after Premarin® injection, we developed an LC-MS/MS assay for the simultaneous detection and quantification of estrone (E1), 17ɑ-estradiol (ɑE2), and 17β-estradiol (βE2). We also validated the method according to US FDA guidelines. The assay revealed that all three endogenous estrogens appeared in the serum. As expected, E1 was present at the highest concentration, 1892±73 pg/mL when measured 30 min after drug administration, since E1-sulfate is the major constituent of Premarin®. The corresponding ɑE2 and βE2 concentrations were 82±13 pg/mL and 1134±75 pg/mL, respectively. At the same time, Premarin® treatment not only produced significant serum estrogen levels, but 3554 ± 109 pg/g of E1, 237 ± 25 pg/g of ɑE2, and 633 ± 25 pg/g of βE2 were also measured in rat brain samples 30 min after drug treatment. Conclusions: The injection of Premarin® not only increased circulating serum estrogen levels, but significant brain uptakes of bioactive estrogens have also been demonstrated. However, our study is the first to show that serum estrogen concentrations are not indicative of brain levels thereby implicating the role of localized steroid-metabolizing enzymes.