Methods to quantify sex steroid hormones in bone: applications to the study of androgen ablation and administration

Yarrow JF, Conover CF, Lipinska JA, Santillana CA, Wronski TJ, Borst SE. Methods to quantify sex steroid hormones in bone: applications to the study of androgen ablation and administration. Am J Physiol Endocrinol Metab 299: E841-E847, 2010. First published August 24, 2010; doi: 10.1152/ajpendo.00384.2010.-Bone may contain an intraskeletal reservoir of sex steroids that is capable of producing biological effects. The purposes of these experiments were to 1) establish and validate methods to extract and measure intraskeletal sex hormones, 2) compare serum and intraskeletal sex hormone abundance, and 3) determine the impact of testosterone-enanthate administration and orchiectomy on intraskeletal sex hormone concentrations. Tibiae from male F344 rats were crushed, suspended in an aqueous buffer, disrupted mechanically and sonically, extracted with organic solvents, dried, and reconstituted in assay buffer appropriate for measurement of testosterone, dihydrotestosterone, and estradiol by immunoassay. Prior to extraction, bone homogenate was spiked with [H-3] testosterone, [H-3] dihydrotestosterone, or [H-3] estradiol, and >80% of each H-3-labeled sex hormone was recovered. Extracted bone samples were also assayed with and without known amounts of unlabeled sex hormones, and >97% of the expected hormone concentrations were measured. Administration of testosterone-enanthate increased intraskeletal testosterone 11-fold and intraskeletal dihydrotestosterone by 82% without altering intraskeletal estradiol (P < 0.01). Conversely, orchiectomy did not alter intraskeletal testosterone or estradiol but increased intraskeletal dihydrotestosterone by 39% (P < 0.05). In intact rats, intraskeletal testosterone and dihydrotestosterone were directionally higher than in serum, whereas intraskeletal estradiol was directionally lower than serum. Serum androgens were positively correlated with intraskeletal androgens (r = 0.74-0.96, P < 0.001); however, neither serum nor intraskeletal androgens nor serum estradiol were correlated with intraskeletal estradiol. We report the validation of a novel method for measuring intraskeletal sex hormones. Our findings demonstrate that the intraskeletal sex steroid reservoirs are modifiable and only partially influenced by circulating sex hormones.