UC Berkeley

We report a high-performance, liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay to quantify without derivatizaton dehyroepiandrosterone (DHEA), 17β-estradiol (E2), testosterone (T), and their sulfates in serum and tissues. This assay functions well with multiple adipose depots, a previously unattained analysis. To delipidate and facilitate recovery, tissues were homogenized in acetonitrile, and the homogenate was frozen. The supernatant was evaporated, resuspended in an aqueous acetate buffer, and extracted with hexane to separate free (unconjugated) from sulfated steroids. Sulfated steroids in the aqueous medium were then hydrolyzed with sulfatase and extracted with hexane. Each extract was analyzed separately. HPLC resolution combined with the sensitivity and specificity of MS/MS allowed quantification of DHEA, E2, and T with 10, 10, and 5 fmol lower limits of quantification and linear ranges to 1 pmol. Application of the method to mouse serum and tissues reveals ranges of DHEA, E2, and T and their sulfates, and tissue-specific differences in steroid profile, especially white versus brown adipose. In addition, marginal decreases of T in all tissues and considerable increases in DHEA in male iWAT and eWAT in response to a high-fat diet further strengthen the inference regarding the role of steroid metabolism in adipogenesis. This assay permits detailed studies of interactions between adiposity and sex steroids in serum and tissues, including adipose.