Human liver cytosolic sulfotransferase 2A1-dependent dehydroepiandrosterone sulfation assay by ultra-high performance liquid chromatography–tandem mass spectrometry

    loading  Checking for direct PDF access through Ovid

Abstract

Sulfotransferase 2A1 (SULT2A1) is a major catalyst of the sulfation of dehydroepiandrosterone (DHEA) to dehydroepiandrosterone sulfate (DHEA-S) in human liver cytosol. However, there is a lack of a sensitive and fast analytical method for the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. Therefore, we developed and validated an ultra-high performance liquid chromatography–tandem mass spectrometric (UPLC–MS/MS) method to quantify DHEA-S and used it to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. DHEA-S and cortisol (internal standard) eluted at 2.95 and 2.75 min, respectively. Negative multiple reaction monitoring was used to quantify DHEA-S (m/z 367.3 → 97.0) and cortisol (m/z 407.2 → 331.3). No interfering peaks were observed in blank samples. The lower limit of quantification was 0.2 pmol DHEA-S and the calibration curve was linear from 0.2 to 200 pmol. The intra-day and inter-day accuracy and precision was <11.7%. DHEA-S in the quality control samples was stable at room temperature, 4 °C, and −20 °C. The cytosolic matrix (20–100 μg cytosolic protein) did not affect DHEA-S quantification. Our UPLC–MS/MS method was applied to optimize the human liver cytosolic SULT2A1-dependent DHEA sulfation assay. The optimal levels of MgCl2 and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) cofactor were 2.5 mM and 20 μM, respectively. Reducing agents, including 2-mercaptoethanol and DL-dithiothreitol, did not affect the enzyme activity. A linear relationship existed between DHEA sulfation and amount of human liver cytosol (20–200 μg cytosolic protein) or incubation time (5–30 min). This UPLC–MS/MS approach is safer, easier, and faster than existing radiometric-based sulfotransferase enzyme assays, and it is the first UPLC–MS/MS method for determining SULT2A1-dependent DHEA sulfation in human liver cytosol.

Related Topics

    loading  Loading Related Articles