title: Human liver cytosolic sulfotransferase 2A1-dependent dehydroepiandrosterone sulfation assay by ultra-high performance liquid chromatography-tandem mass spectrometry.
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 2.75min, 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 0.2pmol DHEA-S and the calibration curve was linear from 0.2 to 200pmol. 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 (20-100μg 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 2.5mM and 20μM, 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 (20-200μg cytosolic protein) or incubation time (5-30min). 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.
authors.affiliation: Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore.
authors.forename: Sumit
authors.initials: S
authors.lastname: Bansal
authors.affiliation: Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore; Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore. Electronic address: aikjiang.lau@nus.edu.sg.
authors.forename: Aik Jiang
authors.initials: AJ
authors.lastname: Lau
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D008099:Liver / Q000201:enzymology*; D015238:Sulfotransferases / Q000032:analysis* / Q000378:metabolism; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D015238:Sulfotransferases; C019998:dehydroepiandrosterone sulfotransferase; C028152:alcohol sulfotransferase
keywords: Dehydroepiandrosterone; Dehydroepiandrosterone sulfate; Human liver cytosol; Sulfotransferase 2A1; Ultra-high performance liquid chromatography–tandem mass spectrometry