title: An improved HPLC method for the quantitation of 3'-phosphoadenosine 5'-phosphate (PAP) to assay sulfotransferase enzyme activity in HepG2 cells.
abstract: Sulfotransferase-catalyzed (SULT-catalyzed) sulfation is responsible for hormone regulation and xenobiotic detoxification. In the current study, a sensitive and widely applicable method of reversed-phase HPLC-UV was developed for the determination of 3'-phosphoadenosine 5'-phosphate (PAP), a common product of different subtypes of sulfation reactions, in HepG2 cells. The analyte was separated on a ZORBAX Extend-C18 column with the mobile phase of methanol and water containing 75 mM KH(2)PO(4), 100mM NH(4)Cl, and 1mM 1-octylamine (pH 4.55), at a flow rate of 1.0 ml/min. The assay exhibited linearity over the range of 0.1-20 μM for PAP with a correlation coefficient of 0.9995. The total time per run was under 10 min. The intra- and inter-day precision was less than 7.2%, with accuracy in the range 82.6-102.0%. The method was applied to the determination of kinetic parameters K(m), V(m), and K(cat), of three different SULTs (SULT1A1, SULT2A1, and SULT1E1) in HepG2. This universal HPLC-UV method was easy to perform, economically feasible, and suitably efficient for the investigation of the enzyme kinetics of the SULT family using multiplex substrates.
authors.affiliation: Department of Pharmaceutics, School of Pharmaceutical Science, Peking University Health Science Center, Beijing 100191, China.
authors.forename: Jiaojiao
authors.initials: J
authors.lastname: Xu
authors.forename: Ye
authors.initials: Y
authors.lastname: Chen
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.forename: Zaiquan
authors.initials: Z
authors.lastname: Li
authors.forename: Chenai
authors.initials: C
authors.lastname: Wang
authors.forename: Tianyan
authors.initials: T
authors.lastname: Zhou
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
mesh_terms: D000244:Adenosine Diphosphate / Q000032:analysis*; D002138:Calibration; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007700:Kinetics; D012015:Reference Standards; D015203:Reproducibility of Results; D015238:Sulfotransferases / Q000378:metabolism*
chemical_list: D000244:Adenosine Diphosphate; C010840:adenosine 3'-phosphate-5'-phosphate; D015238:Sulfotransferases