title: Determination of thiopurine methyltransferase activity in human erythrocytes by high-performance liquid chromatography: comparison with the radiochemical method.
abstract: The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate with recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/100 microL packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine and S-adenosyl-L-methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radiochemical method, a linear correlation was obtained between both procedures ( y = 0.99x + 0.33; x-axis, radiochemical assay; y-axis, HPLC-linked assay; r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and simpler than the previously described ones.
authors.affiliation: Department of Biochemistry and Molecular Biology, Universidad de Alcalá, Alcalá de Henares, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Menor
authors.forename: J A
authors.initials: JA
authors.lastname: Fueyo
authors.forename: O
authors.initials: O
authors.lastname: Escribano
authors.forename: C
authors.initials: C
authors.lastname: Cara
authors.forename: M D
authors.initials: MD
authors.lastname: Fernández-Moreno
authors.forename: I D
authors.initials: ID
authors.lastname: Román
authors.forename: L G
authors.initials: LG
authors.lastname: Guijarro
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics; D001379:Azathioprine / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D016014:Linear Models; D015122:Mercaptopurine / Q000031:analogs & derivatives* / Q000097:blood; D008780:Methyltransferases / Q000097:blood*; D011854:Radiochemistry; D012436:S-Adenosylmethionine / Q000097:blood; D013866:Thioguanine / Q000097:blood
chemical_list: D000964:Antimetabolites, Antineoplastic; C010240:6-methylthiopurine; D012436:S-Adenosylmethionine; D015122:Mercaptopurine; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D013866:Thioguanine; D001379:Azathioprine