title: High-performance liquid chromatographic assay of human red blood cell thiopurine methyltransferase activity.
abstract: An assay is described for the determination of red blood cell (RBC) thiopurine methyltransferase (TPMT) activity. TPMT S-methylates the antileukaemic drugs 6-mercaptopurine (6-MP) and 6-thioguanine and enzyme activity is inherited as a genetic trait. The assay is based on the TPMT-catalysed conversion of 6-MP to 6-methylmercaptopurine (methyl-MP) with non-radioactive S-adenosyl-L-methionine as the methyl donor. The methyl-MP metabolite is extracted into toluene as a phenyl-mercury adduct and back-extracted into 0.1 M HCl. Methyl-MP is quantitated by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet detection using a C18 Resolve cartridge and a mobile phase of methanol-water (20:80, v/v) with 100 mM triethylamine adjusted to pH 3.2 with orthophosphoric acid. There was a strong correlation between the HPLC assay and the established radiochemical assay (P < 0.0001). TPMT activities were measured by both methods in a population study of 111 children. There was no significant difference between the two frequency distribution histograms (P > 0.6).
authors.affiliation: Department of Medicine and Pharmacology, Royal Hallamshire Hospital, Sheffield, UK.
authors.forename: L
authors.initials: L
authors.lastname: Lennard
authors.forename: H J
authors.initials: HJ
authors.lastname: Singleton
mesh_terms: D000704:Analysis of Variance; D002138:Calibration; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D004912:Erythrocytes / Q000166:cytology / Q000201:enzymology*; D005260:Female; D006801:Humans; D016014:Linear Models; D008297:Male; D008780:Methyltransferases / Q000032:analysis / Q000097:blood* / Q000302:isolation & purification; D011786:Quality Control
chemical_list: D008780:Methyltransferases; C022745:thiopurine methyltransferase