title: Thiopurine methyltransferase activity: new conditions for reversed-phase high-performance liquid chromatographic assay without extraction and genotypic-phenotypic correlation.
abstract: Thiopurine methyltransferase (TPMT) is a cytosolic enzyme involved in the metabolism of thiopurine drugs. A genetic polymorphism is responsible for large inter-individual differences observed in TPMT activity. We report a new HPLC technique, which avoids an extraction step and the use of radioactive reagents, based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). Intra- and inter-assay variation, within-day, within-run, between-day, and between-run variations showed high precision. The formation of 6-MMP was linear with respect to the lysate concentration and time. In a blinded assay of 61 samples, the results of HPLC method correlated with those of the radiochemical method (r2=0.82, P<0.0001). Using a cut-off point of 8.5 nmol/h/ml packed RBC, positive predictive value of HPLC was 100% for heterozygous patients. Because of the absence of extraction step, this new HPLC technique of TPMT activity determination reduces analysis variation and is time-saving. This rapid, sensitive, and reproducible method is suitable for routine monitoring of TPMT activity and for fundamental studies.
authors.affiliation: INSERM U490, Centre Universitaire des Saints Pères, 45 rue des Saints Pères, F-75270 Paris Cedex 06, France.
authors.forename: Dany
authors.initials: D
authors.lastname: Anglicheau
authors.forename: Sylvia
authors.initials: S
authors.lastname: Sanquer
authors.forename: Marie Anne
authors.initials: MA
authors.lastname: Loriot
authors.forename: Philippe
authors.initials: P
authors.lastname: Beaune
authors.forename: Eric
authors.initials: E
authors.lastname: Thervet
mesh_terms: D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D017931:DNA Primers; D005838:Genotype; D006801:Humans; D007700:Kinetics; D008780:Methyltransferases / Q000097:blood*; D010641:Phenotype; D011854:Radiochemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D017931:DNA Primers; D008780:Methyltransferases; C022745:thiopurine methyltransferase