OBJECTIVES: To develop a non-radioactive assay to measure thiopurine S-methyltransferase (TPMT) activity. The assay was used to study the distribution of TPMT activity in a healthy German population. METHODS: The assay is based on the conversion of 6-thioguanine (6-TG) to 6-methylthioguanine (6-MTG) using non-radiolabelled S-adenosyl-L-methionine (SAM) as the methyl donor. At the end of the incubation period (60 min) 6-MTG is extracted into chloroform/2-propanol and quantitated by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection at Ex 315 nm and Em 390 nm. RESULTS AND DISCUSSION: The method is rapid, sensitive and reproducible, with an interassay CV of 6.7% (quality control sample with TPMT activity of 43 nmol 6-MTG x g(-1) Hb x h(-1)) and thus suitable for routine monitoring of TPMT activity. The TPMT activity of 219 healthy German blood donors showed the known trimodal distribution with a range from 1.3 to 68.3 nmol 6-MTG x g(-1) Hb x h(-1) with a median value of 38.8 nmol 6-MTG x g(-1) Hb x h(-1). When the cut-off value for intermediate to high activity was set at 23.5 nmol 6-MTG x g(-1) Hb x h(-1), 14.1% belonged to the group with intermediate and 83.6% to the group with high TPMT activity. Five individuals had a very low TPMT activity of <2 nmol 6-MTG x g(-1) Hb x h(-1). Genetic analysis revealed that these persons were found either homozygote for the variant allele *3A (n = 3) or they were compound heterozygotes for the variant alleles *3A/*3C (n = 2). With these alleles for low TPMT activity they would run an increased risk of myelosuppression in case of treatment with standard doses of thiopurine drugs.
OBJECTIVES: To develop a non-radioactive assay to measure thiopurine S-methyltransferase (TPMT) activity. The assay was used to study the distribution of TPMT activity in a healthy German population. METHODS: The assay is based on the conversion of 6-thioguanine (6-TG) to 6-methylthioguanine (6-MTG) using non-radiolabelled S-adenosyl-L-methionine (SAM) as the methyl donor. At the end of the incubation period (60 min) 6-MTG is extracted into chloroform/2-propanol and quantitated by reversed-phase high-performance liquid chromatography (HPLC) with fluorescence detection at Ex 315 nm and Em 390 nm. RESULTS AND DISCUSSION: The method is rapid, sensitive and reproducible, with an interassay CV of 6.7% (quality control sample with TPMT activity of 43 nmol 6-MTG x g(-1) Hb x h(-1)) and thus suitable for routine monitoring of TPMT activity. The TPMT activity of 219 healthy German blood donors showed the known trimodal distribution with a range from 1.3 to 68.3 nmol 6-MTG x g(-1) Hb x h(-1) with a median value of 38.8 nmol 6-MTG x g(-1) Hb x h(-1). When the cut-off value for intermediate to high activity was set at 23.5 nmol 6-MTG x g(-1) Hb x h(-1), 14.1% belonged to the group with intermediate and 83.6% to the group with high TPMT activity. Five individuals had a very low TPMT activity of <2 nmol 6-MTG x g(-1) Hb x h(-1). Genetic analysis revealed that these persons were found either homozygote for the variant allele *3A (n = 3) or they were compound heterozygotes for the variant alleles *3A/*3C (n = 2). With these alleles for low TPMT activity they would run an increased risk of myelosuppression in case of treatment with standard doses of thiopurine drugs.
Authors: B A Kaskas; E Louis; U Hindorf; E Schaeffeler; J Deflandre; F Graepler; K Schmiegelow; M Gregor; U M Zanger; M Eichelbaum; M Schwab Journal: Gut Date: 2003-01 Impact factor: 23.059
Authors: David Gurwitz; Cristina Rodríguez-Antona; Katherine Payne; William Newman; Javier P Gisbert; Emma Gutiérrez de Mesa; Dolores Ibarreta Journal: Eur J Hum Genet Date: 2009-02-18 Impact factor: 4.246