Thiopurine S-methyltransferase activity in human erythrocytes: a new HPLC method using 6-thioguanine as substrate.

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.