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


CONCLUSIONS
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.
Kröplin
T
T
Department of Pharmacology, Lübeck Medical University, Germany.
Weyer
N
N
Gutsche
S
S
Iven
H
H
D000483:Alleles; D002851:Chromatography, High Pressure Liquid; D004912:Erythrocytes / Q000737:chemistry / Q000201:enzymology*; D005260:Female; D014644:Genetic Variation; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D007700:Kinetics; D008297:Male; D008745:Methylation; D008780:Methyltransferases / Q000097:blood* / Q000235:genetics; D015203:Reproducibility of Results; D012436:S-Adenosylmethionine / Q000378:metabolism; D013379:Substrate Specificity; D013866:Thioguanine / Q000378:metabolism
D012436:S-Adenosylmethionine; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D013866:Thioguanine