Thiopurines are an important group of active substances used, among others, in the treatment of acute lymphoblastic leukemia (ALL). Many enzymes are involved in their metabolism, leading to the formation of active metabolites, 6-thioguanosins. The deactivation pathway, in which the enzyme thiopurine S-methyltransferase (TPMT) is a key player, leads to the formation of methylated products. The activity of this enzyme is impaired in about 11% of individuals and even undetectable in 0,3%. Reduced TPMT activity is due to genetic single nucleotide polymorphisms, the most common of which are polymorphisms in TPMT*3 alleles. Knowledge of TPMT activity in patients on thiopurine therapy is important to determine the right dose, which will achieve the desired therapeutic outcome while avoiding serious adverse effects. As part of our Master's thesis, we evaluated a method for measuring TPMT activity using 6-thioguanine (6-TG) as a substrate. We were interested in whether there is a correlation between the results of the new method (based on the formation of 6 methylthioguanine (6-MTG)) and the already established method using 6 mercaptopurine (6-MP) as a substrate. We examined how TPMT activity differs between groups of individuals with different TPMT genotypes. In ALL patients on thiopurine therapy, we investigated, whether there is a correlation between TPMT activity and cumulative thiopurine doses, 6-MP doses, methotrexate doses, leukocyte counts and liver transaminase activities. With the usage of reversed-phase high-performance liquid chromatography, we measured TPMT activity in 109 haemolysates from healthy volunteers from the Estonian Genome Bank using 6-TG as a substrate. The enzyme activity was also measured in haemolysates from ALL patients via the formation of 6-MTG. The TPMT activity measured via the formation of 6-MTG correlated with the TPMT activity measured via the formation of 6-methylmercaptopurine (6-MMP). After classifying healthy volunteers from the Estonian Genome Bank into TPMT*3C/TPMT*3A heterozygotes and non-variant homozygotes, we discovered that TPMT activity differs between subjects with different TPMT genotypes and is significantly lower in the group with the variant allele than in the group without the variant allele. In ALL patients, we found no statistically significant association between TPMT activity and cumulative doses of thiopurines, 6-MP, MTX or liver transaminase activity. There is a weak but statistically significant association between TPMT activity and leukocyte counts, which we consider not clinically relevant. Our results confirm that the method for measuring TPMT activity using 6-TG as substrate is rapid, sensitive, and suitable for routine monitoring of TPMT activity. The method will need some further optimization to improve overall reproducibility before it can be introduced into routine clinical practice, and further validation of the method is still needed.
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