Inhibition of thiopurine S-methyltransferase activity by impurities in commercially available substrates: A factor for differing results of TPMT measurements

Objectives: Thiopurine S-methyltransferase (TPMT) activity, when measured in red blood cells (RBC) with a recently published TPMT activity assay using 6-thioguanine (6-TG) as substrate, could not be reproduced in another laboratory. We investigated factors which could influence the results of the TPMT activity measurement. Methods: We tested twelve 6-TG and four 6- mercaptopurine (6-MP) compounds from different suppliers as substrates and determined the enzyme kinetic parameters K(m) and V(max). Furthermore, we studied the influence of different 6-TG compounds on the affinity of the methyl donor S-adenosyl-L-methionine (SAM) to the TPMT enzyme. Results: All 6-TG products were of equal purity (declared > 98% by the supplier); this was ascertained by HPLC. However, the rate of methylation obtained following incubation with 6-TG from different suppliers ranged from 10% to 100% when incubated with the same RBC lysate. The lowest apparent K(m) value for a 6- TG was 22.3 gmol · 1^-1, while the product with the highest methylation rate showed a K(m) of 156 μmol · 1^-1. From these results we assume that there is a contaminant in some 6-TG products, which acts as a strong inhibitor of TPMT activity. Compounds possibly used for the synthesis of 6- TG (guanine, pyridine, 6-chloroguanine) did not affect the methylation rate. Thioxanthine, which is known to be a strong inhibitor of TPMT when added to the assay system to give a 2% contamination, reduced TPMT activity from 100% to 72%. Using 6-MP from different suppliers as substrate resulted in K(m) values ranging from 110 to 162 (max)mol · 1^-1 and V(max) values ranging from 54 to 68 nmol 6-MMP · g^-1Hb · h^-1. The K(m) value for the methyl donor SAM was similar to and independent from the thiopurine substrates tested (range 4.9-11 (max)mol · 1^-1 SAM). In contrast to other investigators, we found nonenzymatic S-methylation, which was negligible under our assay conditions (3% with 128 (max)mol · 1^-1 SAM), but could become relevant in experiments using higher SAM concentrations. Conclusions: TPMT enzyme activity determined with 6-TG as substrate may be strongly inhibited by a contaminant in some of the 6-TG lots distributed.