ISSN:
1432-1041
Keywords:
Key words Thiopurine methyltransferase polymorphism
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
,
Medicine
Notes:
Abstract 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 Km and Vmax . 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 Km value for a 6-TG was 22.3 μmol · l−1, while the product with the highest methylation rate showed a Km of 156 μmol · l−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 Km values ranging from 110 to 162 μmol · l−1 and Vmax values ranging from 54 to 68 nmol 6‐MMP · g−1Hb · h−1. The Km value for the methyl donor SAM was similar to and independent from the thiopurine substrates tested (range 4.9–11 μmol · l−1 SAM). In contrast to other investigators, we found non-enzymatic S-methylation, which was negligible under our assay conditions (3% with 128 μmol · l−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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002280050630
