title: Inhibition of thiopurine S-methyltransferase activity by impurities in commercially available substrates: a factor for differing results of TPMT measurements.
abstract: OBJECTIVE
  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 micromol x l(-1), while the product with the highest methylation rate showed a Km of 156 micromol x 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 micromol x l(-1) and Vmox values ranging from 54 to 68 nmol 6-MMP x g(-1)Hb x h(-1). The Km value for the methyl donor SAM was similar to and independent from the thiopurine substrates tested (range 4.9-11 micromol-l(-1) SAM). In contrast to other investigators, we found non-enzymatic S-methylation, which was negligible under our assay conditions (3% with 128 micromol x 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.
authors.affiliation: Department of Pharmacology, Lübeck Medical University, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Kröplin
authors.forename: C
authors.initials: C
authors.lastname: Fischer
authors.forename: H
authors.initials: H
authors.lastname: Iven
mesh_terms: D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D015122:Mercaptopurine / Q000737:chemistry; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000097:blood*; D011786:Quality Control; D013866:Thioguanine / Q000737:chemistry
chemical_list: D004791:Enzyme Inhibitors; D015122:Mercaptopurine; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D013866:Thioguanine