Development and validation of a UPLC-UV method for the quantification of thiopurine methyltransferase enzyme activity in human erythrocytes.

Thiopurines are drugs widely used for the treatment of autoimmune conditions, inflammatory bowel disease or acute lymphoblastic leukemia. Determination of thiopurine methyltransferase activity (TPMT), a major determinant of thiopurines toxicity, has been suggested before implementing thiopurine treatment. An ultraperformance liquid chromatography (UPLC) method was developed and validated for the quantification of TPMT enzyme activity based on the conversion of 6-mercaptopurine (6-MP) to 6-methylmercaptopurine (6-MMP) using S-adenosyl-L-methionine (SAM) as methyl donor in red blood cell lysates (RBC). This method was improved from a previous laborious high performance liquid chromatography (HPLC) method, using a lower volume of injection and with a shorter runtime. After incubation and protein precipitation 6-MMP was separated on a HSS-T3 (2.1 × 50 mm, 1.8 μm) column and monitored by UV detection (290 nm). A change on the organic solvent used to dissolve 6-MP resulted in a reduction of interference by endogenous or non-enzymatic methylated 6-MMP. A full validation of the 6-MMP assay was performed according to the FDA and EMA guidelines. The method was linear from 0.125 to 2 nmol/mL, with acceptable values of accuracy and precision. The method was applied in 106 patients treated with thiopurines whose TPMT activity was previously quantified by HPLC. Evaluation through Bland-Altman plot showed that TPMT activities were in agreement between both methods.

Illamola SM ，Echaabi AK ，Mazeron C ，Deshayes S ，Loriot MA ，Pallet N ... -  《-》

Comparison of three methods for measuring thiopurine methyltransferase activity in red blood cells and human leukemia cells.

Thiopurine efficacy is partly reflected by the genetic polymorphism of the thiopurine methyltransferase (TPMT) enzyme, which is responsible for variation in the metabolism, toxicity and therapeutic efficacy of the thiopurines azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Determination of TPMT activity before administration of thiopurines is thus crucial for individualized dosing in order to prevent toxicity in TPMT deficient individuals. These individuals must be treated with markedly lower (eg, 5-10% of the standard) doses of the prescribed medications. This paper describes a comparison of three different methods for the quantification of TPMT activity in red blood cells (RBC) and cultured human cell lines. We succeeded to perform the measurement of TPMT activity in a minimum amount of 1×10(6) cultured cells with an HPLC-UV system modified and optimized in our laboratory. The TPMT activity was linearly correlated with the cell concentration of the cultured cell line in a range of 1-10×10(6) cells. A significant correlation of determination of TPMT activity in RBC between radiometric detection by HPLC, classic radiochemical detection and UV detection by HPLC, was observed, correlation coefficient (r) were 0.72 and 0.73, respectively. The within-day and day-to-day coefficients of variation of the HPLC-UV-based method were 8% and 16%, respectively. The evaluation of the methods was demonstrated by studying the TPMT activity in RBC isolated from 198 patients, as well as in MOLT4 leukemic cell line and its sub-cell lines with acquired resistance to 6-MP and 6-TG.

Karim H ，Appell ML ，Fotoohi A 《-》

Validation of a high-performance liquid chromatography method for thiopurine S-methyltransferase activity in whole blood using 6-mercaptopurine as substrate.

Rieger H ，Schmidt P ，Schaeffeler E ，Abe M ，Schiffhauer M ，Schwab M ，von Ahsen N ，Zurek G ，Kirchherr H ，Shipkova M ，Wieland E ... -  《-》

Determination of thiopurine methyltransferase activity in human erythrocytes by high-performance liquid chromatography: comparison with the radiochemical method.

Menor C ，Fueyo JA ，Escribano O ，Cara C ，Fernández-Moreno MD ，Román ID ，Guijarro LG ... -  《-》

Comparison of 6-mercaptopurine with 6-thioguanine for the analysis of thiopurine S-methyltransferase activity in human erythrocyte by LC-MS/MS.

Thiopurines (TPDs) are first-line drugs in treating neuromyelitis optica spectrum disorders (NMOSD). Evaluation of thiopurine S-methyltransferase activity (TPMT), a major determinant of TPD toxicity, before TPD treatment using 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) as substrate was suggested. However, the equivalent of the two substrates in TPMT activity evaluation was unknown, and an alternative substrate was required in TPMT activity evaluation in patients who were already taking 6-MP or 6-TG. Before evaluating the agreement of 6-MP and 6-TG in TPMT activity measurement in patients with NMOSD, the affinity of the two substrates for the active center of TPMT should be established. A computer-based simulation indicated that 6-MP and 6-TG had similar affinities for the two active sites of TPMT. According to the guidelines, an LC-MS/MS method was developed and validated to evaluate the TPMT activity in human erythrocyte hemolysate using 6-MP or 6-TG as substrates via 1 h incubation at 37°C. The method was applied in 81 patients with NMOSD. Evaluated by Bland-Altman plot, 6-methylmercaptopurine and 6-methylthioguanine represented TPMT activities were in agreement with each other. Further studies are warranted to confirm the results.

Mei S ，Li X ，Gong X ，Zhang X ，Li X ，Yang L ，Zhu L ，Zhou H ，Liu Y ，Zhou A ，Zhang X ，Zhao Z ... -  《-》