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Determination of Thiopurine Methyltransferase Activity in Human Erythrocytes by High-Performance Liquid Chromatography: Comparison With the Radiochemical Method

2001; Lippincott Williams & Wilkins; Volume: 23; Issue: 5 Linguagem: Inglês

10.1097/00007691-200110000-00007

1536-3694

César Menor, Jesús Fueyo, Óscar Escribano, Carlos Cara, María Dolores Fernández-Moreno, Irene D. Román, Luis Gonzales Guijarro,

Folate and B Vitamins Research

The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate with recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/100 μL packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine and S-adenosyl- l -methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radiochemical method, a linear correlation was obtained between both procedures (y = 0.99 x + 0.33;x-axis, radiochemical assay;y-axis, HPLC-linked assay;r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and simpler than the previously described ones.