Determination of thiopurine methyltransferase activity in human erythrocytes by high-performance liquid chromatography: comparison with the radiochemical method.
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 microL 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 = 0.99x + 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.
Menor
C
C
Department of Biochemistry and Molecular Biology, Universidad de Alcalá, Alcalá de Henares, Spain.
Fueyo
J A
JA
Escribano
O
O
Cara
C
C
Fernández-Moreno
M D
MD
Román
I D
ID
Guijarro
L G
LG
D000964:Antimetabolites, Antineoplastic / Q000493:pharmacokinetics; D001379:Azathioprine / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016903:Drug Monitoring; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D016014:Linear Models; D015122:Mercaptopurine / Q000031:analogs & derivatives* / Q000097:blood; D008780:Methyltransferases / Q000097:blood*; D011854:Radiochemistry; D012436:S-Adenosylmethionine / Q000097:blood; D013866:Thioguanine / Q000097:blood
D000964:Antimetabolites, Antineoplastic; C010240:6-methylthiopurine; D012436:S-Adenosylmethionine; D015122:Mercaptopurine; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D013866:Thioguanine; D001379:Azathioprine