Measurement of thiopurine S-methyltransferase activity in human blood samples based on high-performance liquid chromatography: reference values in erythrocytes from children.
BACKGROUND
Monitoring 6-thiopurine S-methyltransferase (TPMT; EC 2.1.1.67) activity is especially important when patients are treated with 6-thiopurine drugs, since severe bone marrow toxicity may be induced if patients have deficient TPMT activity.


METHODS
We have developed a method based on high-performance liquid chromatography (HPLC) for the measurement of TPMT activity in various cell types: erythrocytes (RBC), human peripheral blood mononuclear cells (pMNC) and human malignant lymphoblasts (Molt-F4). The enzymatic activity is measured by the amount of 6-methylmercaptopurine formed, using 6-mercaptopurine (6MP) as substrate and S-adenosylmethionine as co-substrate.


RESULTS
The K(m) values calculated for 6MP were 0.54 (RBC), 0.85 (pMNC) and 0.65 (Molt-F4 cells) mmol/L. The K(m) values for S-adenosylmethionine were 11.9 (RBC), 16.4 (pMNC) and 6.65 (Molt-F4 cells) micro mol/L. The assay variation was 8.2-17%. TPMT activity was determined in a control group of 103 children and young adults (44 female, 59 male). The values observed were (mean +/- standard deviation): female children and young adults, 15.1 +/- 4.8 pmol/10(7) cells per h (n = 44); male children and young adults, 15.8 +/- 6.4 pmol/10(7) cells per h (n = 59). No gender or age differences were found.


CONCLUSIONS
The HPLC-based method enables the rapid screening of TPMT activities in large groups of patients treated with 6-thiopurines.
Keizer-Garritsen
Jenneke J
JJ
Center for Pediatric Oncology, SE Netherlands, Department of Pediatrics, University Medical Center St Radboud, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Brouwer
Connie
C
Lambooy
Lambert H J
LH
Ter Riet
Patricia
P
Bökkerink
Jos P M
JP
Trijbels
Frans J M
FJ
De Abreu
Ronney A
RA
D001853:Bone Marrow / Q000187:drug effects; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D015122:Mercaptopurine / Q000031:analogs & derivatives* / Q000097:blood; D008780:Methyltransferases / Q000097:blood*; D012016:Reference Values; D015203:Reproducibility of Results; D013997:Time Factors
C010240:6-methylthiopurine; D015122:Mercaptopurine; D008780:Methyltransferases; C022745:thiopurine methyltransferase