Validation of a high-performance liquid chromatography method for thiopurine S-methyltransferase activity in whole blood using 6-mercaptopurine as substrate
| Autor: | Elke Schaeffeler, Manabu Abe, Hannah Rieger, Matthias Schwab, Mira Schiffhauer, Eberhard Wieland, Maria Shipkova, Patrik Schmidt, Nicolas von Ahsen, Hartmut Kirchherr, Gabriela Zurek |
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| Rok vydání: | 2018 |
| Předmět: |
Genotype
Clinical Biochemistry Hematocrit 030226 pharmacology & pharmacy High-performance liquid chromatography Substrate Specificity 03 medical and health sciences 0302 clinical medicine medicine Humans Chromatography High Pressure Liquid Whole blood Chromatography medicine.diagnostic_test Thiopurine methyltransferase biology Mercaptopurine Chemistry Biochemistry (medical) Substrate (chemistry) Methyltransferases General Medicine Healthy Volunteers 3. Good health Phenotype 030220 oncology & carcinogenesis Toxicity biology.protein Pharmacogenetics medicine.drug |
| Zdroj: | Clinical Chemistry and Laboratory Medicine (CCLM) |
| ISSN: | 1437-4331 1434-6621 |
| DOI: | 10.1515/cclm-2017-0670 |
| Popis: | Background:Variation in metabolism, toxicity and therapeutic efficacy of thiopurine drugs is largely influenced by genetic polymorphisms in the thiopurine S-methyltransferase (TPMT) gene. Determination of TPMT activity is routinely performed in patients to adjust drug therapy.Methods:We further optimized a previously established high-performance liquid chromatography (HPLC) method by measuring TPMT activity in whole blood instead of isolated erythrocytes, which is based on conversion of 6-mercaptopurine to 6-methylmercaptopurine using S-adenosyl-methionine as methyl donor.Results:The simplified TPMT whole-blood method showed similar or better analytical and diagnostic performance compared with the former erythrocyte assay. The whole-blood method was linear for TPMT activities between 0 and 40 nmol/(mL·h) with a quantification limit of 0.1 nmol/(mL·h). Within-day imprecision and between-day imprecision were ≤5.1% and ≤8.5%, respectively. The optimized method determining TPMT activity in whole blood (y) showed agreement with the former method determining TPMT activity in erythrocytes (x) (n=45, y=1.218+0.882x; p>0.05). Phenotype-genotype concordance (n=300) of the whole-blood method was better when TPMT activity was expressed per volume of whole blood (specificity 92.2%), whereas correction for hematocrit resulted in lower genotype concordance (specificity 86.9%). A new cutoff for the whole-blood method to distinguish normal from reduced TPMT activity was determined at ≤6.7 nmol/(mL·h).Conclusions:This optimized TPMT phenotyping assay from whole blood using 6-MP as substrate is suitable for research and routine clinical analysis. |
| Databáze: | OpenAIRE |
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