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

Hannah Rieger; Patrik Schmidt; Elke Schaeffeler; Manabu Abe; Mira Schiffhauer; Matthias Schwab; Nicolas von Ahsen; Gabriela Zurek; Hartmut Kirchherr; Maria Shipkova; Eberhard Wieland

doi:10.1515/cclm-2017-0670

Article

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

Hannah Rieger , Patrik Schmidt , Elke Schaeffeler , Manabu Abe , Mira Schiffhauer , Matthias Schwab , Nicolas von Ahsen , Gabriela Zurek , Hartmut Kirchherr , Maria Shipkova and Eberhard Wieland

Published/Copyright: December 1, 2017

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From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 56 Issue 5

Abstract

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.

Keywords: 6-MP; drug therapy; genetic polymorphism; hematocrit; HPLC; pharmacogenetics; phenotype-genotype concordance; thiopurine; thiopurine methyltransferase (TPMT); whole-blood

Corresponding author: Dr. Hannah Rieger, Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Kriegsbergstr. 62, 70174 Stuttgart, Germany, Phone: +49-711-278 34850

aPresent address: University Medical Center Göttingen, Institute for Clinical Chemistry, Georg-August-University, 37099 Göttingen, Germany

Acknowledgments

We gratefully acknowledge Andrea Jarmuth for excellent technical assistance.

Author contributions: All the authors have accepted responsibility for the entire content of the submitted manuscript and approved submission. HPLC data are part of the bachelor thesis from Patrik Schmidt written in 2016 at the Reutlingen University, Germany.
Research funding: The work was supported by the Robert Bosch Foundation (Stuttgart, Germany) and European Commission Horizon 2020 UPGx grant 668353.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Received: 2017-7-28

Accepted: 2017-10-31

Published Online: 2017-12-1

Published in Print: 2018-4-25

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Articles in the same Issue

https://doi.org/10.1515/cclm-2017-0670

Keywords for this article

6-MP; drug therapy; genetic polymorphism; hematocrit; HPLC; pharmacogenetics; phenotype-genotype concordance; thiopurine; thiopurine methyltransferase (TPMT); whole-blood