Reference intervals for thiopurine S-methyltransferase activity in red blood cells using 6-thioguanine as substrate and rapid non-extraction liquid chromatography

Ann Clin Biochem 2004;41:303-308
doi:10.1258/0004563041201617
© 2004 Royal Society of Medicine

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Original Articles


Loretta T Ford,
Sheldon C Cooper,
Matthew Jv Lewis and
Jonathan D Berg


Clinical Biochemistry Department, City Hospital, Sandwell and West Birmingham, Hospitals NHS Trust, Birmingham, B18 7QH, UK;
Department of Gastroenterology, City Hospital, Sandwell and West Birmingham, Hospitals NHS Trust, Birmingham, B18 7QH, UK;
Department of Gastroenterology, City Hospital, Sandwell and West Birmingham, Hospitals NHS Trust, Birmingham, B18 7QH, UK;
Clinical Biochemistry Department, City Hospital, Sandwell and West Birmingham, Hospitals NHS Trust, Birmingham, B18 7QH, UK


Background: Although widely used, thiopurine drugs have a narrow therapeutic index and treatment can result in life-threatening toxicity, the basis being pharmacogenetic variation in thiopurine metabolism by thiopurine S-methyltransferase (TPMT). We recentlydeveloped a modified phenotyping assay to determine TPMT activityin red blood cells. Here we describe improvements to the methodand establish reference intervals in a large prospective study.

Methods: A modified enzyme assay for TPMT activity is reported.It uses 6-thioguanine as substrate with heat treatment of theincubate to stop the reaction and precipitate protein priorto high-performance liquid chromatographic (HPLC) analysis.Measurement of the reaction product, 6-methylthioguanine (6-MTG),uses HPLC with fluorimetric detection.

Results: The assay shows excellent characteristics, with cleardiscrimination of patients who are deficient in TPMT activity(< 5 nmol 6-MTG per g Hb per h) from heterozygotes (5-24nmol 6-MTG per g Hb per h) and patients with normal activity(>25 nmol 6-MTG per g Hb per h).

Conclusion: A modified TPMT assay is described which is suitedfor routine analysis in a regional centre. The method overcomesthe need for extraction and has speeded up the chromatographicdetermination of 6-MTG, enabling large numbers of samples tobe analysed. A prospective study of 1000 individuals has establishedthe distribution of TPMT activity using the assay.

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