Original ArticleDetermination of 6-Thioguanosine Diphosphate and Triphosphate and Nucleoside Diphosphate Kinase Activity in Erythrocytes: Novel Targets for Thiopurine Therapy?Karner, Susanne*; Shi, Shaojun MD*†; Fischer, Christine PhD*; Schaeffeler, Elke PhD*; Neurath, Markus F MD‡; Herrlinger, Klaus R MD§; Hofmann, Ute PhD*; Schwab, Matthias MD*‖Author Information From the *Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart and University of Tuebingen, Tuebingen, Germany; ‡Department Internal Medicine III, University Hospital Erlangen, Erlangen, Germany; §Department of Gastroenterology, Hepatology and Endocrinology, Robert Bosch Hospital, Stuttgart, Germany; and ‖Department of Clinical Pharmacology, Institute of Experimental and Clinical Pharmacology and Toxicology, University Hospital Tuebingen, Tuebingen, Germany. Received for publication September 18, 2009; accepted December 23, 2009. †Present address: Department of Pharmacy of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China. Susanne Karner and Shaojun Shi contributed equally to this work. This work was in part supported by the Robert-Bosch Foundation (Stuttgart, Germany) and the Federal Ministry for Education and Research (BMBF, Berlin, Germany) grant 03 IS 2061C. Dr. Shaojun Shi was funded by the China Scholarship Council, China. Correspondence: Matthias Schwab, MD, Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Auerbachstraße 112, D-70376 Stuttgart, Germany (e-mail: email@example.com). Therapeutic Drug Monitoring: April 2010 - Volume 32 - Issue 2 - p 119-128 doi: 10.1097/FTD.0b013e3181d12f19 Buy Metrics Abstract 6-Thioguanine nucleotides are the sum of 6-thioguanosine 5′-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5′-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 ± 3.88 nmol·min−1·mg−1 for NDPK A versus 41.2 ± 1.05 nmol·min−1·mg−1 for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80°C is crucial for prevention of degradation of 5′-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol·min−1·mg−1 hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5′-phosphates levels has to be investigated in a prospective large-scale study. © 2010 Lippincott Williams & Wilkins, Inc.