title: Validation and application of a high-performance liquid chromatographic-based assay for determination of the inosine 5'-monophosphate dehydrogenase activity in erythrocytes.
abstract: Thiopurine drug monitoring has become an important issue in treating children with acute lymphoblastic leukaemia (ALL). In this population, a genetic polymorphism causes wide differences in the activity of thiopurine S-methyletransferase (TPMT)--the rate-limiting enzyme of the thiopurine degradation metabolism--leading to the necessity of drug dose adjustments. It is not yet known if similar differences exist in the inosine 5'-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205), the rate-limiting enzyme of the thiopurine synthesis. To test this, we established and validated a high-performance liquid chromatographic (HPLC)-based assay to determine the IMPDH enzyme activity in erythrocytes. The remarkable features of this assay are its simple erythrocyte separation/haemolysis and assay conditions and a distinct segregation of xanthosine 5'-monophosphate (XMP) from the clear supernatant after precipitation. The probes were processed without a time-consuming extraction and heating procedure and the assay demonstrated a good intra- and interday stability as well as a recovery rate of approximately 100%. The IMPDH enzyme activity was measured in erythrocytes of 75 children with diagnosis of ALL before starting antileukaemic therapy and their activity compared to those of 35 healthy adult controls. The measured enzyme activity was wide ranging in both groups. The individual enzyme activity differences observed in children with ALL might led to differences in the thionucleotide levels in those undergoing the standard thiopurine dose regimen.
authors.affiliation: Department of Surgery, Downtown Medical Centre, Ludwig-Maximilians-University, D-80336 Munich, Germany.
authors.forename: Philipe N
authors.initials: PN
authors.lastname: Khalil
authors.forename: Norbert
authors.initials: N
authors.lastname: Erb
authors.forename: Maurice N
authors.initials: MN
authors.lastname: Khalil
authors.forename: Gabriele
authors.initials: G
authors.lastname: Escherich
authors.forename: Gritta E
authors.initials: GE
authors.lastname: Janka-Schaub
mesh_terms: D000328:Adult; D016022:Case-Control Studies; D002648:Child; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007168:IMP Dehydrogenase / Q000097:blood*; D007700:Kinetics; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007168:IMP Dehydrogenase