Determination of inosine triphosphate pyrophosphatase phenotype in human red blood cells using HPLC.
BACKGROUND
Thiopurine drugs, widely used in cancer chemotherapy, inflammatory bowel disease, and autoimmune hepatitis, are responsible for common adverse events. Only some of these may be explained by genetic polymorphism of thiopurine S-methyltransferase. Recent articles have reported that inosine triphosphate pyrophosphatase (ITPase) deficiency was associated with adverse drug reactions toward thiopurine drug therapy. Here, we report a weak anion exchange high-performance liquid chromatography method to determine ITPase activity in red blood cells and to investigate the relationship with the occurrence of adverse events during azathioprine therapy.


METHODS
ITPase activity was assessed by the enzymatic conversion of inosine triphosphate (ITP) to inosine monophosphate (IMP). The reaction was stopped by heating for 3 minutes at 120°C. IMP, inosine diphosphate, and ITP were analyzed on a Hypersil APS-2 column, a weak anion exchange phase that exhibits both ionic and hydrophobic properties.


RESULTS
The chromatographic method reported allows the analysis of IMP, inosine diphosphate, and ITP in a single run in <12.5 minutes. The method was linear in the range 5-1500 μmole/L of IMP. Intraassay and interassay precisions were <5% for red blood cell lysates supplemented with 50, 500, and 1000 μmole/L IMP. Km and Vmax evaluated by Lineweaver-Burk plot were 677.4 μmole/L and 19.6 μmole·L·min, respectively. The frequency distribution of ITPase from 73 patients was investigated.


CONCLUSIONS
The method described is useful to determine the ITPase phenotype from patients on thiopurine therapy and to investigate the potential relation between ITPase deficiency and the occurrence of adverse events.
Institut des Sciences Parmaceutiques et Biologiques, Pharmacie Clinique, Pharmacocinétique et Évaluation du Médicament, Université de Lyon, Université Lyon 1, Lyon, France.
Antony
A
Citterio-Quentin
Jean-Paul
JP
Salvi
Roselyne
R
Boulieu
D001379:Azathioprine / Q000009:adverse effects / Q000627:therapeutic use; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007293:Inosine Triphosphate / Q000097:blood / Q000737:chemistry; D010641:Phenotype; D011755:Pyrophosphatases / Q000097:blood* / Q000737:chemistry; D000097630:Inosine Triphosphatase
D001379:Azathioprine; D007293:Inosine Triphosphate; D011755:Pyrophosphatases; D000097630:Inosine Triphosphatase