Measurement of Erythrocyte Inosine Triphosphate Pyrophosphohydrolase (ITPA) Activity by HPLC and Correlation of ITPA Genotype-Phenotype in a Caucasian Population

M. Shipkova
2005 Clinical Chemistry  
Inosine triphosphate (ITP) pyrophosphohydrolase (ITPA) catalyzes the pyrophosphohydrolysis of ITP/dITP and xanthosine triphosphate to prevent incorporation of unusual nucleotides into RNA and DNA. Important mutations leading to enzyme deficiency are 94C>A and IVS2 ؉ 21A>C. An association between ITPA 94C>A and adverse reactions during azathioprine treatment has been shown. To investigate the ITPA-phenotype, an HPLC procedure was developed and phenotype-genotype correlations were assessed.
more » ... ere assessed. Methods: The enzymatic conversion of ITP to inosine monophosphate (IMP) was terminated by perchloric acid and saturated dipotassium hydrogenphosphate. We quantified the product IMP at 262 nm after separation on an aqua perfect C18 column, using 20 mmol/L, pH 2.5 phosphate buffer. We also genotyped samples for ITPA 94C>A and IVS2 ؉ 21A>C using real-time fluorescence PCR. Results: The assay was linear to 3 mmol/L IMP (ϳ500 mol/(g Hb ⅐ h)) with an LLQ of 4 mol/L (ϳ0.5 mol/(g Hb ⅐ h)). With IMP-enriched samples, imprecision was <3.6% within a day and <4.9% from one day to the next, and the inaccuracy was <5.2%. Imprecision using pooled erythrocytes was 3.8% within a day and 7.5% from one day to the next. ITPA activity in 130 healthy controls ranged between <0.5 and 408 mol IMP/(g Hb ⅐ h). Mutant allele frequencies were 0.062 (94C>A) and 0.131 (IVS2 ؉ 21A>C). Using a cutoff at 125 mol,
doi:10.1373/clinchem.2005.059501 pmid:16384889 fatcat:nwlbfoviqfef5ekzfqurrw6nha