Determination of inosine 5’-monophosphate dehydrogenase activity by high performance liquid chromatography in comparison with normalization methods

Mycophenolic acid (MPA) is a potent, selective and uncompetitive inhibitor of the inosine 5’ -monophophate dehydrogenase (IMPDH), which is the rate-limiting enzyme in the de novo synthesis of guanosine nucleotides being required during T and B lymphocyte proliferation. In this study, HPLC method was developed and validated to determine the IMPDH activity in human peripheral blood mononuclear cells (PBMCs) for the purpose of investigating the effect of MPAon lymphocytes. PBMCs were isolated from total blood samples. IMPDH activity in PBMCs was expressed according to two normalisation methods, namely as the ratio of produced xanthosine monophosphate (XMP) (micromoles) versus either adenosine monophosphate (AMP) (moles), or protein (mg) concentration per incubation time. IMPDH activities were assessed in 41 healthy volunteers. The total HPLC run time was 14 min. This method was linear in the range of 0.5-80 μmol/L for both AMP and XMP. Mean intra-assay and inter-assay precision and accuracy at quality control (QC) levels were > 94% for AMP and XMP. The recovery was > 75% for AMP and XMP. The lower limit of quantification of both AMP and XMP was 0.5 μmol/L. No interference was identified in the assay. Both IMPDH activity normalisation methods in our study displayed good and similar results, however, the protein normalisation method displayed slightly better reproducibility in our population cohort. Keyword: inosine 5’-monophosphate dehydrogenase activity, mycophenolic acid, HPLC, peripheral blood mononuclear cells