Non-radioactive determination of inosine 5′-monophosphate dehydro-genase (IMPDH) in peripheral mononuclear cells

Abstract

Background: The immunosuppressive activity of mycophenolate mofetil (MMF) is based on the reversible inhibition of inosine 5′-monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA). It was the aim of this study to develop a nonradioactive method for specific measurement of IMPDH activity in isolated peripheral mononuclear cells (MNC). Methods: The procedure is based on the incubation of lysed MNC with inosine 5′-monophosphate (IMP) followed by direct chromatographic determination of produced xanthosine 5′-monophosphate (XMP). IMPDH activity was measured in MNC of MMF-treated patients and nontreated volunteers. Results: The within-run ( n = 10) and between-run ( n = 20) coefficients of variation (CV) for IMPDH activity were < 8% and < 10%, respectively. IMPDH activity in 60 healthy volunteers (19–63 yr) ranged from 4.72 to 32.92 nmol/h/mg protein (mean = 18.39 ± 6.24). The IC 50 for in vitro inhibition of IMPDH activity was about 2 to 3 μg/L. Application of a single dose of 1 g MMF in dialysis patients resulted in a significant inhibition (by 47–95%; p < 0.05) of IMPDH activity in lysed MNC. Conclusions: The proposed assay specifically and reliably measures IMPDH activity in MNC. The procedure is applicable to evaluate pharmacodynamic activity in MMF-treated patients. The observed interindividual variability of IMPDH activity may reflect pharmacodynamic differences in MMF-treated patients.