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

Clin Biochem. 2001 Oct;34(7):543-9. doi: 10.1016/s0009-9120(01)00267-3.

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 microg/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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Chromatography, High Pressure Liquid / methods
  • Enzyme Inhibitors / pharmacology
  • Female
  • Humans
  • IMP Dehydrogenase / analysis*
  • Leukocytes, Mononuclear / enzymology*
  • Male
  • Middle Aged
  • Mycophenolic Acid / analogs & derivatives*
  • Mycophenolic Acid / pharmacology
  • Reproducibility of Results
  • Ribonucleotides / analysis

Substances

  • Enzyme Inhibitors
  • Ribonucleotides
  • xanthosine monophosphate
  • IMP Dehydrogenase
  • Mycophenolic Acid