Influence of mycophenolic acid on inosine 5'-monophosphate dehydrogenase activity in human peripheral blood mononuclear cells.
BACKGROUND
Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyses the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP). Thus, this enzyme plays an important role in the rate-limiting step of the de novo guanine nucleotide biosynthesis, making it a potent target for immunosuppressive drugs. Mycophenolic acid (MPA) is the most potent and specific inhibitor of IMPDH.


METHODS
IMPDH activity is determined via evaluation of XMP formation and the inhibitory influence of MPA in human peripheral blood mononuclear cells (PBMCs) is assessed by means of high-performance liquid chromatography (HPLC). For this objective, we have optimised a method based on solvent-generated ion exchange chromatography by cautiously varying mobile phase parameters.


RESULTS
The optimised method renders it possible to separate 18 analytes in 54 min in a single isocratic experiment and to measure the IMPDH activity in the lysate of human PBMCs in dependence on incubation time, substrate, co-substrate and inhibitor concentrations. In this way, we have determined the Michaelis-Menten constants K(M) and V(max) for IMP and beta-NAD+ and the inhibitor constant K(i) for MPA.


CONCLUSIONS
The chromatographic method presented in this report allows a rapid, reliable and reproducible quantification of IMPDH activity in PBMCs and therefore represents an attractive tool for the pharmacodynamic monitoring of the effects of MPA in patients under immunosuppressive therapy.
Daxecker
Heide
H
Institute of Laboratory Diagnostics and Ludwig-Boltzmann-Institute for Cardiothoracic Research, Kaiser-Franz-Josef Hospital, Kundratstrasse 3, A-1100 Vienna, Austria. other_hmd@yahoo.de
Raab
Markus
M
Müller
Mathias M
MM
D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors / Q000097:blood*; D007202:Indicators and Reagents; D007700:Kinetics; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology*; D009173:Mycophenolic Acid / Q000494:pharmacology*; D009243:NAD / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet
D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D009243:NAD; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid