Heide Daxecker1, Markus Raab, Mathias M Müller. 1. 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
Abstract
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. METHOD: 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.
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. METHOD: 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.
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