Non-radioactive determination of inosine 5'-monophosphate dehydro-genase (IMPDH) in peripheral mononuclear cells.
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.
Glander
P
P
Department of Internal Medicine, Nephrology, University Medical Center Charité, Campus Mitte, Humboldt University, Schumannstr. 20/21, 10098 Berlin, Germany. petra.glander@charite.de
Braun
K P
KP
Hambach
P
P
Bauer
S
S
Mai
I
I
Roots
I
I
Waiser
J
J
Fritsche
L
L
Neumayer
H H
HH
Budde
K
K
D000328:Adult; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D007168:IMP Dehydrogenase / Q000032:analysis*; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008297:Male; D008875:Middle Aged; D009173:Mycophenolic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D015203:Reproducibility of Results; D012265:Ribonucleotides / Q000032:analysis; D019820:Xanthine
D004791:Enzyme Inhibitors; D012265:Ribonucleotides; D019820:Xanthine; C011141:xanthosine monophosphate; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid