Improved Assay for the Nonradioactive Determination of Inosine 5'-Monophosphate Dehydrogenase Activity in Peripheral Blood Mononuclear Cells

Abstract

Mycophenolic acid (MPA) inhibits the enzyme inosine 5'-monophosphate dehydrogenase (IMPDH). Thus, the measurement of IMPDH activity could serve as a specific pharmacodynamic (PD) tool for monitoring MPA therapy. At present, however, monitoring of pharmacokinetic parameters is preferred over that of PD parameters because, in general, PD assays are labor-intensive and poorly reproducible. Currently, cell count or protein concentration is widely accepted as methods to normalize enzyme activity. In the present study, we have attempted to further improve a method for the determination of IMPDH activity to increase the robustness and reproducibility of the IMPDH activity assay itself, without making the assay more labor-intensive. Therefore, several aspects of the IMPDH method were investigated regarding their influence on the reproducibility and also modified to increase the feasibility and consistency of the assay. The isolation of peripheral blood mononuclear cells (PBMCs) of whole blood samples was found to be the most variable step. Normalization on cell count is labor-intensive and at the same time has a poor reproducibility. Determination of the protein content in cell extracts is impaired by contamination with extracellular proteins and non-PBMCs. Alternatively, the intracellular substance adenosine monophosphate (AMP) was investigated to normalize the newly generated xanthosine monophosphate. Among various subject groups, no significant differences in mean AMP concentration were found. To simplify the procedure, PBMCs were diluted to a fixed volume after isolation from sample of whole blood, and the IMPDH activity was normalized to the AMP concentration quantified in the same high-performance liquid chromatography run as xanthosine monophosphate was quantified. The within-run and total imprecision (coefficient of variation) ranged from 4.2% to 10.6% and from 6.6% to 11.9%, respectively. In conclusion, the modified method described here for the measurement of IMPDH activity can be used reliably in multicenter trials and in longitudinal studies to evaluate the additional value of any PD monitoring among a diversity of patients treated with MPA.

Inosine monophosphate dehydrogenase activity in paediatrics: age-related regulation and response to mycophenolic acid

“…The median IC 50 values in our study are substantially higher and correspond with MPA-C max rather than MPA predose levels. This difference may be explained by the use of an improved assay for determination of IMPDH activity [22] in the present study that, in contrast to the former assay [32], is no longer potentially influenced by the MNC count that could lead to falsely low measurements of IMPDH activity.…”

“…The newly generated XMP was normalised to the adenosine monophosphate (AMP) concentration of the cells, which was quantified in the same high-performance liquid chromatography run as XMP. A detailed description and validation of this procedure have been previously published [22]. Using AMP concentrations for normalisation of the newly generated XMP results in a smaller variability when compared with that of other normalisation factors used previously, such as protein concentrations and cell counts.…”

“…Using AMP concentrations for normalisation of the newly generated XMP results in a smaller variability when compared with that of other normalisation factors used previously, such as protein concentrations and cell counts. The within-run and within-day variabilities of the method are <11% [22]. Furthermore this modified assay consists of only one analytical operation without the potential burden of another previous operation (e.g.…”

Consensus Report on Therapeutic Drug Monitoring of Mycophenolic Acid in Solid Organ Transplantation

“…Glander et al (123) developed an HPLC method that measures the rate of xanthine monophosphate production by peripheral blood mononuclear cells under controlled in vitro conditions, assuming that this reflects the target immunologic cells involved in graft rejection. Recently, this assay was further optimized (124). Typically, considerable interpatient variability in IMPDH activity combined with relatively small intraindividual variability has been found, and part of the variability is related to a polymorphism in the IMPDH type II gene (125).…”

Pharmacokinetics and Pharmacodynamics of Intensified versus Standard Dosing of Mycophenolate Sodium in Renal Transplant Patients

“…Blood (collected in heparin-containing tube) was taken before the morning dose and at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, and 12 hours before dose. Peripheral mononuclear cells were isolated within 24 hours, and prepared cell extracts were frozen and analyzed in a central laboratory (20). The pharmacodynamic parameters derived from each enzyme activity-time profile were calculated using WinNonLin 5.2 software and included the areaunder-enzyme-activity time curve (AEC; calculated by linear trapezoid method) and maximum (A max ), minimum (A min ), and average (A avg ) daytime enzyme activity during dosing interval.…”