Measurement of IMPDH activity

We used a linear trapezoidal model to calculate manually the AEC_0–12h, including six points (0, 0.5, 1, 2, 6 and 12 h). A validated nonradioactive HPLC method by Glander et al. 29 was used to measure the IMPDH activity. The rate of xanthosine 5′‐monophosphate (XMP) production by IMPDH from peripheral blood mononuclear cells (PBMCs) was measured and was normalized to the measured intra‐cellular adenosine monophosphate (AMP). PBMCs were isolated with Ficoll‐Paque (Greiner Bio‐One, Alphen a/d Rijn, The Netherlands) according to the manufacturer's protocol. The aliquot was resuspended in 250 μl ice‐cold water and stored at −20°C after one washing step. To 50 μl mononuclear cells lysate 130 μl reaction buffer with IMP and β‐NAD⁺ was added to start the incubation of enzyme reaction. The reaction tubes were placed in a Thermomixer (Eppendorf Ltd, Cambridge, UK) and were incubated at 37°C and 1 × g for 2.5 h. The reaction was stopped by adding 20 μl ice‐cold perchloric acid (4 mol l⁻¹). After centrifugation, 10 μl of 5 mol l⁻¹ potassium carbonate was added to 170 μl supernatant to neutralize the solution. The supernatant was transferred into HPLC for the determination of AMP and of produced XMP. Enzyme activity was expressed as produced XMP (μmol) per time unit (s) per amount of AMP (mol). The precision of the IMPDH activity assay was 6.6–11.9% (quality control samples) and the precision for patient samples ranged from 0.6 to 3.4%.