Measurement of PBMC IMPDH enzyme activity

Measurement of IMPDH activity from lysed PBMCs was based on previously published HPLC methods for the quantification of IMPDH activity in PBMCs 16, 29. In brief, after thawing at 4°C, the PBMC pellets were resuspended in 900 μl ice‐cold Millipore water and insoluble fragments of disrupted cells were removed by centrifugation at 15 800 g at room temperature (RT) for 2 min. The PBMC lysate was used for protein content (20 μl) and IMPDH enzymatic activity (50 μl) determinations. The measurement of lysate protein concentration was performed with Bio‐Rad Protein Assay Reagent (Bio‐Rad Laboratories, California, USA) using bovine serum albumin as standard according to the manufacturer's protocol. IMPDH activity in PBMCs was determined from the conversion of inosine 5′‐monophosphate (IMP) to xanthosine 5′‐monophosphate (XMP) based on methods described previously 16, 29. Briefly, the IMPDH incubation mixture (pH 7.4) consisted of 1 mmol l^–1 IMP, 0.5 mmol l^–1 NAD⁺, 40 mmol l^–1 NaH₂PO₄ and 100 mmol l^–1 KCl. The enzymatic reaction was initiated by the addition of 50 μl of the PBMC lysate to 120 μl of reaction mixture and incubated at 37°C for 2.5 h. After incubation, the reaction was terminated by adding 20 μl of 4 mol l^–1 ice‐cold HCIO₄, vortexing for 10 s, and the deproteinised solution was centrifuged at 15 800 g at RT for 2 min. Subsequently, 170 μl of supernatant was neutralised by adding 17 μl of 5 mol l^–1 ice‐cold K₂CO₃, vortexing for 10 s, and storing the samples for 30 min at –80°C. After thawing and centrifugation at 15 800 g at RT for 2 min, 25 μl of the supernatant was immediately injected onto the HPLC column for analysis.

Chromatographic detection of XMP production was achieved using a Synergi HydroRP 80A column (4 μmol l^–1, 250 × 3 mm; Phenomenex, Lane Cove, NSW, Australia) maintained at 45°C on an Agilent HPLC system, with two mobile phases: A) 50 mmol l^–1 potassium phosphate (KH₂PO₄) and 7 mmol l^–1 TBA buffer (pH 5.5); and B) 100% MeOH. The mobile phases were pumped at a flow rate of 0.7 ml min^–1 using a semigradient programme of: 94% A and 6% B for 0–13.0 min; 80% A and 20% B for 13.1–23.0 min; and 95% A and 5% B for 23.1–40.0 min. Injection volume was 25 μl with ultraviolet detection at a wavelength of 254 nm.

Specificity was tested in control incubations containing IMP in the absence of cosubstrate NAD⁺, or containing NAD⁺ in the absence of IMP. No endogenous XMP was detected in samples incubated without IMP or NAD⁺, and no interfering peaks were observed at the retention time of XMP. Linearity of XMP formation with protein content was confirmed for protein concentrations up to 2.7 mg ml^–1 and time of incubation up to 200 min. IMPDH activity was expressed as XMP produced (nmol) per incubation time (h) per mg protein (nmol h^–1 mg protein^–1).