Plasma Metabolite Profiling Workflow

Plasma samples were prepared following a procedure described previously^{7 (link)}. Please refer to Table S1 for patient characteristics. The samples were thawed at 4 °C. In all, 400 μl of ice-cold methanol/ethanol 50:50 [v/v] was added to 100 μl of plasma and mixed using a vortex mixer for 15 s. For the samples used for metabolic profiling the ice-cold methanol/ethanol mixture contained five internal standards (caffeine-d3 0.88 μmol/L, hippuric-d5 acid 0.22 μmol/L, nicotinic-d4 acid 0.88 μmol/L, octanoyl-l-carnitine-d3 0.22 μmol/L, l-phenyl-d5-alanine 0.44 μmol/L (all from C/D/N Isotopes, Pointe-Claire, Canada)). Next, these dilutions were incubated for 20 min at 4 °C and subsequently centrifuged at 18,600 × g for 15 min at 4 °C. In all, 350 μl of the supernatant was transferred into new tubes and dried in a centrifugal vacuum evaporator (Eppendorf). The sample was reconstituted in 100 ml 0.1% formic acid in deionised water/methanol 90:10 [v/v], mixed using a vortex mixer for 15 s and centrifuged at 18,600 × g for 15 min at room temperature. In all, 90 μl of the supernatant was transferred to an autosampler vial and used for LC-MS analysis.