Whole blood was collected in non-fasted state by dried blood sampling (DBS), a biosampling method where capillary blood was obtained by a fingerprick using a disposable lancet. Complete DBS cards were allowed to dry overnight (maximum 12 h) in a room without light exposure, before being stored in a sealed aluminium bag with a desiccant pack at -20 degrees Celsius (°C) until analysis. Methylmalonic acid (MMA) was analyzed from the whole blood (VITAS™ Analytical Services, Norway). A sufficient volume of blood for analysis of MMA was available for 65 participants. MMA was extracted from 8 mm DBS punches using an isotope dilution GC-MS assay. Four punches were eluted in an aqueous alkaline solution containing stable isotope-labelled MMA prior to derivatization using propyl chloroformate. The liquid extraction of derivatized MMA was performed before injection into the GC-MS system. The GC-MS system utilized was an Agilent 6890 GC (Agilent Technologies, Palo Alto, CA, USA) equipped with an Agilent 5973 N mass-selective detector operated in EI-SIM mode. Separation was performed on a Zebron ZB-AAA GC column supplied by Phenomenex Inc (Torrance, CA, USA) and quantification was performed against a 5-point standard curve prepared from standards with known concentrations.
Haemoglobin (Hb) was measured from the same finger prick as the whole blood for DBS, using Hemocue® HB801 Analyzer and Microcuvettes (Sweden). In total 164 participants provided blood for analysis of Hb.