Plasma Metabolites and Oxidative Status

Glucose (enzymatic-colorimetric method, sensitivity: 0.06 mmol/L) and urea (kinetic method, sensitivity: 0.056 mmol/L) concentrations were determined in plasma with an automatic analyzer (Gernon, RAL S.A, Barcelona, Spain). The mean intra- and interassay CV were 1.5% and 1.9% for glucose and 3.2% and 4.8% for urea, respectively. Plasma BHB (kinetic enzymatic method, sensitivity: 0.100 mmol/L) and NEFA (colorimetric method, sensitivity: 0.072 mmol/L) were determined using Randox kits (Randox Laboratories Ltd., Country Antrim, UK). The mean intra- and interassay CV were respectively 3.3% and 3.7% for NEFA and 6.2% in both cases for BHB. Oxidative status was determined using MDA as a biomarker of lipid peroxidation. This indicator was determined by liquid chromatography using an Acquity UPLC H-Class liquid chromatograph (Waters, Milford, MA, USA) equipped with a silica-based bonded phase column (Acquity UPLC HSS PFP, 100 mm × 2.1 mm × 1.8 μm, Waters), an absorbance detector (Acquity UPLC Photodiode Array PDA eλ detector, Waters) and a fluorescence detector (2475 Multi λ Fluorescence Detector, Waters). The quantification of MDA was done by fluorescence detection at ʎ_excitation = 530 nm and ʎ_emission = 550 nm following the chromatographic conditions described in Bertolín et al. (2019) (link). The mean intra- and interassay CV were 4.6% and 7.3%, respectively.