Immunoassay analyser

The amount of β-hydroxybutyrate (CAS No. 150-83-4, Sigma-Aldrich, USA) in serum was measured by LC-MS/MS using an Ultimate 3000 UHPLC and Q-Extractive Orbitrap Plus (Thermo Fisher Scientific, USA). Standard concentrations ranged from 0.625 to 50 μg/mL. To measure the total ketone (acetoacetate + β-hydroxybutyrate) concentration, the ketone body assay kit (Sigma-Aldrich, USA) was used according to the manufacturer’s instructions. Absorbance was measured at 340 nm using a Tecan GENios multi-functional plate reader (Infinite®F500, TecanGrödig, Austria). Serum insulin levels were measured using an electrochemiluminescence immunoassay with an Insulin Reagent kit (Roche, Germany) and an immunoassay analyser (Roche, Japan). Insulin sensitivity was 0.20 μU/mL. Serum glucose was determined using an enzymatic method (Asan Diagnostics, Korea) according to the manufacturer’s instructions. Serum tumour necrosis factor-α (TNF-α) levels were measured using enzyme-linked immunosorbent assay CyMAX human TNF-α (AbFrontier, Korea) according to the manufacturer’s instructions. The absorbance was measured at 450 nm on a microplate reader (Infinite® 200 PRO, Tecan Trading AG, Switzerland).

Serum samples were used for the determination vitamin D and two parameters of bone turnover: osteocalcin (OC), a marker of bone formation and β-CrossLaps (β-CTx) marker of bone resorption. The electrochemiluminescence immunoassay “ECLIA” was used for the determination of markers using commercially kits for OC (N-MID Osteocalcin, Cobas-Roche diagnostic Ltd., Basel, Switzerland) and Beta CrossLaps (β-CTx, Cobas-Roche diagnostic Ltd., Basel, Switzerland). Total vitamin D (25-Hydroxy vitamin D) was analysed by the immunoassay analyser (Cobas-Roche, Basel, Switzerland). The measuring range for vitamin D, OC and CTXs is 3.00–70.0 ng/mL, 0.500–300 ng/mL and 10–6000 pg/mL, respectively.