Ketone body assay

Blood glucose concentrations were measured through tail veil bleeding with glucose analyzer (GM9, Analox Instruments Ltd, UK). The concentrations of serum triacylglycerides (TAGs) and nonesterified fatty acids (NEFAs) were measured using TAG reagent (Sigma-Aldrich, T2449) and free glycerol reagent (Sigma-Aldrich, F6248), and a Lab Assay NEFA kit (Wako Pure Chemical Industries, Japan, 294-63601), respectively. Serum ketone body concentrations and serum alanine were determined by Ketone Body Assay (Sigma-Aldrich, MAK134) and Alanine assay kit (Sigma-Aldrich, MAK001), respectively. Milk lactose was measured by Lactose Assay Kit (Sigma-Aldrich, MAK017). Pyruvate carboxylase activity was determined by Pyruvate Carboxylase Activity Assay Kit (Boxbio, AKSU070M). Brain ATP levels were determined by ATP Assay Kit (Beyotime, S0026).

Total serum ketone body (TKB) level was obtained using a ketone body assay (Sigma-Aldrich, St. Louis, MO, USA). Briefly, 5 μl of blood sample and standard solution were added to each of the wells. The levels of acetoacetic acid (AcAc) and 3-hydroxibutyric acid (BOH) were determined using the reaction catalyzed by 3-hydroxybutyrate dehydrogenase (HBDH) in which the change in absorbance, measured at 340 nm, is directly related to AcAc and BOH concentration [17 (link)]. TKB concentration was calculated using the following formula: TKB = (AcAc) + (BOH).