β hbdh

Plasma β-HB and acetoacetate were measured by an automated colorimetric assay as previously described (9 (link)). Briefly, for acetoacetate, 25 μL plasma was mixed with 330 μL fresh reagent [Tris buffer, pH 7.0, 100 mmol/L; 20 mmol sodium oxamate/L; 0.15 mmol NAD(H)/L, and 1 U β-HB dehydrogenase (β-HBDH)/mL]. For β-HB, the reagent was Tris buffer (pH 9.0; 20 mM sodium oxamate, 1 mmol NAD/L; and 1 U β-HBDH/mL). Tris, oxamic acid, DL-β-HB sodium salt, Li-acetoacetate standard, and NAD were purchased from Sigma; NAD(H) was purchased from Roche; and β-HBDH was purchased from Toyobo. The change in absorbance at 340 nm between 15 and 120 s after the addition of the reagent was measured by using an automated clinical chemistry analyzer (Dimension Xpand Plus; Siemens). The assay was calibrated with freshly diluted standards from frozen aliquots of a 10-mmol/L standard of Li-acetoacetate or DL-β-HB sodium salt, which are stable at −20°C for 2 and 6 mo, respectively. Calibrations and quality controls were performed for each assay to ensure the precision of the kits (CV between tests: 5% ± 1%). Plasma glucose, lactate, TGs, cholesterol (Siemens Medical Solutions USA, Inc.), and FFAs (Randox Laboratories Ltd.) were analyzed by using commercial kits. Glycated hemoglobin was measured by HPLC-723G7, a fully automated HPLC instrument-reagent system (Tosoh Bioscience).