Wako autokit 3 hb

Blood samples were collected by venipuncture of the tail vessels using evacuated tubes containing EDTA as the anticoagulant at 4, 7, 10, and 14 DIM. Blood samples were placed on ice until processing. Within 6 h of collection, samples were centrifuged and plasma was harvested and frozen at -80°C until analysis. Commercial kits were used to determine plasma concentrations of NEFA (NEFA-C kit; Wako Diagnostics Inc., Richmond, VA; as modified by Johnson and Peters, 1993) and BHB (Wako Autokit 3-HB; Wako Diagnostics Inc.) as previously reported (Martinez et al., 2012) . Inter-and intraassay coefficients of variation were 6.3 and 4.5% for NEFA, and 8.5 and 3.6% for BHB, respectively. Plasma concentration of haptoglobin was determined using a colorimetric method based on peroxidase activity as previously described (Cooke and Arthington, 2013) . Inter-and intraassay coefficients of variation were 3.2 and 9.0%.

All assays followed the initial randomization with blocks, such that samples from each block were analyzed in the same assay. Plasma concentrations of NEFA (NEFA-C kit; Wako Diagnostics Inc., Richmond, VA; according to Johnson and Peters, 1993) and BHB (Wako Autokit 3-HB; Wako Diagnostics, Inc.) were analyzed using colorimetric enzymatic assays. The intra-and interassay coefficients of variation (CV) were, respectively, 3.4 and 8.4% for NEFA and 1.0 and 4.0% for BHB. Concentrations of glucose in plasma were determined by colorimetric continuous flow analysis (Autoanalyzer II, Seal Analytical, Southampton, UK) using a modification of the method described by Gochman and Schmitz (1972) . Plasma concentrations of tCa and tMg were analyzed by atomic absorption using a spectrophotometer equipped with Ca-and Mg-specific hollow cathode lamps (AAnalyst 200, Perkin-Elmer Inc., Waltham, MA) as described by Martinez et al. (2012) . Intra-and interassay CV were, respectively, 1.8 and 5.1% for tCa and 1.6 and 4.4% for tMg. Concentrations of tP were quantified in plasma using the molybdenum blue method (Quinlan and DeSesa, 1955) . The intra-and interassay CV were, respectively, 3.4 and 10.1%.

Plasma concentrations of NEFA (NEFA-C kit; Wako Diagnostics Inc., Richmond, VA; according to Johnson and Peters, 1993) and BHB (Wako Autokit 3-HB; Wako Diagnostics Inc.) were analyzed using colorimetric enzymatic assays. The intra-and interassay coefficients of variation (CV) were, respectively, 9.2 and 4.8% for NEFA, and 9.8 and 5.9% for BHB. Concentrations of glucose in plasma were determined by colorimetric continuous flow analysis (Autoanalyzer II, Seal Analytical, Southampton, UK) using a modification of the method described by Gochman and Schmitz (1972) ). The intra-and interassay CV were 4.5 and 4.7%, respectively. Details of assays of vitamin D metabolites in plasma and iCa in whole blood are presented in a companion paper (Rodney et al., 2018) .