Wako nefa kit

Concentrations of the enzymes aspartate transaminase (AST) and alanine transaminase (ALT) as well as cholesterol, triglycerides, bile acids and β-hydroxybutyrate were measured by ASAP laboratories, Melbourne, Australia using a Dade Behring Dimension^® Xpand^® Integrated Chemistry System. Non-esterified fatty acids (NEFA) were measured using the WAKO NEFA kit (Wako Chemicals, Japan) according to manufacturer’s instructions.

Plasma glucose concentrations were measured using the YSI Glucose Analyzer. Plasma insulin was measured by radioimmunoassay. Plasma NEFA concentration was measured spectrophotometrically using a Wako NEFA kit (Wako Diagnostics). cAMP was measured by a cAMP ELISA kit (Enzo Life Science) in accordance with manufacturer protocol. PKA activity was measured by a PKA colorimetric activity kit (Invitrogen).

Blood glucose and β-hydroxybutyrate (BHB) concentrations were measured using the FreeStyle Optium glucometer (Abbot Diabetes Care Ltd., Oxfordshire, UK) [21 (link)]. Plasma samples were isolated weekly from 5 mL of freshly drawn blood as described in [17 (link)] and stored at −20 °C until analysis of non-esterified fatty acids (NEFA) concentration using the Wako NEFA kit (Wako Chemicals, GmbH, Neuss, Germany).
Before the experiment and at the end of each nutritional phase, 5 mL of blood samples were collected into serum vacutainers (CAT serum Sep Clot Activator, VACUETTE, Greiner Bio-One, Kremsmünster, Austria) via venipuncture for serum biochemical analysis. Blood samples were left to clot at room temperature for ~3 h and then centrifuged at 1800× g for 15 min at 20 °C. Biochemical measurements of the ensuing sera were done using a Cobas Integra 400 Plus (Roche Diagnostics, Rotkruez, Switzerland).