Plasma was analyzed for NEFA using an enzymatic colorimetric procedure, glucose by a colorimetric kit insulin by a bovine-specific sandwich ELISA, haptoglobin by a bovine-specific ELISA and BHBA using an enzymatic reaction.
Plasma concentrations of NA and nicotinamide were determined by HPLC with a method adapted from . Plasma samples were acidified with 200 µL of 1.5 M perchloric acid ,mixed and centrifuged at 10000 x g for 10 min. The supernatant was recovered and 200 µL of 1 M K2CO3 was added. The samples were mixed and then centrifuged at 10000 x g for 10 min, and the resulting supernatant was collected for injection. A Discovery BIO Wide Pore C18 column and Discovery BIO Wide Pore C18 guard column were used for all analyses. The photochemical reaction was carried out in a mobile phase consisting of… in a polytetrafluoroethylene tube wound around a black light Detection was carried out with a scanning fluorescence detector that operated at excitation and emission wavelengths of 322 nm and 380 nm, respectively. The injection volume was 20 µL and the flow rate was 0.8 mL/min.
Caffeine was analyzed using HPLC as described by Lakritz et al. Briefly 250 µL of plasma was added to 250 µL of 0.8 M perchloric acid and centrifuged at 14000 x g for 20 min at 21 °C . A 200- µL aliquot of clarified supernatant was transferred into an autosampler vial containing 10 µL of 4 M NaOH. Vials were capped and 20 µL was injected . The column and guard column were the same as described above, and the mobile phase was 20 mM phosphate buffer – acetonitrile,85:15,at flow rate of 1 mL/min. Absorbance was read at 273 nm using an Acutect 500 UV/Vis detector.