Uni kit 3

Plasma glucose (Uni Kit III, 07367204; Roche) concentrations were analyzed with a COBAS-FARA semiautomatic analyzer (Roche). Insulin was analyzed by using a radioimmunoassay (Insulin RIA kit; LINCO Research Inc). Plasma (100 mL) for amino acid analyses was deproteinized on ice with 10 mg dry 5-sulphosalicylic acid, mixed, and the clear supernatant fluid was collected after centrifugation. Plasma amino acid concentrations were determined by using HPLC after precolumn derivatization with o-phthaldialdehyde [23 (link)]. For plasma enrichment measurements, plasma Phe and Tyr were derivatized to their t-butyldimethylsilyl derivatives and analyzed by using gas chromatography–mass spectrometry (GC-MS) (Agilent 6890N GC/5973N MSD; Agilent) by using selected ion monitoring of masses 336 and 341 for unlabeled and labeled (ring-²H₅) Phe, respectively; and masses 466, 468, and 470 for unlabeled and labeled (ring-²H₂ and ring-²H₄) Tyr, respectively [24 ]. Thereafter, ratios of labeled: unlabeled derivatives were analyzed by using gas chromatography–combustion isotope ratio mass spectrometry (FinniganMAT 252; ThermoFisher Scientific). Standard regression curves were applied in all isotopic enrichment analyses to assess the linearity of the mass spectrometer and to control for the loss of tracer.

At all time points, 8 mL blood was collected in pre-chilled tubes with 200 μL of 0.2 M EDTA (Sigma, Dorset, UK). After collection, blood samples were centrifuged immediately at 4 °C for 10 min at 1000 g and frozen at −80 °C until analysis. Additionally, 500 μL of the cell free plasma supernatant was combined with exactly 500 μL stabilization buffer at ambient temperature for the determination of EGCG concentration by HPLC. Plasma glucose (Uni Kit III, Roche, Basel, Switzerland), lactate, FFA (NEFA-C, Wako Chemicals, Neuss, Germany), TAG and free glycerol (148270, Roche Diagnostics, Indianapolis, IN, USA) concentrations were analyzed with a COBAS FARA semi-automatic analyzer (Roche). Insulin was analyzed by radioimmunoassay (Human Insulin RIA Kit, LINCO Research Inc, St. Charles, MO).
Glycerol, glucose and lactate concentrations from the microdialysates were measured by bioluminescence after enzymatic oxidation of L-Lactate53 (link). Ethanol concentrations were measured spectrophotometrically using a standard enzymatic technique (R-Biopharm AG, Darmstadt, Germany).