30 series amino acid analyzer

The amino acid content in collagen was determined according to Sotelo et al. (2016 ). The samples were hydrolysed at 110 °C for 24 h in 6 N HCl containing 0.1% w/v phenol. HCl was vaporized, and the residues were resuspended in 20 to 50 µl of 0.2 M sodium citrate buffer at pH 2.2. Norleucine was used as an internal standard. The samples were analysed using an automated amino acid analyser (Biochrom 30 series Amino Acid Analyzer, Cambridge, UK).

To assess the impact of the test meal on glucose and amino acid levels, plasma samples (2 mL) were taken from venous blood of volunteers at time 0 (baseline in the fasting state) and at 30 min-intervals during 120 min after meal ingestion. For serum glucose analysis, 1 drop of blood (ca. 1 µL) was assayed using the Accu-Chek Active Meter (Roche Diagnostics, Indianapolis, Indiana, USA), and a reflectometric glucose meter with plasma-corrected results. For amino acid analysis, venous blood was collected in EDTA monovettes, and plasma was immediately separated by centrifugation (4000 rpm for 8 min at 4 °C) and stored at −20 °C. Total free amino acids were determined through the Biochrom 30 series Amino Acid Analyzer [19 (link),51 (link)].