Measuring Muscle Protein Turnover Using Stable Isotopes

Studies were approved by the Institutional Animal Care and Use Committee (IACUC) and the Mayo Clinic Institutional Review Board. Mixed muscle protein samples were obtained from two independent studies. In the first study, healthy volunteers received a primed continuous infusion of L-[ring-¹³C₆]phenylalanine (0.75 mg/kg/h with 0.75 mg/kg priming dose) ^{18 (link)}. Muscle samples were obtained by percutaneous needle biopsy from the vastus lateralis muscle ^{8 (link)}. In the second study, male rats were injected with a flooding dose of L- [ring-¹³C₆]phenylalanine (15 mg/kg). Final sample set taken for analysis included muscle tissues from both experiments and with mixed-muscle-protein enrichment values ranging from 0.0091 molar percent excess (MPE) to 0.1321MPE (as measured by GC/C/IRMS). MPE represents [¹³C] label above the background (MPE=¹³C/¹²C +¹³C)×100). All samples were prepared as follows. Tissue samples weighing approximately 10mg were taken. Mixed muscle proteins were isolated as previously described ^{19 (link)}. The mixed muscle proteins precipitate from the isolation was hydrolyzed overnight at 110°C using 6M HCl. Liberated amino acids were purified using Biorad AG-50W×8 cation exchange resin. The final column eluent containing the amino acids was evaporated to dryness using a SpeedVac (Thermo, USA) and subsequently dissolved in 100μl of 0.1M HCL.