Supporting Information

Abstract

The details of quantifying the L-[ring 2 H 5 ]-phenylalanine enrichment in plasma and in the skeletal muscle bound protein compartment as well as amino acid assays are as below. Sample preparation: 100 µl plasma was deproteinized with 10µl trichloroacetic acid solution (TCA, 50% w/v), centrifuged at 50,000g at 4°C for 5 min. 20 µl of the supernatant was used for the analyses of the plasma tracer (L-[ring 2 H 5 ]-phenylalanine) tracee (natural occurring phenylalanine) ratio (t/T). Muscle biopsies, for t/T analyses of protein bound phenylalanine, were homogenized and deproteinized in 250 µl 0.098M TCA, containing 0.1 gram glass beads using a mini-BeadBeater (Biospec Products, Bartlesville, OK, USA) for 30 seconds and then centrifuged at 8000g at 4°C for 5 min. The protein pellet was isolated, washed 3 times with 0.3 M TCA. The proteins were hydrolyzed with 1 ml 6N hydrochloride solution at 100°C for 24 hours. Then 5 ml HPLC grade water was added and the samples were dried down with a vacuum concentrator (SpeedVac, Thermoscientific, Waltham, MA, USA). Samples were then dissolved in 20µl water per mg wet tissue, centrifuged at 50,000g 4°C for 5 min. 20µl of the supernatant was used for the t/T analyses of protein-bound phenylalanine with LCMS. LCMS analyses: t/T ratio of phenylalanine in plasma and muscle protein were determined on a fully automated LC-ESI-MS system (QTrap 5500 MS; AB Sciex, Foster City, CA, USA) with ExpressHT Ultra LC (Eksigent Div., AB Sciex, Foster City, CA, USA). Supernatant (20 µl) of centrifuged TCA deproteinized plasma or muscle protein hydrolysate was added to a 0.1 N hydrochloric acid containing internal standard (20 µl) of a stable isotopomer. For t/T measurement, the internal standard contained L-[D8]-valine. Samples and internal standard containing enriched standards with t/T ratios of L-[ring-2