Agilent 7890a gc 5975c

Plasma glucose and insulin concentrations were measured using commercially available kits (GLUC3, Roche, Ref: 05,168,791 190, and Immunologic, Roche, Ref: 12,017,547 122, respectively). Plasma amino acid concentrations were quantified using UPLC-MS as described previously (1 (link)). Plasma phenylalanine concentrations and L-[ring-¹³C₆]-phenylalanine enrichments were determined by GC-MS (Agilent 7890A GC/5975C; MSD) as described previously (17 (link)). For measurement of muscle protein-bound L-[ring-¹³C₆]-phenylalanine enrichments, muscle protein was first extracted as described previously (2 (link)). Next, muscle protein-bound L-[ring-¹³C₆]-phenylalanine enrichments were measured using GC-isotope ratio MS (GC-IRMS) as described previously (17 (link)).

Plasma glucose and insulin concentrations were analyzed using commercially available kits (GLUC3, Roche, Ref: 0,516,8791 190, and Immunologic, Roche, Ref: 12,017,547 122, respectively). Plasma amino acid concentrations and enrichments were determined by GC-MS analysis (Agilent 7890A GC/5975C; MSD). Myofibrillar protein-bound l-[ring-²H₅]-phenylalanine enrichments were determined by GC-MS analysis, whereas the l-[1-¹³C]-phenylalanine and l-[1-¹³C]-leucine enrichments were determined by GC-C-isotope ratio mass spectrometer analysis (Trace GC Ultra, IRMS model MAT 253; Thermo Scientific). For complete details, see the Supplementary Data.

To analyze the fatty acid (FA) content and composition, we used a protocol previously applied to macroalgae and seagrasses [88 (link),89 (link),90 (link)] based on the analysis of fatty acid methyl esters (FAMEs) by direct transmethylation. Before the initial extraction process, freeze-dried biomass was powdered using a Beadmill 4—Fisher Scientific (Hampton, NH, USA) machine (five ms⁻¹ for 2 min). Then, we carried out extractions with 2 mL of dry methanol, containing 2% (v/v) H₂SO₄, which we carefully pipetted into each separate vial containing 25 mg (±10 mg) of dry biomass. Then, 10 μL of the FA C15 (pentadecanoic acid) was added to each transmethylation vial as an internal standard. To prevent FA oxidation and degradation, vials were closed under nitrogen atmosphere, and vials containing the samples were heated at 80 °C for 1.5–2 h. After transmethylation, we extracted the FAME which was analyzed using an Agilent 7890A GC/5975C (Santa Clara, CA, USA) mass selective detector (MSD). We identified the FAME using co-chromatography with authentic commercially available FAME standards (Supelco™ 37 Component FAME Mix, catalogue no. 47885-U, Supelco, Bellefonte, PA, USA) and FAME of fish oil (Menhaden Oil, catalogue no. 47116, Supelco).