L phenylalanine

Glycine anhydride (99.0%, Alfa Aesar, Kandel, Germany), L-Alanine (99.0%, Alfa Aesar, Kandel, Germany), L-Methionine (pure, pharma grade, AppliChem, Barcelona, Spain), L-Valine (99.0%, Alfa Aesar, Kandel, Germany), L-Phenylalanine (98.5%–101.0%, specified according to the requirements of USP, Acros Organics, Barcelona, Spain), zinc sulfate monohydrate (99.0%, Acros Organics, Barcelona, Spain) were used in experiments. All the media (BHIB: Brain Heart Infusion Broth; MHA: Muller Hinton Agar; SDB: Sabouraud Dextrose Broth) were procured from HiMedia™ Laboratories Pvt. Ltd., Thane West, India and all other reagents and chemicals (sodium hydroxide, ethanol) used were of analytical grade. For dilution, highly purified water was used, obtained using the Milli-Q^® purification system (Merck, Darmstadt, Germany).

Fresh beetroots were obtained from a supermarket in Greensboro, NC, USA. Methanol, HPLC grade chloroform and hexane were purchased from Fisher-Scientific, Waltham, MA, USA. Deuterated chloroform (CDCl₃) with 1% v/v 3-(Trimethylsilyl)propionic-2, -3-(Trimethylsilyl)prop-98 atom % D (TSP) was obtained from Acros Organics, Morris Plains, NJ, USA. Optima grade water and Acetonitrile for LCMS were obtained from Fisher-Scientific, Waltham, MA, USA. Sodium phosphate dibasic (Na₂HPO₄, 99.0%) was obtained from Alfa Aesar, Tokyo, Japan; sodium phosphate monobasic (NaH₂PO₄, 99.0%), and sodium azide 99% extra pure were obtained from Acros Organics, Morris Plains, NJ, USA. L-lysine (C₆H₁₄N₂O₂, 98.5%), L-leucine (C₆H₁₃NO₂, 98.5%), L-histidine (C₆H₉N₃O₂, 98.5%), L-phenylalanine (C₉H₁₁NO₂, 98.5%) and sucrose (C₁₂H₂₂O₁₁) were purchased from Fisher BioReagents, Pittsburgh, PA, USA.

L-Proline (>85%), ninhydrin, choline chloride, and (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) were purchased from Acros Organics (Geel, Germany). Lactic acid was acquired from Panreac (Barcelona, Spain), and gallic acid, Folin–Ciocalteu (F-C) reagent, and sodium carbonate from VWR (Leuven, Belgium). Sodium hydroxide (NaOH), ascorbic acid, potassium iodide, and acetic acid were supplied by Merck (Darmstadt, Germany). Trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), hydrogen peroxide (H₂O₂), L-Proline (≥99%), indole-3-acetic acid, toluene, Bradford reagent, glucose, shikimic acid, periodic acid, cinnamic acid, Bovine Serum Albumin (BSA), luteolin, protocatechuic acid, and epigallocatechin gallate, HPLC-MS-grade water, HPLC-MS grade acetonitrile, and formic acid were purchased from Sigma–Aldrich (Steinheim, Germany). Quercetin and rosmarinic acid were acquired from Extrasynthese (Genay, France). Catechin, caffeic, and ρ-coumaric acids were supplied by AASC Ltd. (Southhampton, UK). Phenol, glycine, sulfuric acid, 6-benzyladenine were acquired from Fluka (Buchs, Switzerland), and L-phenylalanine was obtained from Fisher Scientific (Leicestershire, UK).

Penicillin–streptomycin 100,000 UI/mL, poly(ethylenimine) (PEI) 1300~50% in H₂O, sodium citrate, cytochrome c from equine heart, transferrin, fibrinogen, human serum albumin, bovine serum albumin, staurosporine from streptomyces, and 3-(4,5-dimethulthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Slide-A-Lyzer^® Dialysis Cassette–2000 MWCO 3.0–12 mL Capacity and DRAQ5™ were purchased from Thermo Scientific, Rockford, IL, USA. l-Valine, l-Serine, l-Methionine, l-Leucine, l-Aspartic Acid, l-Tryptophan, l-Asparagine monohydrate, l-Phenylalanine were purchased from ThermoFischer (Kandel, Germany). Fetal bovine serum and phosphate saline buffer were purchased from Gibco, Grand Island, NY, USA. Etoposide was purchased from Fresenius Kabi, Bad Homburg, Germany. CytoPainter Mitochondrial Staining Kit Red Fluorescence was purchased from Abcam, Cambridge, UK. Phalloidin-FITC Acti-stainTM 488 Fluorescent Phalloidin was purchased from Cytoskeleton Inc., Denver, CO, USA. Prolong^® Gold Antifade Reagent Molecular Probes was purchased from Cell Signaling Technology, Danvers, CO, USA. Double distilled water (18.2 MΩ) was used as a solvent.

Commercial fish feed (L2 Alterna^® Skretting, Burgos, Spain), with a size of 2.2 mm, was used as the control diet. L-phenylalanine and L-tyrosine (dry powder) were purchased from ThermoFisher (Kandel, Germany). The commercial control diet was finely ground and mixed with AA and later water (300 mL Kg^–1 dry feed). The amount of phenylalanine and tyrosine in each experimental diet was 5% (on dry feed) except for the control diet which did not have any amino acid. The mixture was thread pelleted into 2 mm diameter and 20–25 cm length strips. These were cut to get 2–3 mm size pellets. Finally, these food strips were dried at 30°C for 48 h and stored at 4°C.
Analysis of AA was performed as reported previously (Herrera et al., 2020 (link); Salamanca et al., 2020 (link)). According to the above method, the final amino acid concentrations in the experimental diets are shown in Table 1.

All drugs and supplemented metabolites were added at initiation of starvation, unless otherwise stated. BHAM (150 μM–1.5 mM), oligomycin (10 μM), FCCP (100 nM–5 μM), rotenone (100 nM–1 μM), succinate (10 mM), ascorbate (10 mM), TMPD (100 μM), glutamate (10 mM), malate (10 mM), antimycin A (10 μM), ADP (4 mM), l-cysteine (100 μM–2 mM), cystine, (0.05–1 mM) N-acetyl cysteine (0.5–10 mM), reduced GSH (1–20 mM), 2-deoxyglucose (1–10 mM), koningic acid (10–20 μM), glucose (1–20 mM), NH₃ (1–5 mM), NH₄Cl (1–10 mM), 3-methyladenine (1–10 mM), MG132 (10 μM), l-buthionine sulfoximine (100 μM–1 mM) and erastin (100 μM) were all from Sigma. Bafilomycin (10–100 nM) was from Cell Signaling. Rapamycin (20–500 nM) was from LC Laboratories. Sulfasalazine (1 mM) was from Tocris Bioscience. MitoParaquat (1–10 μM) was from Abcam. We diluted 10× essential amino acids from 50× MEM amino acids solution (ThermoFisher), for final concentrations of 6 mM l-arginine hydrochloride, 1 mM l-cystine, 2 mM l-histidine hydrochloride-H₂O, 4 mM l-isoleucine, 4 mM l-leucine, 4 mM l-lysine hydrochloride, 1 mM l-methionine, 2 mM l-phenylalanine, 4 mM l-tryptophan, 2 mM l-tyrosine and 4 mM l-valine.

Gibco™ DMEM High Glucose and Gibco™ DMEM No Glucose cell culture media were purchased from Life Technologies Europe (Bleiswijk, The Netherlands) via Merck (Darmstadt, Germany). HyClone ™ Fetal Bovine Serum was acquired from GE Healthcare Life Scienes (Logan, UT, USA). MEM Non-essential Amino Acid Solution was supplied by Sigma^® Life Science (Gillingham, UK). D (+)-Glucose was supplied by Carl Roth^® (Karlsruhe, Germany). Aspartame was purchased from Sigma Aldrich^® (St. Louis, MO, USA). L-aspartic acid and L-phenylalanine were acquired from Alfa Aesar by Thermo Fisher Scientific (Kandel, Germany). Methanol and high quality HPLC water were purchased from Fisher Chemicals^® (Loughborough, UK).