L alanine

To induce wasting via growth factor deprivation, cells were washed once in serum free DMEM (Life Technologies, Australia) and then incubated in DMEM (i.e., standard amino acid composition) containing 1% L-glutamine and 1% antibiotic solution (Life Technologies) but lacking 2% horse serum for 48 h (SF) (9 (link)). SF was supplemented with an additional 2.5 mM glycine (Sigma-Aldrich, Castle Hill, NSW, Australia) or L-alanine (Sigma-Aldrich). To induce wasting via nutrient starvation, cells were washed once in HEPES buffered saline (HBS; 20 mM HEPES/Na pH 7.4, 140 mM NaCl, 2.5 mM MgSO₄, 5 mM KCl, and 1 mM CaCl₂, no amino acids present), then incubated in HBS (9 (link), 10 (link)) with glycine or equimolar concentrations of L-alanine for up to 5 h. L-alanine serves as an isonitrogenous control as it does not modulate cell size and protein turnover in cell and animal models (4 (link)–6 (link), 9 (link), 10 (link)).
Rapamycin (100 nM, Sigma-Aldrich) was used to inhibit mTORC1 activation (10 (link)). We have previously reported that these atrophy models are not associated with altered myotube viability as assessed by Trypan Blue staining (9 (link)).

Reagents and instrumentsRacemic propranolol, S-(-)-propranolol and R-(+)-propranolol were purchased from Sigma-Aldrich (USA). L-alanine, Cu (CH₃COO) ₂.H₂O, Cu (NO₃)₂.3H₂O, Cu (SO₄)₂.5H₂O, Cu (Cl) ₂.2H₂O and metanol were obtained from Merck (Germany). Chromatographic separations were carried out using a Cecil 1100 HPLC instrument, equipped with columns (125 mm Length× 4.0 mm I.D., particle size 5 μm, HiCHROM) packed with LiChrosorb RP8 and LiChrosorb RP18. Detection of eluted species was carried out by a UV detector (Cecil 1100, λ=275 nm). The mobile phase used was a mixture of methanol/water containing Cu (II) salts and L-alanine (pH=5.0). Injection volume of 15 μL was utilized in the chromatographic experiments.
Analysis of propranolol enantiomers in human blood serum In order to analyze propranolol enantiomers in plasma sample, µL amount of chiral propranolol standard solutions was injected in 5 mL plasma sample in order to adjust the propranolol concentrations in the desired levels. Then, the described plasma samples were mixed with 0.5mL of 30% ammonium hydroxide and 3 mL of 10% chloroform in n-heptane, shaken for 6 min, and centrifuged; the upper phase was evaporated to dryness under a stream of nitrogen. The residues were dissolved in 50 μL of methanol and injected to HPLC system for the analysis.

A pure standard solution of n‐alkanes (from n‐C7 to n‐C30) for linear retention indices (I^T) calibration and system quality control was from Merck (Milan, Italy) and prepared in toluene at the concentration of 100 mg L⁻¹.
The internal standard (IS) 1,4-dibromobenzene (from Merck, Milan, Italy) solution was prepared in toluene at a concentration of 10 g L⁻¹.
Pure reference standards used for identity confirmation of pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, 2-ketoglutaric acid, 3-hydroxybutyric acid, fumaric acid, 2-keto-3-metilvaleric acid, aspartic acid, hippuric acid, citric acid, uric acid, l-alanine, l-valine, l-leucine, l-proline, glycine, l-threonine, l-tyrosine, l-phenylalanine, l-isoleucine, l-methionine, l-cysteine, l-ornithine, l-tryptophan, xylitol, ribitol, fructose, galactose, glucose, mannitol, myo-inositol, glycerol, palmitic acid, stearic acid, and creatinine were from Merck (Milan, Italy).
Derivatizing agents O-methyl hydroxylamine hydrochloride (MOX), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and LC-grade pyridine, n-hexane, dichloromethane, and toluene used as solvents were all from Merck (Milan, Italy).