Alanine

Standards of amino acids (AA) namely phenylalanine (Phe), tryptophan (Trp), isoleucine (Ile), leucine (Leu), asparagine (Asn), methionine (Met), valine (Val), proline (Pro), tyrosine (Tyr), alanine (Ala), threonine (Thr), glycine (Gly), serine (Ser), glutamic acid (Glu), aspartic acid (Asp), arginine (Arg), glutamine (Gln), lysine (Lys), histidine (His), cysteine (Cys) and cystine (Cys2),were purchased from Sigma-Aldrich (Steinheim, Germany). Acetonitrile (ACN) and water (LC-MS grade) was purchased from Carlo Erba (Milan, Italy). Formic acid (LC-MS additive), TGA (thioglycolic acid; ≥98%), and ammonium formate (NH₄HCO₂) were purchased from Sigma-Aldrich (Steinheim, Germany). Analytical grade hydrochloric acid (HCl) 37% w/w was also supplied from Carlo Erba (Milan, Italy). The chromatographic column HILIC amide BEH, Acquity UPLC 1.7 m, 2 × 150 mm (Waters) were used.

Adult male Sprague-Dawley rats weighing 230 ± 10 g were obtained from the Animal Breeding Centre of Beijing Vital River Laboratories Company (Beijing, China). The project identification code was 20162005. All animals were housed at 22 ± 2°C with a relative humidity of 50 ± 10% and a 12 h light/12 h dark cycle. The animals had free access to water and fodder (Beijing Keaoxieli Co. Ltd.). All experimental animal procedures were approved by the China Academy of Chinese Medical Sciences' Administrative Panel on Laboratory Animal Care and performed in accordance with institutional guidelines and ethics of the committee as part of the China Academy of Chinese Medical Sciences (February 1^st, 2016).
Ten AA standards including alanine, valine, taurine, leucine, isoleucine, glutamine, glutamate (glutamic acid), phenylalanine, (S)-tyrosine, and tryptophan (purity > 99%, all) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile and methanol were provided from Fisher Scientific (Shanghai, China). Formic acid, ammonium formate, and phenylalanine-d5 (internal standard (IS)) were purchased from Toronto Research Chemicals (YTO, Canada). All chemicals used throughout this study were of HPLC grade unless stated otherwise. BNC (batch number: 140156) was provided from Buchang Pharma Co. Ltd.

Acrylamide (>99.8%), asparagine, glutamine, aspartic acid, alanine, valine, gamma-aminobutyric acid (GABA), NaCl, KCl, MgCl₂, CaCl₂, thiamine hydrochloride (vitamin B1), nicotinic acid (vitamin B3), pyridoxal hydrochloride (vitamin B6), sucrose, D-glucose, D-fructose, glacial acetic acid (≥99%), and citric acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). Analytical-standard-grade (97%) 5-hydroxymethylfurfural (HMF) was purchased from Thermo Fisher (Kandel GmbH, Kandel, Germany).

HPLC-grade formic acid was obtained from Sigma-Aldrich (USA). HPLC/MS-grade formic acid, ammonium formate, and acetonitrile were acquired from Fisher Scientific (USA). The internal standards Labeled Carnitine Standards (L-carnitine (N-trimethyl-D₉), L-acetylcarnitine (N-methyl-D₃), L-propionylcarnitine (Nimethyl-D₃), L-butyrylcarnitine (N-methyl-D₃), L-isovalerylcarnitine (N-trimethyl-D₉), L-octanoylcarnitine (N-methyl-D₃), L-myristoylcarnitine (N-trimethyl-D₉), L-palmitoylcarnitine (N-methyl-D₃)) were attained from Cambridge Isotope Laboratories. Seventeen amino acid standards (alanine, valine, leucine, isoleucine, phenylalanine, methionine, proline, glycine, serine, threonine, cysteine, tyrosine, histidine, lysine, arginine, aspartic acid, glutamic acid) were purchased from Sigma-Aldrich. Eight amino acid standards (tryptophan, asparaginate, glutamine, ornithine, taurine, citrulline, cysteine, γ-aminobutyric acid) were gained from J&K Scientific. Ultrahigh purity water was prepared by Millipore-Q Water Purification System (Millipore, Germany).

The cell line AGE1.CR.pIX was provided by ProBioGen AG and cultivated in 125 mL vented and baffled shaking flasks (wv, working volume 50 mL) at 185 rpm, 37 °C, and 5% CO₂. The chemically defined CD-U3 medium (Biochrom/Merck) was supplemented with 2 mM glutamine (Sigma), 2 mM alanine (Sigma), and 10 ng/mL recombinant insulin-like growth factor (LONG³ IGF, Sigma). Cells were passaged twice a week by dilution to the starting cell concentration of 8 × 10⁵ cells/mL using fresh medium.
Adherent MDCK cells (ECACC, #84121903), used for the innocuity test (see Results, UV irradiation for inactivation of standard virus) and TCID₅₀ assay (see Measurement of virus titers), were maintained in T75 flasks containing 50 mL GMEM (GIBCO) supplemented with 10% (v/v) fetal calf serum (PAN Biotech) and 1% (v/v) peptone (Lab M) at 37 °C in a 5% CO₂ atmosphere.