Norleucine

Sample preparations for analysis of total amino acids were performed as described by Mæhre et al. [11 ]. For the raw material samples, approximately 200 mg of fish and shrimp samples and approximately 50 mg of flour and dulse samples, were dissolved in 0.7 mL distilled H₂O and 0.5 mL 20 mM norleucine (internal standard). For the protein extract samples, 500 µL extract was mixed with 50 µL 20 mM norleucine. Subsequently, for all samples, concentrated hydrochloric acid (HCl, 12 M) was added, to a final concentration of 6 M. The sample mixtures were flushed with nitrogen gas for 15 s in order to minimize oxidation, before hydrolysis at 110 °C for 24 h according to Moore and Stein [12 ]. Following hydrolysis, 100 µL aliquots of the hydrolysates were evaporated under nitrogen gas until complete dryness and re-dissolved to a suitable concentration in lithium citrate buffer at pH 2.2. All amino acids were analyzed chromatographically using an ion exchange column followed by ninhydrin post column derivatization on a Biochrom 30 amino acid analyzer (Biochrom Co., Cambridge, UK). Amino acid residues were identified using the A9906 physiological amino acids standard (Sigma Chemical Co., St. Louis, MO, USA) as described previously [13 (link)]. Protein content was calculated as the sum of individual amino acid residues (the molecular weight of each amino acid after subtraction of the molecular weight of H₂O).

All of the marine and terrestrial samples were collected in 2001–2013 from a stony shore and a farm in Yugawara (35°08′N, 139°07′E), Japan, respectively. The stony shoreline surveyed represented ∼0.2 hectares and ranged in depth from 0 to 5 m, where brown and red macroalgae are dominant primary producers but seagrass is absent. The farm was also approximately 0.2 hectares with cultivation of fruits and vegetables, all of which were C₃ plants. Green leaves and/or nuts were collected for higher plants, and whole samples of 1–15 individuals within a single stage were collected for the other species. The collected samples were cleaned with distilled water to remove surface contaminants and stored at −20°C. For most terrestrial species and marine macroalgae, whole-organism samples were prepared for isotopic analyses. For the remaining marine specimens, small samples of muscle tissue were taken. Shell samples were taken from several gastropod and lobster specimens, and scales were dissected from most of the fish species (Appendices A1 and A2). There was no substantial effect on the trophic position estimates among these different tissue types within a single animal specimen (e.g., Chikaraishi et al. 2010 , 2011 ; Ogawa et al. 2013 ). The bulk-carbon and bulk-nitrogen isotopic compositions of representative samples (40 coastal marine and 69 terrestrial samples, Appendices A1 and A2) were determined using a Flash EA (EA1112) instrument coupled to a Delta^plusXP IRMS instrument with a ConFlo III interface (Thermo Fisher Scientific, Bremen, Germany). Carbon and nitrogen isotopic compositions are reported in the standard delta (δ) notation relative to the Vienna Peedee Belemnite (VPDB) and to atmospheric nitrogen (AIR), respectively.
The nitrogen isotopic composition of amino acids was determined by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) after HCl hydrolysis and N-pivaloyl/isopropyl (Pv/iPr) derivatization, according to the procedure in Chikaraishi et al. (2009 ) (which are described in greater detail at http://www.jamstec.go.jp/biogeos/j/elhrp/biogeochem/download_e.html). In brief, samples were hydrolyzed using 12 Mol/L HCl at 110°C. The hydrolysate was washed with n-hexane/dichloromethane (3/2, v/v) to remove hydrophobic constituents. Then, derivatizations were performed sequentially with thionyl chloride/2-propanol (1/4) and pivaloyl chloride/dichloromethane (1/4). The Pv/iPr derivatives of amino acids were extracted with n-hexane/dichloromethane (3/2, v/v). The nitrogen isotopic composition of amino acids was determined by GC/C/IRMS using a 6890N GC (Agilent Technologies, Palo Alto, CA) instrument coupled to a Delta^plusXP IRMS instrument via a GC-C/TC III interface (Thermo Fisher Scientific, Bremen, Germany). To assess the reproducibility of the isotope measurement and obtain the amino acid isotopic composition, reference mixtures of nine amino acids (alanine, glycine, leucine, norleucine, aspartic acid, methionine, glutamic acid, phenylalanine, and hydroxyproline) with known δ¹⁵N values (ranging from −25.9‰ to +45.6‰, Indiana University, SI science co.) were analyzed after every four to six samples runs, and three pulses of reference N₂ gas were discharged into the IRMS instrument at the beginning and end of each chromatography run for both reference mixtures and samples. The isotopic composition of amino acids in samples was expressed relative to atmospheric nitrogen (AIR) on scales normalized to known δ¹⁵N values of the reference amino acids. The accuracy and precision for the reference mixtures were always 0.0‰ (mean of Δ) and 0.4–0.7‰ (mean of 1σ) for sample sizes of ≥1.0 nmol N, respectively.
The δ¹⁵N values were determined for the following 10 amino acids: alanine, glycine, valine, leucine, isoleucine, proline, serine, methionine, glutamic acid, and phenylalanine (Appendices A1 and A2). These amino acids were chosen because their peaks were always well separated with baseline resolution in the chromatogram (Chikaraishi et al. 2009 ). Also, it should be noted that glutamine was quantitatively converted to glutamic acid during acid hydrolysis; as a result, the α-amino group of glutamine contributed to the δ¹⁵N value calculated for glutamic acid.
The TP_Glu/Phe value (and its potential uncertainty calculated by taking into account the propagation of uncertainty on each factor in the Eq. (1)) was calculated from the observed δ¹⁵N values (as 1σ = 0.5‰) of glutamic acid and phenylalanine in the organisms of interest, using eq. (1) with the β value of −3.4 ± 0.9‰ for coastal marine and +8.4 ± 1.6‰ for terrestrial samples, and with the TDF value of 7.6 ± 1.2‰ for both ecosystems, according to Chikaraishi et al. (2009 , 2010 , 2011 ). The TP_Tr/Scr values were not calculated, because we did not measure the δ¹⁵N values of lysine and tyrosine for all investigated samples and of serine for approximately a half of samples.

Diet samples were analyzed for dry matter by oven drying at 135°C for 2 h (method 930.15; AOAC Int., 2007 ) and for ash (method 942.05; AOAC Int., 2007 ). The gross energy in diets was measured using an isoperibol bomb calorimeter (model 6400, Parr Instruments, Moline, IL, USA) with benzoic acid used as the standard for calibration. Total starch was analyzed by the glucoamylase procedure (method 979.10; AOAC Int., 2007 ), which yields the enzymatically hydrolyzed starch in the sample. Diets were analyzed for N (method 990.03; AOAC Int., 2007 ) using a Leco Nitrogen Determinator (model FP628, Leco Corp., St. Joseph, MI, USA) and crude protein was calculated as N × 6.25. Diets were analyzed for insoluble dietary fiber and soluble dietary fiber on an Ankom Total Dietary Fiber Analyzer (Ankom Technology, Macedon, NY, USA) using method 991.43 (AOAC Int., 2007 ). Total dietary fiber was calculated as the sum of analyzed insoluble and analyzed soluble dietary fiber. Acid-hydrolyzed ether extract in diets was measured by crude fat extraction using petroleum ether (Ankom^XT15, Ankom Technology, Macedon, NY, USA) following hydrolysis using 3N HCl (Ankom^HCl, Ankom Technology, Macedon, NY, USA). Amino acids were analyzed at the University of Missouri Agricultural Experiment Station (Columbus, MO). Diets were analyzed for amino acids on a Hitachi Amino Acid Analyzer, Model No. L8800 (Hitachi High Technologies America, Inc; Pleasanton, CA, USA) using ninhydrin for post-column derivatization and norleucine as the internal standard. Prior to analysis, samples were hydrolyzed with 6N HCl for 24 h at 110°C [method 982.30 E(a); AOAC Int., 2007 ]. Methionine and Cys were determined as Met sulfone and cysteic acid after cold performic acid oxidation overnight before hydrolysis [method 982.30 E(b); AOAC Int., 2007 ]. Tryptophan was determined after NaOH hydrolysis for 22 h at 110°C [method 982.30 E(c); AOAC Int., 2007 ]. The corn and hybrid rye used in the experiment were analyzed for mycotoxins at Trilogy Analytical Laboratories (Washington, MO, USA) using liquid chromatography-tandem mass spectroscopy. The detection limit was 0.1 mg/kg for 15-acetyl deoxynivalenol, 3-acetyl deoxynivalenol, deoxynivalenol, fumonisin (B1, B2, and B3), fusarenon X, and nivalenol. Citrinin and diacetoxyscirpenol had a detection limit of 0.05 mg/kg, neosolaniol had a detection limit of 0.02 mg/kg. The detection limit for zearalenone was 0.0125 mg/kg, the detection limits for HT-2 and T-2 toxins were 0.005 mg/kg, and detection limits for aflatoxin B1, B2, G1, and G2 and for ochratoxin A was 0.001 mg/kg. Analysis of ergot alkaloids in hybrid rye was conducted by refractive index high-performance liquid chromatography using Phenomenex Strata-X-CW weak cation exchange and a reversed-phase column with a detection limit of 10 µg/kg (Phenomenex, Inc., Torrance, CA, USA).

LCL cells were placed in T‐25 flasks upright at 1 × 10⁶ cells per ml in 10 ml RPMI ± Asn per replicate. After 24 h, the media were collected, cleared by centrifugation, and stored frozen at −80°C until processing. The cells were washed with cold 0.9% saline, then flash frozen in liquid nitrogen, and stored at −80°C as well. Cell pellets and media samples were processed and analyzed by gas chromatography‐mass spectrometry (GC‐MS), as reported previously.²⁴, ³¹, ³² GC‐MS data acquisition was accomplished with a Thermo Scientific Single Quadrupole Mass Spectrometer (ISQ) and Gas Chromatograph (Trace 1310). Amino acid peak areas were processed with XCalibur Quan Browser software and normalized to the peak area of a DL‐norleucine internal standard. Asn concentrations were quantified with an external calibration curve.

Indomethacin (IMC) was purchased from Sigma Aldrich (purity ≥ 99%). It is a model drug which presents a rich and very original through polymorphism through the α–γ monotropic system. Indeed, the commercial and more stable phase, namely the γ form [8 (link)], is not the denser phase. The long-range order is induced by the formation of dimers via H-bonding of the carboxylic acid group. The metastable α phase is denser than the γ form because it is composed of more compact trimers [9 (link)]. It was shown that devitrification of IMC occurs in the γ or α phase depending on whether the temperature of recrystallization is below or above the T_g, respectively [10 (link)]. The recrystallized α phase remains stable by cooling down to room temperature. The α phase can also be prepared by dissolution in methanol and precipitation in water at room temperature [11 (link)]. The molecular structure of IMC and the molecular packing distinctive of the α and γ phases are plotted in Figure S2.
Amino acids (AAs), i.e., L-leucine (LEU, C₆H₁₃NO₂, purity ≥ 98%), L-norleucine (NLE, C₆H₁₃NO₂, purity ≥ 98%), L-tert-leucine (TLE, C₆H₁₃NO₂, purity ≥ 99%) and L-arginine (ARG, C₆H₁₃NO₂, purity ≥ 98%) were purchased from Sigma Aldrich (St. Louis, MO, USA) and used as received without any further purification.