Biochrom 30 amino acid analyser

Urinary cystine and COLA concentrations were determined by qualitative cyanide-nitroprusside test [44 (link)] and quantitative amino acid analyzer (Biochrom 30+ Aminoacid Analyser, Biochrom Ltd., Cambridge, UK) [45 ]. Quantitative crystallographic analysis of calculi was performed by the Hill’s Minnesota Urolith Center, University of Minnesota [46 (link)].

Ramos wild type and ADPGK KO cells were seeded at 1 million cells/ml in 6-well plates and stimulated with PMA for two days and seven days, at the end of which, cells were harvested, and media was collected in separate 15 ml falcons. Media was further centrifuged at high speed and additionally passed through 0.35 µm filters to remove residual cells. 200 µl of the sample was mixed with 50 µl Sulfosalicylic acid and centrifuged at maximum speed on a table-top centrifuge. Supernatant was collected, and amino acid analysis was performed by injecting the samples into a Biochrom 30 + Amino acid analyser (Biochrom, UK) based on ion exchange chromatography, with resulting data expressed as µmol/litre. For analysis of cellular amino acids, cells were lysed using RIPA buffer and centrifuged at 13,000 g on a table top centrifuge to remove the cell debris. Supernatant was collected and analysed for amino acids as described above.

The extraction of free amino acids was done as described by Hacham et al. (2002) (link). Samples of 50 mg of frozen leaf powder were homogenized with 600 μl of water:chloroform:methanol (3:5:12 v/v/v). After centrifugation at 12,000 rpm for 2 min, the supernatant was collected and the residue was re-extracted with 600 μl of the same mixture, pooling the two supernatants. A mixture of 300 μl of chloroform and 450 μl of water were added to the supernatants, and after centrifugation the upper water:methanol phase was collected and dried in the SpeedVac. The samples were dissolved on 100 μl of sodium citrate loading buffer pH 2.2 (Biochrom, United States) and 10 μl were injected on a Biochrom 30 Amino Acid Analyser (Biochrom, United States) at the Protein Chemistry Service at CIB (CSIC, Madrid, Spain).

The feed was dried and ground prior to analysis, and analyses were performed in duplicate for dry matter by drying to a constant weight at 104 °C, for ash by combustion at 550 °C, for crude protein by Kjeldahl nitrogen × 6.25 according to Commission Regulation (EC) No 152/2009, and for starch as described in McCleary et al. [25 (link)]. Lipid was determined after extraction with petroleum ether and acetone (70/30) on an accelerated solvent extractor (ASE 200) (Dionex Corp, Sunnyvale, CA, USA), while gross energy was established with a PARR 1281 Adiabatic bomb calorimeter (Parr Instruments, Moline, IL, USA) according to ISO 9831. Amino acids were analysed according to Commission Regulation (EC) No 152/2009, for all amino acids except tryptophan, on a Biochrom 30 amino acid analyser (Biochrom Ltd,. Cambridge, UK). Tryptophan was analysed according to Commission Regulation (EC) No 152/2009 with a Dionex Ultimate 3000 HPLC system (Dionex Softron GmbH, Germering, Germany) and a Shimadzu RF-535 fluorescence detector (Shimadzu Corporation, Kyoto, Japan).

Carbohydrates and fermentation metabolites were extracted from a homogeneous suspension of flour or sourdough (5 g) and H₂SO₄ 10 mM (45 ml) (Minervini et al., 2012a (link)). Glucose, fructose, maltose, sucrose, lactic acid (only sourdough samples), acetic acid (only sourdough samples) and ethanol (only sourdough samples) were quantified using enzymatic Assay Kits (Megazyme International Ireland Limited, Bray, Co. Wicklow, Ireland), following the manufacturer’s instructions. A second water-soluble extract was prepared for analysis of free amino acids (FAA), using Tris-HCl 50 mM pH 8.8 as homogenizing solution (Minervini et al., 2012a (link)), and analyzing the water-soluble extract through the Biochrom 30 Amino Acid Analyser (Biochrom LTD, Cambridge, United Kingdom) (De Angelis et al., 2007 (link)).

To analyse the amino acid profile of medium from cell cultures, 100 μL of the medium sample was mixed with 100 μL of internal standard (12 mg of norleucine mixed with 15 g sulphosalicylic acid in 250 mL of water). The analysis was performed according to the method of Moore, Spackman and Stein [60] on a Biochrom 30™ Amino acid Analyser (Biochrom.co.uk). Acquisition and data handling were done with Thermo Scientific™ Chromeleon™ 7.2 Chromatography Data System software (Thermo Fisher Scientific).