Biochrom 30 amino acid analyzer

Free amino acids were analyzed by a Biochrom 30 amino acid analyzer (Biochrom Co. Ltd., Holliston, MA, USA). The extraction and analysis method was according to Song with modification [30 (link)]. One gram of defatted sesame cake (accurate to 0.0001 g) was weighed into a 50 mL grinding triangle flask, and 20 mL lithium loading buffer (Biochrom Co. Ltd., USA) was added. Ultrasonic extraction (300 W) was carried out for 30 min. After centrifugation (10,000 rpm) and filtration, the samples were detected by an automatic analyzer.

The concentration of mouse or human RAGE was determined by quantitative amino acid analysis. The analyses were performed in triplicate with approximately 2 µg of purified mouse or human soluble RAGE. The samples were dried in 500-µl polypropylene vials, the lids were punctured and the vials placed in a 25-ml glass vial equipped with a MinInert valve (Pierce Biotechnology). A total of 200 µl 6 N HCl containing 0.1% phenol was placed in the bottom and the glass was purged with argon before vacuum was applied. The samples were incubated at 110°C for 18 h. The samples were subsequently re-dissolved in 50 µl of 0.2 M sodium citrate loading buffer at pH 2.2 (Biochrom, Cambridge, UK), transferred into microvials and loaded onto a BioChrom 30 amino acid analyzer (Biochrom). Data analysis was performed using in-house developed software. The extinction coefficients were calculated based on the determined protein concentrations and the UV absorbance at 280 nm.

Both commercial samples and reference Fontina PDO samples were analyzed. Twenty-one free amino acids (FAAs) were determined using a Biochrom 30+ amino acid analyzer (Biochrom Ltd., Cambridge, UK) according to the procedure described by Hogenboom et al. [15 (link)] with minor modifications. Briefly, 3 g of grated cheese was added to 40 mL of 0.2 N sodium citrate buffer, homogenized with an UltraTurrax (Ika T25, Sigma Aldrich, Milan, Italy), deproteinated with 7.5% 5-sulfosalicilic acid and filtered through a 0.22 µm membrane filter prior to injection. The chromatographic separation was achieved by a 7-buffer gradient, including the column washing step, and multipoint calibration was used. Analyses were carried out in duplicate.

The amino acid content of heart and skeletal muscle homogenates was analyzed by high-performance liquid chromatography (HPLC) using a methodology certified by the Spanish National Accreditation Entity ENAC (Entidad Nacional de Acreditación) (ISO15189) used for the analysis of clinical samples in the Clinical Biochemistry Department of the Hospital 12 de Octubre. Briefly, homogenate samples containing 1.7 and 2.0 mg of total protein for heart and skeletal muscles homogenates, respectively, were deproteinized with 50% sulfosalicylic acid and analyzed by ion exchange chromatography with post-column derivatization with ninhydrin on a Biochrom 30+ amino acid analyzer (Biochrom Ltd.; Cambridge, UK). Amino acid peaks were detected and quantified with OpenLAB EZChrom Edition software A.04.10 (Siegwerk Druckfarben AG & Co. KGaA; Siegburg, Germany).

Twenty plasma amino acids will be analyzed by using the high-performance liquid chromatography (Biochrom30 amino acid analyzer, Biochrom).

Amino acids were determined in the BSCH, which showed higher molecular weight in the commercial hydrolyzed collagen. For this purpose, acid hydrolysis was performed using 6 N HCl containing 0.1% phenol under an inert atmosphere by heating at 110 °C for 24 h. Then, HCl was removed by vacuum. The hydrolysate was resuspended in 20–50 µL of 0.2 M sodium citrate buffer (pH 2.2), to which a known amount of norleucine was added as an internal standard and applied to an automated amino acid analyzer (Biochrom30 Amino Acid Analyzer, Biochrom, Cambridge, UK).

Colon digesta SCFA and biogenic amines were quantified as described previously [32 (link),33 (link)]. Briefly, SCFA analysis of digesta was performed by acidifying the samples with oxalic acid, followed by centrifugation for 3 min at 14,000 g and adding the internal standard (caproic acid). A gas chromatograph (Agilent Technologies 6890N, autosampler G2614A and injection tower G2613A; Network GC Systems, Böblingen, Germany) was used. Ion exchange chromatography was performed with a Biochrom 30 Amino Acid Analyzer (Biochrom) to analyse biogenic amines (putrescine, cadaverine, spermidine, spermine, propylamine, tyramine). Trichloroacetic acid (10%) was added to the digesta samples. After homogenisation and filtering (0.2 µm pore size), samples (25 µL) were injected onto a 10 cm polyamine ion-exchange column (Laborservice Onken GmbH, Gründau, Germany). The eluent was sodium citrate buffer (pH 7.2). Amines were quantified after post-column ninhydrin derivatisation by photometric detection at 570 nm [33 (link)].